Liposome-assisted penetration and antiaging effects of collagen in a 3D skin model.

BACKGROUND: Collagen is a major component of the extracellular matrix that supports the epidermal layers of the skin; thus, many strategies have been made to enhance the topical delivery of collagen for antiaging purposes. In addition, our previous study indicated that liposome can help the penetration of active ingredients into the skin. AIMS: To produce stable collagen-encapsulated liposomes to improve the topical delivery of collagen. METHODS: Collagen-encapsulated liposomes were fabricated using high-pressure homogenization method. The colloidal stability and adhesion ability were confirmed using dynamic light scattering, and spectrofluorophotometer, respectively. Keratinocyte differentiations of 3D skin before and after treatment with collagen-encapsulated liposomes were confirmed by real-time PCR. RESULTS: In comparison with native collagen, collagen-encapsulated liposomes enhanced collagen retention in artificial membranes by twofold, even after repeated washings with water. In addition, real-time PCR results indicated that 3D skin treated with collagen-encapsulated liposomes exhibited higher levels of collagen, keratin, and involucrin, even after ethanol treatment. CONCLUSION: Liposomes could serve as efficient delivery vehicles for collagen, thereby enhancing its antiaging effects.

Reactive Oxygen Species (ROS)-Assisted Nano-Therapeutics Surface-Decorated with Epidermal Growth Factor Fragments for Enhanced Wound Healing.

In this study, stimuli-responsive liberation of an epidermal growth factor fragment (EGFfr) is accomplished using nanofibrous meshes to improve wound healing effects. Electrospun nanofibers are fragmented by mechanical milling, followed by aminolysis to fabricate powdered nanofibrils (NFs). EGFfrs are covalently immobilized on NFs via thioketal linkers (EGFfr@TK@NF) for reactive oxygen species (ROS)-dependent liberation. EGFfr@TK@NF exhibits ROS-responsive liberation of EGFfr from the matrix at hydrogen peroxide (H2 O2 ) concentrations of 0-250 mm. Released EGFfr is confirmed to enhance the migration of HaCaT cell monolayers, and keratinocytic gene expression levels are significantly enhanced when H2 O2 is added to obtain the released fraction of NFs. An in vivo study on the dorsal wounds of mice reveals that EGFfr-immobilized NFs improve the expression levels of keratin1, 5, and 14 for 2 weeks when H2 O2 is added to the wound sites, suggesting that the wounded skin is re-epithelized with the original epidermis. Thus, EGFfrs-immobilized NFs are anticipated to be potential nanotherapeutics for wound treatment in combination with the conventional disinfection process with H2 O2 .

Spectroscopic techniques for monitoring stem cell and organoid proliferation in 3D environments for therapeutic development.

Spectroscopic techniques for monitoring stem cell and organoid proliferation have gained significant attention in therapeutic development. Spectroscopic techniques such as fluorescence, Raman spectroscopy, and infrared spectroscopy offer noninvasive and real-time monitoring of biochemical and biophysical changes that occur during stem cell and organoid proliferation. These techniques provide valuable insight into the underlying mechanisms of action of potential therapeutic agents, allowing for improved drug discovery and screening. This review highlights the importance of spectroscopic monitoring of stem cell and organoid proliferation and its potential impact on therapeutic development. Furthermore, this review discusses recent advances in spectroscopic techniques and their applications in stem cell and organoid research. Overall, this review emphasizes the importance of spectroscopic techniques as valuable tools for studying stem cell and organoid proliferation and their potential to revolutionize therapeutic development in the future.

Korean Amberjack Skin-Inspired Hyaluronic Acid Bioink for Reconstruction of Human Skin.

Decellularized extracellular matrix (dECM) has been extensively employed as tissue engineering scaffolds because its components can greatly enhance the migration and proliferation of cultivating cells. In this study, we decellularized Korean amberjack skin and incorporated soluble fractions in hyaluronic acid hydrogels with 3D-printed tissue engineering hydrogels to overcome any limitation of animal-derived dECM. The hydrolyzed fish-dECM was mixed with methacrylated hyaluronic acid and chemically crosslinked to 3D-printed fish-dECM hydrogels, where fish-dECM contents affected both printability and injectability of the hydrogels. Swelling ratios and mass erosion of the 3D-printed hydrogels were dependent on fish-dECM contents, where higher fish-dECM in the hydrogel increased swelling ratios and mass erosion rates. The higher content of fish-dECM considerably enhanced the viability of the incorporated cells in the matrix for 7 days. Artificial human skin was constructed by seeding human dermal fibroblasts and keratinocytes in the 3D-printed hydrogels, and a formation of a bilayered skin was visualized with tissue staining. Thus, we envision that 3D-printed hydrogels containing fish-dECM can be an alternative bioink composed of a non-mammal-derived matrix.

Reactive oxygen species-responsive clicked assembly of gold nanoparticles to enhance photothermal therapy.

To enhance the efficacy of photothermal therapy (PTT) at tumor sites, we designed a reactive oxygen species (ROS)-responsive gold nanoparticle (AuNP)-based nanosystem in which azide-decorated AuNPs (N3@AuNPs) and diselenide-coated alkyne-decorated AuNPs (Se/Ak@AuNPs) were separately prepared for selective clicking into nanoclusters when exposed to ROS. Se/Ak@AuNPs were dual-functionalized with alkyne moieties and diselenide linkers embedded in a long chain of polyethylene glycol (PEG) to enable the alkyne moieties of Se/Ak@AuNPs to be inaccessible to the azide moieties of N3@AuNPs owing to steric hindrance. At tumor sites where the ROS level is elevated due to the increased metabolic activity, cellular receptor signaling, mitochondrial dysfunction, and oncogene activity, the diselenide linkers were cleaved, leading to the liberation of the long PEG chains tethered to AuNPs, and the alkyne moieties could be recognized by the surrounding azide moieties to generate a click reaction. The clicked AuNPs formed clustered nanoparticles with increased size. Upon 808 nm laser irradiation, these large clusters of AuNPs significantly enhanced the photothermal conversion efficiency compared with that of isolated AuNPs. In vitro studies revealed that the AuNP clusters exhibited a noticeably higher apoptosis rate than AuNPs. Therefore, ROS-responsive clicked AuNP clusters can be a potential tool for PTT enhancement in cancer treatment.

Cell-directed assembly of luminal nanofibril fillers in nerve conduits for peripheral nerve repair.

Graphene and its derivatives, graphene oxide (GO) and reduced graphene oxide (rGO), have attracted significant attention in the field of tissue engineering, particularly in nerve and muscle regeneration, owing to their excellent electrical conductivity. This paper reports the fabrication of cell-mixable rGO-decorated polycaprolactone (PCL) nanofibrils (NFs) to promote peripheral nerve repair with the assistant of electron transmission by rGO and cytokine paracrine by stem cells. Oxidized GO (GO-COOH) and branched polyethylenimine are layer-by-layer coated on hydrolyzed PCL NFs via electrostatic interaction, and the number of layering is manipulated to adjust the GO-COOH coating amount. The decorated GO-COOH is reduced in situ to rGO for electrical conductivity retrieval. PC12 cells cultivated with rGO-coated NF demonstrate spontaneous cell sheet assembly, and neurogenic differentiation is observed upon electrical stimulation. When transplant nerve guidance conduit containing the assembly of rGO-coated NF and adipose-derived stem cell to the site of neurotmesis injury of a sciatic nerve, animal movement is enhanced and autotomy is ameliorated for 8 weeks compared to transplanting the hollow conduit only. Histological analysis results reveal higher levels of muscle mass and lower levels of collagen deposition in the triceps surae muscle of the rGO-coated NF-treated legs. Therefore, the rGO-layered NF can be tailored to repair peripheral nerve injuries in combination with stem cell therapy.

Highly selective and quantitative in situ monitoring of cell surface proteins by SERS immunoassay system.

Due to their pivotal roles in many biological functions, cell surface proteins (CSPs) are often used for cancer prognosis, as evidenced by a number of studies that have reported significant changes in the expression levels of specific surface proteins depending on the stage of tumorigenesis and selection/variety of reprogrammed cells during cell fate conversion. Current CSP detection strategies suffer from poor selectivity and lack the ability for in situ analysis but maintain the spatial information between cells. Here, we have fabricated nanoprobes for surface-enhanced Raman scattering (SERS) immunoassays by conjugating a specific antibody onto silica-coated gold nanoparticles incorporating an individual Raman reporter (Au-tag@SiO2-Ab NPs) for highly sensitive and selective in situ detection in different types of cells. When multiple HEK293 cell lines stably expressing different levels of the CSP, ACE2, were investigated by the SERS immunoassay, we demonstrated that the level of ACE2 expression in each cell line could be statistically distinguished from that in the other cell lines, indicating the quantitative feature of this biosensing system. When detecting living cells without cell lysis or fixation, as well as fixed cells, the levels of the epithelial CSPs, EpCAM (epithelial cell adhesion molecule) and E-cadherin, were successfully determined using our Au-tag@SiO2-Ab NPs and SERS immunoassay system in a highly selective and quantitative manner without significant cytotoxicity. Hence, our work provides technical insight into the development of a biosensing platform for a variety of biomedical applications, such as cancer metastasis prognosis and the in situ monitoring of stem cell reprogramming and differentiation.

Electrospun Nanofibrils Surface-Decorated with Photo-Cross-Linked Hyaluronic Acid for Cell-Directed Assembly.

Hyaluronic acid (HA) was chemically immobilized on the surface of electrospun nanofibrils to form a cell/NF complex. Poly(caprolactone) (PCL) was electrospun into nanofibrous mats that were subsequently aminolyzed into nanofibrils. The aminolyzed nanofibrils were surface-decorated with methacrylated HA via Michael type addtion and by photo-cross-linking. Fourier transform infrared spectroscopy revealed the presence of HA on the surface of the nanofibrils. The thermogravimetric and colorimetric analyses indicate that the degree of HA immobilization could be varied by varying the photo-cross-linking duration. Thus, on increasing the photo-cross-linking duration, the swelling ratios increased gradually, and the surface charge of the decorated nanofibrils decreased. NIH3T3 cells and surface-decorated nanofibrils spontaneously assembled into the cell/NF complex. A higher degree of surface-immobilized HA enhanced cell viability and proliferation compared to nanofibrils without surface-immobilized HA. Thus, we envision that HA-immobilized nanofibrils can be employed as a tissue-engineering matrix to control cell proliferation and differentiation.

Discovery of E3 Ligase Ligands for Target Protein Degradation.

Target protein degradation has emerged as a promising strategy for the discovery of novel therapeutics during the last decade. Proteolysis-targeting chimera (PROTAC) harnesses a cellular ubiquitin-dependent proteolysis system for the efficient degradation of a protein of interest. PROTAC consists of a target protein ligand and an E3 ligase ligand so that it enables the target protein degradation owing to the induced proximity with ubiquitin ligases. Although a great number of PROTACs has been developed so far using previously reported ligands of proteins for their degradation, E3 ligase ligands have been mostly limited to either CRBN or VHL ligands. Those PROTACs showed their limitation due to the cell type specific expression of E3 ligases and recently reported resistance toward PROTACs with CRBN ligands or VHL ligands. To overcome these hurdles, the discovery of various E3 ligase ligands has been spotlighted to improve the current PROTAC technology. This review focuses on currently reported E3 ligase ligands and their application in the development of PROTACs.

Recent Advances in Monitoring Stem Cell Status and Differentiation Using Nano-Biosensing Technologies.

In regenerative medicine, cell therapies using various stem cells have received attention as an alternative to overcome the limitations of existing therapeutic methods. Clinical applications of stem cells require the identification of characteristics at the single-cell level and continuous monitoring during expansion and differentiation. In this review, we recapitulate the application of various stem cells used in regenerative medicine and the latest technological advances in monitoring the differentiation process of stem cells. Single-cell RNA sequencing capable of profiling the expression of many genes at the single-cell level provides a new opportunity to analyze stem cell heterogeneity and to specify molecular markers related to the branching of differentiation lineages. However, this method is destructive and distorted. In addition, the differentiation process of a particular cell cannot be continuously tracked. Therefore, several spectroscopic methods have been developed to overcome these limitations. In particular, the application of Raman spectroscopy to measure the intrinsic vibration spectrum of molecules has been proposed as a powerful method that enables continuous monitoring of biochemical changes in the process of the differentiation of stem cells. This review provides a comprehensive overview of current analytical methods employed for stem cell engineering and future perspectives of nano-biosensing technologies as a platform for the in situ monitoring of stem cell status and differentiation.

Clinical evaluation of the brightening effect of chitosan-based cationic liposomes.

BACKGROUND: Cationic liposomes can enhance the permeability of drugs in 3-D skin. Chitosan is considered a safe material for percutaneous delivery; thus, this study uses chitosan-incorporated cationic liposomes. AIMS: This study investigated the improvement in skin brightness, melanin, and melasma after treatment niacinamide-incorporated chitosan cationic liposomes. METHODS: A skin brightening agent, niacinamide, was formulated into cationic liposomes to facilitate percutaneous absorption and was clinically tested in 21 Korean female subjects. Cationic liposomes were prepared using a high-pressure homogenizer after mixing an oil phase containing lecithin and cholesterol and an aqueous phase containing niacinamide and chitosan. RESULTS: The cationic liposomes exhibited stability over 28 days, with a particle size of 255-275 nm and zeta potential of 10-14 mV. Cationic liposomes containing niacinamide and a control formulation were applied to the left and right side of the face, respectively, twice daily for 28 days. Skin brightness, melanin index, and area of melasma were significantly enhanced where cationic liposomes were used, in comparison with formulations without cationic liposomes, demonstrating a 1.38-2.08-fold improvement. CONCLUSION: Thus, we established that chitosan liposomes augmented the percutaneous absorption of niacinamide and improved the appearance of the skin.

Dual irradiation-triggered anticancer therapeutics composed of polydopamine-coated gold nanoparticles.

The therapeutic efficacy of nanoparticles depends on their ability to release encapsulated photosensitizers. Here, surface-engineered metallic gold nanoparticles (AuNP) were irradiated with dual near-infrared (NIR) light to enhance the release of photosensitizer. Dopamine hydrochloride was surface-polymerized to polydopamine (PDA) layers on AuNP, and chlorin-e6 (Ce6) was chemically tethered to primary amines of PDA. The resulting Ce6-conjugated AuNP were characterized by Raman and X-ray photoelectron spectroscopy and visualized by electron microscopy and light scattering. The generation of reactive oxygen species was increased following dual NIR irradiation at 650 nm and 808 nm, which was attributed to the increased liberation of Ce6. In vitro, dual NIR irradiation significantly enhanced the anticancer effect of Ce6-incorporating AuNP by increasing the population of apoptotic cells. In vivo, tumor xenografted animals exhibited much better tumor suppression when subjected to dual NIR irradiation. Thus, we propose the use of Ce6-incorporating AuNP coupled to dual NIR irradiation for future anticancer treatment of solid tumors.

Fluorescence-Shadowing Nanoparticle Clusters for Real-Time Monitoring of Tumor Progression.

Monitoring tumor progression is important for elucidating appropriate therapeutic strategies in response to anticancer therapeutics. To fluorescently monitor the in vivo levels of tumor-specific enzymes, we prepared matrix metalloprotease (MMP)-responsive gold nanoparticle (AuNP) clusters to sense tumor microenvironments. Specifically, AuNPs and quantum dots (QDs) were surface-engineered with two poly(ethylene glycol) [PEG] shells and cyclooctyne moieties, respectively, for the copper-free click reaction. Upon "peeling off" of the secondary shell from the double-PEGylated AuNPs under MMP-rich conditions, shielded azide moieties of the AuNPs were displayed toward the QD, and those two particles were clicked into nanoparticle clusters. This consequently resulted in a dramatic size increase and fluorescence quenching of QDs via fluorescence energy transfer (FRET) due to the molecular proximity of the particles. We observed that FRET efficiency was modulated via changes in MMP levels and exposure time. Cancer cell numbers exhibited a strong correlation with FRET efficiency, and in vivo studies that employed solid tumor models accordingly showed that FRET efficiency was dependent on the tumor size. Thus, we envision that this platform can be tailored and optimized for tumor monitoring based on MMP levels in solid tumors.

Light-Triggered Structural Modulation of Nanofibrous Meshes to Promote Deep Penetration of Cultured Cells.

Although nanofibrous meshes are considered promising cultivation beds for maintaining cell differentiation, 3D cultivation is not possible because their nanoporous structures impede cell infiltration. To facilitate transverse cell migration across nanofibrous meshes, electrospun nanofibers are prepared with structures that vary in response to red laser light. Polyoxalate (POX), composed of oxalate linkers and oligomeric caprolactone, is synthesized and electrospun into fibrous meshes with a photosensitizer (chlorin e6, Ce6). These meshes exhibit morphological and chemical changes upon laser irradiation, and mass erosion rates of the meshes are faster after laser irradiation. Cell cultivation on POX meshes reveals that red laser effectively facilitates traverse migration of the cells without affecting cell viability. The use of light-triggered change of meshes is envisioned to promote the migration of cells during 3D matrix cultivation.

Process monitoring of photocatalytic degradation of 2,4-dinitrotoluene by Au-decorated Fe3O4@TiO2 nanoparticles: surface-enhanced Raman scattering method.

Recently, the degradation and detection of 2,4-dinitrotoluene (2,4-DNT) capable of producing 2,4,6-trinitrotoluene (TNT) for environmental and human health risks have been developed. We prepared photoresponsive Au-decorated Fe3O4@TiO2 nanoparticles (Fe3O4@TiO2-Au NPs) under sunlight simulated Xe lamp irradiation. The photodegradation process of 2,4-DNT by Fe3O4@TiO2-Au NPs was successfully monitored by surface-enhanced Raman scattering (SERS). Since SERS monitoring shows intrinsic information about the molecular structure, it was possible to predict the photodegradation of 2,4-DNT. The 2,4-DNT photodegradation mechanism based on two-dimensional correlation spectroscopy (2D-COS), which provides very beneficial information for a deeper understanding of systems, has been identified. We confirmed that Fe3O4@TiO2-Au NPs can be widely used in organic pollutant degradation under sunlight. Furthermore, the combination of SERS based process monitoring and 2D-COS can be a convincing analytical technique for photodegradation studies of organic pollutants.

Atom transfer radical-polymerized cationic shells on gold nanoparticles for near infrared-triggered photodynamic therapy of tumor-bearing animals.

Gold nanoparticles (AuNPs) were surface-engineered with a cationic corona to enhance the incorporation of photosensitizers for photodynamic therapy (PDT). The cationic corona composed of poly(2-(dimethylamino)ethyl methacrylate) was atom transfer radical-polymerized on the surface of the AuNPs. The cationic corona of the engineered surface was characterized by dynamic light scattering, electron microscopy, Raman spectroscopy, and mass spectroscopy. Chlorin-e6 (Ce6) incorporated onto the surface-engineered AuNPs exhibited higher cell incorporation efficiency than bare AuNPs. Ce6-incorporated AuNPs were confirmed to release singlet oxygen upon NIR irradiation. Compared to Ce6, Ce6-incorporated AuNPs exhibited higher cellular uptake and cytotoxicity against cancer cells in an irradiation time-dependent manner. Near-infrared-irradiated animals administered Ce6-incorporated AuNPs exhibited higher levels of tumor suppression without noticeable body weight loss. This result was attributed to the higher localization of Ce6 at the tumor sites to induce cancer cell apoptosis. Thus, we envision that engineered AuNPs with cationic corona can be tailored to effectively deliver photosensitizers to tumor sites for photodynamic therapy.

Modulating Mechanism of the LSPR and SERS in Ag/ITO Film: Carrier Density Effect.

Herein, we fabricated a uniform and dispersible Ag/indium tin oxide (ITO) cosputtered film on a two-dimensional ordered polystyrene template and observed distinct localized surface plasmon resonance (LSPR) properties that can be tuned by changing the doping level. The increase in the optical band gap is due to the variation in the metallic Ag content, which can effectively change the accumulation of free electrons in the conduction band, in addition to the near-IR absorbance. Surface-enhanced Raman scattering (SERS) was used to monitor the variations in the band gap and transfer of electrons, which causes variations in the SERS intensity. The presented research provides new insights into the relationships between the carrier density and maximum absorption wavelength, band gap distribution, and charge transfer process. This is the first study on the influence of the carrier density on the properties of Ag/ITO cosputtered films and suggests practical applications of these films.

Comparison Study of the Effects of Cationic Liposomes on Delivery across 3D Skin Tissue and Whitening Effects in Pigmented 3D Skin.

Charged phospholipids are employed to formulate liposomes with different surface charges to enhance the permeation of active ingredients through epidermal layers. Although 3D skin tissue is widely employed as an alternative to permeation studies using animal skin, only a small number of studies have compared the difference between these skin models. Liposomal delivery strategies are investigated herein, through 3D skin tissue based on their surface charges. Cationic, anionic, and neutral liposomes are formulated and their size, zeta-potential, and morphology are characterized using dynamic light scattering and cryogenic-transmission electron microscopy (cryo-TEM). A Franz diffusion cell is employed to determine the delivery efficiency of various liposomes, where all liposomes do not exhibit any recognizable difference of permeation through the synthetic membrane. When the fluorescence liposomes are applied to 3D skin, considerable fluorescence intensity is observed at the stratum cornea and epithelium layers. Compared to other liposomes, cationic liposomes exhibit the highest fluorescence intensity, suggesting the enhanced permeation of liposomes through the 3D skin layers. Finally, the ability of niacinamide (NA)-incorporated liposomes to suppress melanin transfer in pigmented 3D skin is examined, where cationic liposomes exhibit the highest degree of whitening effects.

Preparation of Stretchable Nanofibrous Sheets with Sacrificial Coaxial Electrospinning for Treatment of Traumatic Muscle Injury.

Traumatic muscle injury with massive loss of muscle volume requires intramuscular implantation of proper scaffolds for fast and successful recovery. Although many artificial scaffolds effectively accelerate formation and maturation of myotubes, limited studies are showing the therapeutic effect of artificial scaffolds in animal models with massive muscle injury. In this study, improved myotube differentiation is approved on stepwise stretched gelatin nanofibers and applied to damaged muscle recovery in an animal model. The gelatin nanofibers are fabricated by a two-step process composed of co-axial electrospinning of poly(ɛ-caprolactone) and gelatin and subsequent removal of the outer shells. When stepwise stretching is applied to the myoblasts on gelatin nanofibers for five days, enhanced myotube formation and polarized elongation are observed. Animal models with volumetric loss at quadriceps femoris muscles (>50%) are transplanted with the myotubes cultivated on thin and flexible gelatin nanofiber. Treated animals more efficiently recover exercising functions of the leg when myotubes and the gelatin nanofiber are co-implanted at the injury sites. This result suggests that mechanically stimulated myotubes on gelatin nanofiber is therapeutically feasible for the robust recovery of volumetric muscle loss.