Femtosecond pulsed laser ablation to enhance drug delivery across the skin

Laser poration of the skin locally removes its outermost, barrier layer, and thereby provides a route for the diffusion of topically applied drugs. Ideally, no thermal damage would surround the pores created in the skin, as tissue coagulation would be expected to limit drug diffusion. Here, a femtosecond pulsed fiber laser is used to porate mammalian skin ex vivo. This first application of a hollow core negative curvature fiber (HC‐NCF) to convey a femtosecond pulsed, visible laser beam results in reproducible skin poration. The effect of applying ink to the skin surface, prior to ultra‐short pulsed ablation, has been examined and Raman spectroscopy reveals that the least, collateral thermal damage occurs in inked skin. Pre‐application of ink reduces the laser power threshold for poration, an effect attributed to the initiation of plasma formation by thermionic electron emission from the dye in the ink. Poration under these conditions significantly increases the percutaneous permeation of caffeine in vitro. Dye‐enhanced, plasma‐mediated ablation of the skin is therefore a potentially advantageous approach to enhance topical/transdermal drug absorption. The combination of a fiber laser and a HC‐NCF, capable of emitting and delivering femtosecond pulsed, visible light, may permit a compact poration device to be developed.

Using a femtosecond pulsed, visible laser beam to create an array of micropores in dyed mammalian skin, with little collateral, thermal damage, leads to an enhancement in the percutaneous permeation of caffeine in vitro.     

Cage opening and fragmentation of C60 fullerene induced by an ultrashort laser pulse

Response of C₆₀ fullerene to a 40 fs full-width at half-maximum laser pulse with a photon energy of 2.0 eV and different laser intensities is studied by semiclassical dynamics simulation technique. The simulation results show that soon after the irradiation with a strong laser pulse, many C–C bonds abruptly break but no fragments are produced. The breaking of multiple C–C bonds induces a quick increase in the kinetic energy and potential energy and a decrease in electronic energy. These results suggest that the opening of the C₆₀ cage is an effective channel for the conversion of electronic energy to kinetic energy for the electronically excited C₆₀ fullerene.     

Influence of environmental conditions in bovine bone ablation by ultrafast laser

Ultrafast lasers are promising tools for surgical applications requiring precise tissue cutting. Shallow ablation depth and slow rate as well as collateral damage are common barriers limiting the use of laser in clinical applications. Localized cooling with water and/or air jet is known to reduce collateral thermal damage. We studied the influence of environmental conditions including air, compressed air flow, still water and water jet on ablation depth, ablation rate and surface morphology on bovine bone samples with an 800 nm femtosecond laser. At 15 J/cm², no thermal effect was observed by electron microscopy and Raman spectroscopy. The experimental results indicate that environmental conditions play a significant role in laser ablation. The deepest cavity and highest ablation rate were achieved under the compressed air flow condition, which is attributed to debris removal during the ablation process. The shallowest ablation depth and lowest ablation rates were associated with water flushing. For surface morphology, smooth surface and the absence of microcracks were observed under air flow conditions, while rougher surfaces and minor microcracks were observed under other conditions. These results suggest that ultrafast ablation of bone can be more efficient and with better surface qualities if assisted with blowing air jet.      

In situ tissue classification during laser ablation using acoustic signals

We suggest a novel method to classify the type of tissue that is being ablated, using the recorded acoustic sound waves during pulsed ultraviolet laser ablation. The motivation of the current research is tissue classification during vascular interventions, where the identification of the ablated tissue is vital. We classify the acoustic signatures using Mel‐frequency cepstral coefficients (MFCCs) feature extraction with a Support Vector Machine (SVM) algorithm, and in addition, use a fully connected deep neural network (FC‐DNN) algorithm. First, we classify three different liquids using our method as a preliminary proof of concept. Then, we classify ex vivo porcine aorta and bovine tendon tissues in the presence of saline. Finally, we classify ex vivo porcine aorta and bovine tendon tissues where the acoustic signals are recorded through chicken breast medium. The results for tissue classification in saline and through chicken breast both show high accuracy (>98%), based on tens of thousands of acoustic signals for each experiment. The experiments were conducted in a noisy and challenging setting that tries to imitate practical working conditions. The obtained results could pave the way towards practical tissue classification in various important medical procedures, achieving enhanced efficacy and safety.     

5-氨基酮戊酸光动力疗法对不同疾病来源的耐甲氧西林金黄色葡萄球菌的体外杀伤作用

光动力疗法(photodynamic therapy,PDT)利用光敏剂与光源反应后产生的活性氧,破坏细菌组分,进而致细菌死亡。其多靶位杀伤特性在治疗耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)感染方面有应用前景,但相关研究尚处于起步阶段。本研究MRSA菌株取自烧伤、急性咽炎、鼻窦炎和肺炎4类临床常见MRSA感染性疾病患者,使用5-氨基酮戊酸(5-aminolevulinic acid,ALA)光敏剂、发光二极管光源,于体外检测ALA介导的PDT(ALA-PDT)对MRSA菌株的杀伤作用。结果显示,经5mmol/L ALA孵育1h后,给予360J/cm~2强光[(633±10)nm]照射1h,ALA-PDT对MRSA菌株具有1.80log_(10)cfu的有效杀伤作用。结果提示,在相同实验条件和参数下,ALA-PDT对上述4种疾病来源的MRSA菌株体外杀伤作用无统计学差异。     

Effect of curcumin-nanoemulsion associated with photodynamic therapy in breast adenocarcinoma cell line

Curcumin, a natural compound has several antineoplastic activities and is a promising natural photosensitizer used in photodynamic therapy. However, its low solubility in physiological medium limit the clinical use of curcumin. This study aimed to analyze the action of curcumin-nanoemulsion, a new and well-designed Drug Delivery System (DDS+) molecule, used as a photosensitizing agent in photodynamic therapy in an in vitro breast cancer model, MCF-7 cells. The empty nanoemulsion fulfils all necessary requirements to be an excellent DDS. Furthermore, the use of curcumin-nanoemulsion in photodynamic therapy resulted in a high phototoxic effect after activation at 440?nm, decreasing to <10% viable tumor cells after two irradiations and increasing the reactive oxygen species (ROS) production. The use of curcumin-nanoemulsion associated with photodynamic therapy resulted in an increase in the levels of caspase 3/7 activity for the studied MCF-7 cell model, indicating that this therapy triggers a cascade of events that lead to cell death, such as cellular apoptosis. In conclusion, curcumin-nanoemulsion proved to be efficient as a photosensitizing agent, had phototoxic effects, significantly decreased the proliferation of MCF-7 cells and stimulating the ROS production in combination with photodynamic therapy, so, this formulation has a great potential for use in treatment of breast cancer.     

The detection of elements in larval otoliths from Atlantic herring using laser ablation ICP-MS

Trace element concentrations of otoliths from larval herring Clupea harengus collected from known spawning beds in the Celtic and Irish Seas, were investigated using laser ablation ICP-MS and compared with concentrations in the larval cores of juvenile otoliths from the same populations and year class. A range of elements (Mg, Zn, Sr, Ba and Pb) was detectable in early larval otoliths (20–40 µm diameter). Larval otolith concentrations exceeded the larval core concentrations of juvenile otoliths and also the concentrations reported in the literature, for Mg, Zn, Ba and Pb, indicating that the measurement of elements in larval otoliths was severely affected by post-mortem contamination, most likely due to adherence of tissue and endolymph residue on the otolith surface. Comparison of otolith composition between larvae from two freezing treatments showed that contamination from Mg and Zn was more serious in otoliths that had remained in frozen larvae for prolonged periods. Larval populations from the two seas showed significant differences in otolith Sr concentrations, which were consistent over two sampling years. Similar differences were seen in the corresponding juvenile populations. The results show that while early larval otoliths are extremely susceptible post-mortem contamination, Sr concentrations can be reliably measured using laser ablation ICP-MS and for this element, the detection of region specific differences is possible.     

光动力疗法对体外培养人胰腺癌细胞株PANC-1作用

目的:探讨血卟啉注射液(Hematoporphyrin Derivative,HPD)光动力疗法(Photodynamic Therapy,PDT)对体外培养的人胰腺癌细胞株PANC-1的生物作用。方法:实验分为4组,空白对照组、单纯HPD组、单纯光照组及HPD+PDT组。采用MTT法检测光动力作用后细胞的存活率,并用Annexin V-FITC/P I双染法检测其凋亡率。结果:在光敏剂浓度为5mg/L,光照剂量为10J/cm2时,光动力对PANC-1细胞达到最佳的实验效果,与对照组相比差异有显著性。在此实验参数条件下,流式细胞术(FCM)检测各组人胰腺癌细胞PANC-1凋亡率:HPD+PDT实验组达(36.40±4.21)%,明显高于单纯HPD(6.76±0.44)%,单纯PDT组(8.30±0.32)%及空白组(5.00±0.53)%三个对照组(P<0.05),三个对照组间差异无统计学意义(P>0.05)。结论:PDT光动力作用对体外培养人胰腺癌细胞PANC-1有明确抑制效应,并与HPD浓度及光照强度相关。     

Isolation and characterization of squamous carcinoma cells resistant to photodynamic therapy

Photodynamic therapy (PDT) employing methyl δ-aminolevulinic acid (Me-ALA), as a precursor of the photosensitizer protoporphyrin IX (PpIX), is used for the treatment of non melanoma cutaneous cancer (NMCC). However, one of the problems of PDT is the apparition of resistant cell populations. The aim of this study was to isolate and characterize squamous carcinoma cells SCC-13 resistant to PDT with Me-ALA. The SCC-13 parental population was submitted to successive cycles of Me-ALA-PDT and 10 resistant populations were finally obtained. In parental and resistant cells there were analyzed the cell morphology (toluidine blue), the intracellular PpIX content (flow cytometry) and its localization (fluorescence microscopy), the capacity of closing wounds (scratch wound assay), the expression of cell-cell adhesion proteins (E-cadherin and β-catenin), cell-substrate adhesion proteins (β1-integrin, vinculin and phospho-FAK), cytoskeleton proteins (α-tubulin and F-actin) and the inhibitor of apoptosis protein survivin, in the activated form as phospho-survivin (indirect immunofluorescence and Western blot). The results obtained indicate that resistant cells showed a more fibroblastic morphology, few differences in intracellular content of the photosensitizer, higher capacity of closing wounds, higher number of stress fibers, more expression of cell-substrate adhesion proteins and higher expression of phospho-survivin than parental cells. These distinctive features of the resistant cells can provide decisive information to enhance the efficacy of Me-ALA applications in clinic dermatology.     

Integrated effects of fractional laser microablation and sonophoresis on skin immersion optical clearing in vivo

This study is aimed to find an approach for effective skin optical clearing in vivo using polyethylene glycol 300 (PEG‐300) as an optical clearing agent in combination with physical enhancers: fractional laser microablation (FLMA) and/or low‐frequency sonophoresis. In this study albino outbred rats were used. Light attenuation coefficient and optical clearing potential (OCP) of these approaches were evaluated in upper (from ~70 to ~200 μm) and middle (from ~200 to ~400 μm) dermis separately using optical coherence tomography. In 30 minutes, OCP of sonophoresis in combination with FLMA and PEG‐300 in the upper dermis was the maximal (2.3 ± 0.4) in comparison with other treatments in this time point. The most effective approach for optical clearing of middle dermis was PEG‐300 and sonophoresis; but the maximal value of OCP (1.6 ± 0.1) was achieved only in 90 minutes.     

Tissue post-classification using the measured acoustic signals during 355 nm laser atherectomy procedures

The current laser atherectomy technologies to treat patients with challenging (to-cross) total chronic occlusions with a step-by-step (SBS) approach (without leading guide wire), are lacking real-time signal monitoring of the ablated tissues, and carry the risk for vessel perforation. We present first time post-classification of ablated tissues using acoustic signals recorded by a microphone placed nearby during five atherectomy procedures using 355 nm solid-state Auryon laser device performed with an SBS approach, some with highly severe calcification. Using our machine-learning algorithm, the classification results of these ablation signals recordings from five patients showed 93.7% classification accuracy with arterial vs nonarterial wall material. While still very preliminary and requiring a larger study and thereafter as commercial device, the results of these first acoustic post-classification in SBS cases are very promising. This study implies, as a general statement, that online recording of the acoustic signals using a noncontact microphone, may potentially serve for an online classification of the ablated tissue in SBS cases. This technology could be used to confirm correct positioning in the vasculature, and by this, to potentially further reduce the risk of perforation using 355 nm laser atherectomy in such procedures.     

Effect of intermittency factor on singlet oxygen and PGE2 formation in azulene-mediated photodynamic therapy: A preliminary study

In photodynamic therapy, intermittent irradiation modes that incorporate an interval between pulses are believed to decrease the effect of hypoxia by permitting an interval of re-oxygenation. The effect of the irradiation intermittency factor (the ratio of the irradiation pulse time to the total irradiation time) on singlet oxygen formation and inflammatory cytokine production was examined using azulene as a photosensitizer. Effects of difference intermittency factor on singlet oxygen formation and inflammatory cytokine were examined. Azulene solutions (1/10 μM) were irradiated with a 638-nm 500 mW diode laser in fractionation (intermittency factor of 5 or 9) or continuous mode using 50 mW/cm² at 4 or 8 J/cm². Singlet oxygen measurement was performed using a dimethyl anthracene probe. Peripheral blood mononuclear cells (PBMC) were stimulated by 10 ng/ml rhTNF-α for 6 h, before addition of 1 and 10 μM azulene solutions and irradiation. PGE₂ measurement was undertaken using a human PGE₂ ELISA kit. Kruskal-Wallis with Dunn Bonferroni test was used for statistical analyses at p < 0.05.Irradiation of 1 μM azulene+4 J/cm²+intermittency factor of 9 increased singlet oxygen 3-fold (p < 0.0001). Irradiation of 10 μM azulene at either 4 J/cm²+intermittency of 9 or 8 J/cm²+intermittency factor of 5 reduced PGE₂ expression in PBMCs to non-inflamed levels. Thus, at 50 mW/cm², 10 μM azulene-mediated photodynamic therapy with a high intermittency factor and a low energy density generated sufficient singlet oxygen to suppress PGE₂ in Inflamed PBMCs.     

Structural damage of Bacillus subtilis biofilms using pulsed laser interaction with gold thin films

There is a huge interest in developing strategies to effectively eliminate biofilms due to their negative impact in both industrial and clinical settings. In this study, structural damage was induced on two day‐old B. subtilis biofilms using the interaction of 532 nm pulsed laser with gold thin films. Radiant exposure of 225 mJ/cm² induced distinct changes on the surface structure and overall morphology of the matured biofilms after laser irradiation. Moreover, at the radiant exposure used, changes in the colour and viscosity of the biofilm were observed which may indicate a compromised extracellular matrix. Irradiated biofilms in the presence of gold film also showed strong propidium iodide signal which implies an increase in the number of dead bacterial cells after laser treatment. Thus, this laser‐based technique is a promising approach in targeting and eradicating matured biofilms attached on surfaces such as medical implants.

     

Investigations on the mechanism of photodynamic action of different psoralens with DNA

Investigations on the photodynamic action of psoralens with DNA were performed, using experimental techniques of fluorescence lifetime and NMR-CIDNP, as well as SCF-MO and CNDO molecular orbital calculations. It has been shown that the formation of a biradical through the triplet state is the decisive step for psoralen dimer formation, as well as for cyclobutane addition with thymine, while singlet oxygen production is responsible for enzyme inactivation (e.g., lysozyme and trypsin). The molecular orbital calculations, in agreement with experimental results, indicate that the differences in biological effectivity of different psoralens are based on variations in triplet formation probability.     

In vitro laser ablation of laboratory developed biofilms using an Nd:YAG laser of 532 nm wavelength

We studied the laser ablation of laboratory-developed biofilm on titanium and glass surfaces. Specifically, Pseudoalteromonas carrageenovora, a marine biofilm forming bacterium was used to generate laboratory biofilm. Two fluences, 0.05 and 0.1 J/cm(2) and three durations of irradiation, 30 s, 5 min, and 10 min were tested using an Nd;YAG laser of 532 nm wavelength (in the green light area). Nonirradiated coupons with biofilm served as control. The biofilm removal efficiency increased with the increase in laser fluence and duration of irradiation. The maximum biofilm area cover on control coupons of glass and titanium was 62.5 and 76.0%, respectively. Upon irradiation with fluence 0.1 J/cm(2) for the very short duration of 30 s, this reduced to 5.6 and 12.4% and at 10 min to 2.17 and 0.7% on glass and titanium coupons, respectively, while the controls did not show any reductions (62.5 and 76.0% respectively, for glass and titanium coupons). The biofilm TRC (Total Resuscitated Cells) reduction during this period was even more prominent than the area cover, indicating that the remaining biofilm portions on coupons after irradiation were largely composed of dead bacterial cells. The TRC in the irradiation chamber medium for short durations of irradiation showed a significant increase, indicating that the laser irradiation removed live bacteria from the biofilm. The re-growth of the resuscitated cells showed they could grow like the control cells but with a significant lag. The laser's efficiency in the removal of biofilm was better seen on titanium coupons than on glass. Our results showed that a low-power pulsed laser irradiation could be used to remove biofilm formed on hard surfaces.     

Structure-activity relationship of retinoids on the differentiation of cultured chick foot skin

Summary The influence of retinoids (vitamin A and its analogues) on epithelial differentiation was examined in explants of foot skin from chick embryos. In the presence of retinoic acid (10 M) keratinization and differentiation of scale-like structures, which occurred in tarsometatarsal skin explants, was inhibited and a mucous metaplasia developed. Retinoic acid caused club-shaped feathers in skin explants taken from the anterior surface of the tibia — skin which was determined to differentiate into feathers. In skin explants taken from a breed with feathered feet, the differentiation of tarsometatarsal skin was completely blocked; in tibial skin, club-shaped feathers resulted in response to retinoic acid. These findings indicated that skin of the two origins reacted differently to the retinoid, as was noted in the breed with scaly feet. The structure-activity relationship of 22 retinoids with marked differences in their biological activity was investigated in tarsometatarsal skin explants. Comparing the concentration of various retinoids needed to completely inhibit the differentiation of scale-like structures, retinoids containing tetramethylated indane or tetraline were 100 and 1,000 times more active than retinoic acid. Retinoids with a sulphur-containing end group were also active but less so than the corresponding compound with a carboxylic acid end group. The inactive ethyl, ester analogue, etretinate, was activated in the presence of esterase, indicating that the free carboxylic acid group was important for the activity of retinoids. The retinoid-induced inhibition of keratinization followed by mucous metaplasia in cultured chick embryo skin is a simple and useful model system to test new retinoids which may be helpful in the treatment of dermatological and oncological diseases.     

Evaluation of bone healing following Er:YAG laser ablation in rat calvaria compared with bur drilling

The Er:YAG laser is currently used for bone ablation. However, the effect of Er:YAG laser irradiation on bone healing remains unclear. The aim of this study was to investigate bone healing following ablation by laser irradiation as compared with bur drilling. Rat calvarial bone was ablated using Er:YAG laser or bur with water coolant. Er:YAG laser effectively ablated bone without major thermal changes. In vivo micro‐computed tomography analysis revealed that laser irradiation showed significantly higher bone repair ratios than bur drilling. Scanning electron microscope analysis showed more fibrin deposition on laser‐ablated bone surfaces. Microarray analysis followed by gene set enrichment analysis revealed that IL6/JAK/STAT3 signaling and inflammatory response gene sets were enriched in bur‐drilled bone at 6 hours, whereas the E2F targets gene set was enriched in laser‐irradiated bone. Additionally, Hspa1a and Dmp1 expressions were increased and Sost expression was decreased in laser‐irradiated bone compared with bur‐drilled bone. In granulation tissue formed after laser ablation, Alpl and Gblap expressions increased compared to bur‐drilled site. Immunohistochemistry showed that osteocalcin‐positive area was increased in the laser‐ablated site. These results suggest that Er:YAG laser might accelerate early new bone formation with advantageous surface changes and cellular responses for wound healing, compared with bur‐drilling.      

Predicting the effect of relative humidity on skin temperature and skin wettedness

The purpose of this research is to investigate the relative humidity effects on skin temperature and skin wettedness for different operative temperatures. For this aim, thermal interactions between human body and environment are simulated. In this simulation, Gagge 2-node model is used but includes some significant modifications. The simulation is to apply the Gagge 2-node model to individual segments rather than to whole body. Also, the results of the simulation are compared with present measurements, and available experimental data and simulated results in the literature in order to present reliability of the 16 segments-Gagge 2-node model. It is determined that the simulation results are in good agreement with measured results obtained from present experiments, and simulated results and experimental data in the literature.     

头孢哌酮联合夫西地酸对细菌性皮肤病患者皮肤菌群的影响及疗效

目的 探讨头孢哌酮联合夫西地酸软膏对细菌感染性皮肤病患者皮肤菌群种类的影响及疗效,为细菌感染性皮肤病的治疗提供参考。方法 选择2015年11月至2019年6月在我院诊治的100例细菌感染性皮肤病患者作为研究对象,入选患者随机分为对照组（n=50）与观察组（n=50）。对照组患者采取头孢哌酮/舒巴坦静脉注射治疗,观察组患者采用头孢哌酮/舒巴坦联合夫西地酸软膏治疗。对各组患者治疗第3天和第7天时皮损处菌群进行检测,同时观察两组患者的疗效。结果 与对照组相比,观察组患者治疗第3天和第7天时皮损处的菌群数量及种类数量呈现明显的下降趋势,差异有统计学意义（P＜0.05）。观察组中疗效极好病例的占比为78.0%,治疗的总有效率为100.0%。结论 头孢哌酮联合夫西地酸软膏对细菌感染性皮肤病患者疗效确切,对患者皮损处的细菌具有较强的抑制作用,值得推广使用。