Development of Phosphinate Ligand-Based Low-Affinity Ca2+ Fluorescent Probes and Application to Intracellular Ca2+ Imaging.

Divalent metal cations such as calcium ion (Ca2+) and magnesium ion (Mg2+) are indispensable to the regulation of various cellular activities. In this research, we developed the KLCA series utilizing o-aminophenol-N,N-diacetate-O-methylene-methylphosphinate (APDAP) as a target binding site, which was reported recently as a highly free Mg2+-selective ligand. KLCA-301 with orange fluorescence based on a rhodamine fluorophore and KLCA-501 with near-infrared (NIR) fluorescence based on a Si-rhodamine fluorophore were synthesized, intended for application to multicolor imaging. The evaluation of the fluorescence response to Ca2+ and Mg2+ of the KLCA series indicated the applicability as low-affinity Ca2+ probes. While KLCA-301 mainly localized in the cytosol in cultured rat hippocampal neurons, KLCA-501 localized to the cytosol and granular organelles in neurons. Comparison of the fluorescence response of KLCA-301 and the high-affinity Ca2+ probe Fluo-4 upon stimulation by glutamate in stained neurons revealed that KLCA-301 could reflect the secondary large rise of intracellular Ca2+, which Fluo-4 could not detect. In addition, KLCA-501 showed a fluorescence response similar to the low-affinity Ca2+ probe Fluo-5N upon stimulation by glutamate in stained neurons, concluding that KLCA-301 and KLCA-501 could be used as low-affinity Ca2+ probes. The KLCA series offers new options for low-affinity Ca2+ probes. Moreover, KLCA-501 achieved simultaneous visualization of the change in Ca2+ and ATP concentrations and also in mitochondrial inner membrane potential in neurons. KLCA-501 is expected to be a strong tool that enables simultaneous multicolor imaging of multiple targets and elucidation of their relationship in cells.

Evaluation of cellophane as platform for colorimetric assays on microfluidic analytical devices.

Due to their low cost, simplicity, and pump-free liquid transport properties, colorimetric assays on paper spots and microfluidic paper-based analytical devices (µPADs) are regarded as useful tools for point-of-care testing (POCT). However, for certain types of colorimetric assays, the "non-transparent" and "white" characters of paper can be a disadvantage. In this work, the possibilities of using cellophane as an alternative platform for colorimetric assays have been investigated. Cellophane is a low cost and easy-to-handle transparent film made of regenerated cellulose. Owing to its hydrophilic character, cellophane-based microfluidic channels fabricated through a print-cut-laminate approach enabled pump-free liquid transport into multiple detection areas, similar to µPADs. In addition, the water absorption characteristics of cellophane allowed the stable immobilization of water-soluble colorimetric indicators without any surface modification or additional reagents. The transparency of cellophane provides possibilities for simple background coloring of the substrates, increasing the dynamic signal range for hue-based colorimetric assays, as demonstrated for two model assays targeting H2O2 (46-fold increase) and creatinine (3.6-fold increase). Finally, a turbidity detection-based protein assay was realized on black background cellophane spots. The lowest limits of detection achieved with the cellophane-based devices were calculated as 7 µM for H2O2, 2.7 mg dL-1 for creatinine, and 3.5 mg dL-1 for protein (human serum albumin).

A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level.

Bioluminescence (BL) imaging, which utilizes light emitted through the enzymatic reaction of luciferase oxidizing its substrate luciferin, enables sensitive and noninvasive monitoring of life phenomena. Herein, we developed a series of caged furimazine (FMZ) derivatives by introducing a protective group at the C-3 position and a hydroxy group at the C-6 phenyl ring to realize long-term live-cell BL imaging based on the NanoLuc (NLuc)/NanoKAZ (NKAZ)-FMZ system. The membrane permeability and cytotoxicity of the substrates were evaluated and related to their hydrophobicity. Among the series, the derivative with the bulkiest protective group (adamantanecarbonyl group) and a hydroxy substituent (named Ad-FMZ-OH) showed significantly prolonged and constant BL signal in cells expressing NLuc compared to the native FMZ substrate. This derivative enabled continuous BL imaging at the single-cell level for 24 h. Furthermore, we applied Ad-FMZ-OH to BL imaging of myocyte fusion and succeeded in the consecutive and sensitive monitoring at a single-cell level over a day. In summary, NLuc/NKAZ-caged FMZ derivatives have the potential to be applied to live-cell BL imaging of various life phenomena that require long-term observation.

Colorimetric paper-based sarcosine assay with improved sensitivity.

This manuscript reports on a simple paper-based bienzymatic colorimetric assay for sarcosine as an important urinary biomarker of prostate cancer. All required assay reagents are pre-deposited on hydrophilic filter paper spots surrounded by a hydrophobic barrier. Sarcosine in the sample solution is selectively oxidized in the presence of sarcosine oxidase (SOx), resulting in the formation of hydrogen peroxide, which is subsequently detected through the horseradish peroxidase (HRP)-catalyzed conversion of the colorless indicator 3,3',5,5'-tetramethylbenzidine (TMB) into its blue-colored oxidation product. By the modification of the paper with positively charged poly(allylamine hydrochloride) (PAH), a linear response to sarcosine between 0 and 10 µM and a significant lowering of the limit of detection (LOD) (0.6 µM) compared to the unmodified paper substrate (12.6 µM) has been achieved. The improvement of the LOD was attributed to the fact that the presence of the polymer limits the enzyme-driven colorimetric reaction to the surface of the paper substrate, resulting in stronger color development. In experiments in artificial urine matrix, the bicarbonate anion was identified as an inhibitor of the colorimetric reaction. This inhibition was successfully eliminated through on-device sample pH adjustments with pH-buffer components pre-deposited onto assay devices. The LOD for sarcosine achieved in artificial urine matrix (2.5 µM) is below the 5 µM threshold value for this urinary biomarker required for diagnostic purposes. Finally, good selectivity over all 20 natural amino acids and satisfactory long-term storage stability of reagent-modified paper substrates at - 20 °C for a period of 50 days were confirmed.

Differential Effect of Azetidine Substitution in Firefly Luciferin Analogues.

Replacing an N,N-dimethylamino group in a classical fluorophore with a four membered azetidine ring provides an improved luminescence quantum yield. Herein, we extended this strategy to bioluminescent firefly luciferin analogues and evaluated its general validity. For this purpose, four types of luciferin cores were employed, and a total of eight analogues were evaluated. Among these analogues, unexpectedly, only the benzothiazole core analogue benefited from an azetidine substitution and showed enhanced bioluminescence. In addition, fluorescence measurements revealed that an azetidine substitution improved the fluorescence quantum yield by 2.3-times compared to a N,N-dimethylamino group. These findings clarify the differential effects of azetidine substituents in luciferins and present one possible strategy for enhancing photon output in benzothiazole type luciferins through a synthetic approach.

Interference-based wide-range dynamic tuning of the plasmonic color of single gold nanoparticles.

Dynamic tuning of nanoscale coloration by exploiting localized surface plasmon resonance of gold nanoparticles (AuNPs) combined with an interference coloration mechanism is demonstrated experimentally. When interference between the scattering field from AuNPs and the reflected field from the substrate is observed under back-scattering white-light microscopy, the AuNPs exhibit various colors depending on their distance to the substrate. When the numerical aperture of the microscope objective is optimized, much greater coverage of the color space than was achieved with previously reported plasmon-based approaches is attained. Also, color tunability is examined by exploiting the temperature-induced volume change of a temperature-responsive hydrogel with embedded AuNPs to dynamically modify the distance to the substrate.

Long-term single cell bioluminescence imaging with C-3 position protected coelenterazine analogues.

Bioluminescence is a powerful imaging modality for monitoring biological phenomena both in vitro and in vivo. Bioluminescence imagin (BLI) is becoming a seamless imaging technology covering the range from cells to organs of small animals. Long-term imaging at the single cell level would lead to a true understanding of the dynamics of life phenomena. This work presents a long-term single cell bioluminescence imaging technology accomplished with C-3 position protected furimazines (FMZs), a CTZ analogues, which generate intense blue emission when paired with a highly stable engineered luciferase, Nanoluc. Four types of FMZs protected at the C-3 position have been synthesized. The type and steric bulkiness of the protection group strongly contributed to storage stability and the kinetics of the bioluminescence reactions of the analogues in human living cells. In particular, two developed FMZ analogues resulted in significantly longer bioluminescence emission with higher S/N ratio than FMZ at single cell level. Long-term bioluminescence single cell imaging technology with the developed FMZ analogues will lead to seamless imaging in the range from cells to organs of small animals.

Centrifugal Paperfluidic Platform for Accelerated Distance-Based Colorimetric Signal Readout.

Distance-based readout is one of the most user-friendly and simple colorimetric signaling methods widely applied for paper-based analytical devices (PADs). This work presents the integration of distance readout PADs into a centrifugal platform enabling affordable, rapid, and sample volume-independent colorimetric assays. Centrifugally assisted distance-based PADs (CD-PADs) eliminate the requirement to use micropipets for sample introduction and reduce the overall time for distance-based assays. All device fabrication steps were performed through computer-controlled print, cut, and laminate (PCL) techniques oriented toward mass production. The inexpensive centrifugal platform was built on a recycled DVD player combined with an open source microcomputer (Arduino). Assay protocols, including rotational velocity and rotation time, were optimized to obtain a maximum dynamic range and reproducible results for sample volume metering (coefficient of variation 3.62%). A colorimetric Ni2+ assay chosen to demonstrate measurements on CD-PADs allowed the detection of nickel ions (Ni2+) with naked-eye interpretation within 1.5 min and a limit of detection (LOD) of 44.1 ng of Ni2+, which to the best of our knowledge is the lowest value reported for a distance-based Ni2+ assay.

Ring-Fused Firefly Luciferins: Expanded Palette of Near-Infrared Emitting Bioluminescent Substrates.

Firefly bioluminescence is broadly applied as a noninvasive imaging modality in the biomedical research field. One limitation in firefly bioluminescence imaging is the limited variety of luciferins emitting in the near-infrared (NIR) region (650-900 nm), where tissue penetration is high. Herein, we describe a series of structure-inherent NIR emitting firefly luciferin analogues, NIRLucs, designed through a ring fusion strategy. This strategy resulted in pH-independent structure-inherent NIR emission with a native firefly luciferase, which was theoretically supported by quantum chemical calculations of the oxidized form of each luciferin. When applied to cells, NIRLucs displayed dose-independent improved NIR emission even at low concentrations where the native d-luciferin substrate does not emit. Additionally, excellent blood retention and brighter photon flux (7-fold overall, 16-fold in the NIR spectral range) than in the case of d-luciferin have been observed with one of the NIRLucs in mice bearing subcutaneous tumors. We believe that these synthetic luciferins provide a solution to the longstanding limitation in the variety of NIR emitting luciferins and pave the way to the further development of NIR bioluminescence imaging platforms.

Near-Infrared Fluorescent Probes for Imaging of Intracellular Mg2+ and Application to Multi-Color Imaging of Mg2+, ATP, and Mitochondrial Membrane Potential.

The magnesium ion (Mg2+) is an essential cation to maintain proper cellular activities. To visualize the dynamics and functions of Mg2+, there is a great need for the development of Mg2+-selective fluorescent probes. However, conventional Mg2+ fluorescent probes are falling behind in low selectivity and poor fluorescence color variation. In this report, to make available a distinct color window for multi-color imaging, we designed and synthesized highly Mg2+-selective and near-infrared (NIR) fluorescent probes, the KMG-500 series consisting of a charged ß-diketone as a selective binding site for Mg2+ and a Si-rhodamine residue as the NIR fluorophore, which showed photoinduced electron transfer (PeT)-type OFF-ON response to the concentration of Mg2+. Two types of KMG-500 series probes, tetramethyl substituted Si-rhodamine KMG-501 and tetraethyl substituted Si-rhodamine KMG-502, were synthesized for the evaluation of cell permeability. For intracellular application, the membrane-permeable acetoxymethyl derivative KMG-501 (KMG-501AM) was synthesized and allowed to stably stain cultured rat hippocampal neurons during imaging of intracellular Mg2+. On the other hand, KMG-502 was cell membrane permeable without AM modification, preventing the probe from staying inside cells during imaging. KMG-501 distributed mainly in the cytoplasm and partially localized in lysosomes and mitochondria in cultured rat hippocampal neurons. Mg2+ increase in response to the FCCP uncoupler inducing depolarization of the mitochondrial inner membrane potential was detected in the KMG-501 stained neurons. For the first time, KMG-501 succeeded in imaging intracellular Mg2+ dynamics with NIR fluorescence. Moreover, it allows one to simultaneously visualize changes in Mg2+ and ATP concentration and also mitochondrial inner membrane potential and their interactions. This probe is expected to be a strong tool for multi-color imaging of intracellular Mg2+.

All-printed semiquantitative paper-based analytical devices relying on QR code array readout.

The wide spread of smartphones and QR codes for various end-user applications has had an impact beyond traditional fields of use, recently also reaching point-of-care testing (POCT). This work presents the integration of QR code recognition into paper-based analytical devices (PADs) with "distance-based" colorimetric signalling, resulting in semiquantitative readout fully relying on straightforward barcode reader solutions. PADs consist of an array of QR codes arranged in series inside a paperfluidic channel. A mask dye concept has been developed, which enables utilisation of colour changing indicators by initially hiding QR codes. The colour change of the indicator induced by the presence of an analyte of interest results in gradual unmasking of QR codes, which become recognisable by the smartphone barcode reader app. To reproducibly fabricate devices, all fabrication steps were performed by commercial desktop solid ink and inkjet printing. The QR code masking function was optimised by controlling the amount of printed mask dye through adjusting the opacity of printing patterns during the inkjet deposition process. For proof-of-concept, a model assay in the form of colorimetric copper ion (Cu2+) detection in the concentration range of 0.4 mM to 3.2 mM was evaluated. Consistent results independent of the smartphone model and environmental light condition were achieved with a free barcode reader app. To the best of our knowledge, this work is the first demonstration of a semiquantitative assay approach fully relying on QR code readout without digital colour analysis, customised app or hardware modification.

Inkjet-printed pH-independent paper-based calcium sensor with fluorescence signal readout relying on a solvatochromic dye.

A challenge for paper-based cation sensors relying on classical carrier-based ion-selective optodes (ISOs) is their pH-cross response caused by the use of H+-sensitive chromoionophores as optical signal transducers. This work demonstrates fully pH-independent fluorescence-based calcium detection with a paper-based plasticizer-free ISO. To achieve a pH-independent assay, a solvatochromic dye (SD) instead of a traditional H+-sensitive chromoionophore has been applied to the paper-based ISO by means of inkjet printing technology. The detection principle depends on an ionophore-driven phase-transfer ion-exchange reaction between target cations and the positively charged SD, which no longer involves H+ in the optical signal transduction process. The developed paper-based ISOs with the SD resulted in Ca2+ concentration-dependent response curves not affected by the sample pH (pH 6.0, 7.0, and 8.0). The dynamic range obtained for Ca2+ detection was from 10-5 to 1 mol L-1 with a detection limit of 19.3 µmol L-1. Additionally, excellent selectivity derived from the used ionophore has been confirmed. As a simple practical application, the determination of Ca2+ in mineral water has been achieved without the pH-buffering process required for conventional cation-exchange ISOs.

Biothiol-Activatable Bioluminescent Coelenterazine Derivative for Molecular Imaging in Vitro and in Vivo.

There is a high demand for sensitive biothiol probes targeting cysteine, glutathione, and homocysteine. These biothiols are known as playing essential roles to maintain homeostasis and work as indicators of many diseases. This work presents a bioluminescent probe (named AMCM) to detect biothiols in live mammalian cells and in vivo with a limit of detection of 0.11 µM for cysteine in solution and high selectivity for biothiols, making it suitable for real-time biothiol detection in biological systems. Upon application to live cells, AMCM showed low cytotoxicity and sensitively reported bioluminescence in response to changes of biothiol levels. Furthermore, a bioluminescence resonance energy transfer system consisting of AMCM combined with the near-infrared fluorescent protein iRFP713 was applied to in vivo imaging, with emitted tissue-permeable luminescence in living mice. In summary, this work demonstrates that AMCM is of high practical value for the detection of biothiols in living cells and for deep tissue imaging in living animals.

Adsorption-Desorption Control of Fibronectin in Real Time at the Liquid/Polymer Interface on a Quartz Crystal Microbalance by Thermoresponsivity.

The cell manipulation technique using thermoresponsive polymers is currently attracting much attention for applications in the medical field. To achieve arbitrary and accurate cell control, it is necessary to intensely research fibronectin behavior. A smart surface, which has thermoresponsive wettability and which can adsorb or desorb fibronectin repeatedly without the presence of cells, was fabricated by an electrospinning method. The fabricated coating changed its structure as the temperature was changed, and this transformation could substitute for the pulling force generated by the cytoskeletal contraction of cells. Moreover, a coated quartz crystal microbalance was able to detect the fibronectin behavior as frequency shifts, which could be used in the estimation of the mass shift with the aid of suitable equations. This coating and measurement system can contribute greatly not only to the development in the medical field centered on biomaterial manipulation technologies, but also to the improvement of medical instruments.

Fully inkjet-printed distance-based paper microfluidic devices for colorimetric calcium determination using ion-selective optodes.

Although the determination of calcium ions (Ca2+) is of high importance to monitor water hardness, currently available devices for on-site analysis suffer from a lack of user-friendliness and sensitivity. This work demonstrates fully inkjet-printed and low-cost microfluidic paper-based analytical devices (µPADs) for the simple naked-eye colorimetric determination of calcium ions (Ca2+) in drinking and tap water samples. The quantification of Ca2+ relies on visual readout of the length of a colour-changed detection channel modified with ionophore-doped ion-selective optode nanospheres (nano-optodes), eliminating the requirement of a scanner or a camera. All fabrication steps for deposition of assay reagents have been performed by means of a simple desktop thermal inkjet printer, which is expected to contribute to highly batch-to-batch reproducible device preparation. The detectable Ca2+ concentrations between 0.05 mmol L-1 and 5 mmol L-1 cover the range recommended by the International Organization for Standardization (0.05-2.5 mmol L-1) and the World Health Organization (WHO) guideline for Ca2+ quantification in drinking water (less than 5 mmol L-1). The lowest concentration of Ca2+ detectable by the naked eye was found to be 0.05 mmol L-1, which is below the value achieved with previously reported paper-based devices. µPAD quantified Ca2+ concentrations in tap or drinking waters were within 15% error of the results obtained with a classical complexometric titration. Hence, distance-based µPADs relying on nano-optodes are sensitive and reproducible tools for equipment-free on-site assaying of Ca2+ in real samples.

Crosslinked Poly(N-Isopropylacrylamide)-Based Microfibers as Cell Manipulation Materials with Prompt Cell Detachment.

Stimuli-responsive smart materials are a key to the realization of next-generation medical technologies. Among them, the temperature-responsive polymer poly(N-isopropylacrylamide) (PNIPAAm) is attracting particular attention because it is easy to use in physiological conditions. PNIPAAm-grafted surfaces can undergo temperature-modulated cell adhesion and detachment without proteolytic enzymes, and can be used as cell-separating materials through selective cell adhesion/detachment. However, cell detachment at reduced temperatures is problematic because it takes several hours. A novel thermoresponsive crosslinked microfiber system that can greatly reduce the cell detachment time is introduced in this study. The crosslinked fibers provide temperature-dependent volume change, and enable cell detachment within 10 min of reducing the temperature, which is one-sixth of the time required in previous studies. The prompt cell detachment is thought to arise from a completely new mechanism derived from fiber swelling. This system will make a significant contribution as a novel cell manipulating system for next-generation medical technology.

LAT1-Targeting Thermoresponsive Fluorescent Polymer Probes for Cancer Cell Imaging.

L-type amino acid transporter 1 (LAT1) is more highly expressed in cancer cells compared with normal cells. LAT1 targeting probes would therefore be a promising tool for cancer cell imaging. In this study, LAT1-targeting thermoresponsive fluorescent polymer probes based on poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)) were synthesized and their affinity for LAT1 was evaluated. The synthesized polymer probes interacted with LAT1 on HeLa cells, and inhibition of l-[³H]-leucine, one of the substrates for LAT1 uptake, was investigated. l-Tyrosine-conjugated P(NIPAAm-co-DMAAm) inhibited the uptake of l-[³H]-leucine, while P(NIPAAm-co-DMAAm) and l-phenylalanine-conjugated P(NIPAAm-co-DMAAm) did not. This result indicated that l-tyrosine-conjugated polymer has a high affinity for LAT1. The fluorescent polymer probes were prepared by modification of a terminal polymer group with fluorescein-5-maleimide (FL). Above the polymer transition temperature, cellular uptake of the polymer probes was observed because the polymers became hydrophobic, which enhanced the interaction with the cell membrane. Furthermore, quantitative analysis of the fluorescent probe using flow cytometry indicated that l-tyrosine-conjugated P(NIPAAm-co-DMAAm)-FL shows higher fluorescence intensity earlier than P(NIPAAm-co-DMAAm)-FL. The result suggested that cellular uptake was promoted by the LAT1 affinity site. The developed LAT1-targeting thermoresponsive fluorescent polymer probes are expected to be useful for cancer cell imaging.

Paper-Based Antibody Detection Devices Using Bioluminescent BRET-Switching Sensor Proteins.

This work reports on fully integrated "sample-in-signal-out" microfluidic paper-based analytical devices (µPADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET-based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume-independent and fully reagent-free operation, including on-device blood plasma separation. User operation is limited to the application of a single drop (20-30 µL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti-HIV1, anti-HA, and anti-DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user-friendly point-of-care testing in low-resource environments.

The use of a temperature-responsive column for the direct analysis of drugs in serum by two-dimensional heart-cutting liquid chromatography.

A novel pretreatment method, which was performed using a two-dimensional high-performance liquid chromatography (2D-HPLC) system, was proposed for the direct analysis of drugs in human serum. A temperature-responsive column was used as a pretreatment column. The stationary phase of the temperature-responsive column exhibits temperature-regulated hydrophilic/hydrophobic characteristics. Controlling the ionic strength of the eluent enables human serum albumin (HSA) to pass through the column without retention. When serum samples containing barbiturates or benzodiazepines were injected into the temperature-responsive column using 10 mM of ammonium acetate (pH 6.5) as the mobile phase and in the temperature range of 10-40 °C, HSA was eluted from the column near the dead time, followed by the individual drugs. When the column temperature was changed, the retention times of the drugs were altered owing to surface property changes within the pretreatment column. These closely eluted compounds were subsequently introduced into the analytical column using a column-switching valve, with a minimal gap time to avoid foreign substance contamination. This new 2D-HPLC method afforded high-quality chromatograms of multiple drugs without unwanted peaks from foreign substances. The present technique could be an attractive choice in selecting the analytical method for drug analysis.

Transcutaneous drug delivery by liposomes using fractional laser technology.

OBJECTIVE: Transdermal delivery of hydrophilic peptides remains a challenge due to their poor cellular uptake and transdermal penetration. We hypothesize that combination of a CO2 fractional laser to enhance percutaneous absorption and liposomes as transdermal carriers would improve skin penetration of hydrophilic drugs. STUDY DESIGN: NA. METHODS: Liposomes were prepared using membrane fusion lipid dioleoylphosphatidylethanolamine, and used to deliver 5-carboxyfluorescein (CF) and fluorescein isothiocyanate-conjugated ovalbumin (OVA-FITC) as model hydrophilic peptide drugs. Liposome size was estimated by dynamic light scattering. Liposome uptake into murine macrophage cells and penetration or permeation into Yucatan micropig skin after irradiation by CO2 fractional laser at varying energy levels (laser power and exposure duration) were investigated using Franz cell and fluorescence microscopy. Oxidative damage to the irradiated mouse skin was assessed by electron spin resonance. RESULTS: Size of CF and OVA-FITC encapsulated liposomes was 324 ± 75 nm. Cellular uptake of OVA-FITC delivered by liposomes was 10-fold higher (1,370 relative fluorescence units, RFU) than delivered in solution form (130 RFU). Fractional laser irradiation increased skin permeation rate of CF liposomes (0-10%) and OVA-FITC liposomes (4-40%) in a dose-dependent manner. Although peeling off the stratum corneum facilitated CF liposome penetration at low energy levels (2.69-3.29 J/cm2 ; 10-20 W for 500 µs), drug permeation was similar (7-8%) in peeled or untreated skin at higher laser energy levels (6.06 J/cm2 ; 20 W for 1,500 µs). FITC penetrated deeper in the skin after laser irradiation. However, OH, O2-, and VC reactive oxygen species were generated upon irradiation of the skin with a fractional CO2 laser. CONCLUSIONS: Increasing laser power and irradiation, time increased liposome uptake by cells and penetration of peptide drugs across the skin in a dose-dependent manner. High-energy CO2 fractional laser overcomes the rate-limiting barrier function of the stratum corneum. Further investigations are required to establish the safety and efficacy of fractional laser-irradiation assisted delivery of liposome-encapsulated drugs as a transcutaneous drug delivery system. Lasers Surg. Med. 49:525-532, 2017. © 2016 Wiley Periodicals, Inc.