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.

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.

Atom Transfer Radical Polymerization of Multishelled Cationic Corona for the Systemic Delivery of siRNA.

We propose an effective siRNA delivery system by preparing poly(DAMA-HEMA)-multilayered gold nanoparticles using multiple surface-initiated atom transfer radical polymerization processes. The polymeric multilayer structure is characterized by transmission electron microscopy, matrix-associated laser desorption/ionization time-of-flight mass spectrometry, UV-vis spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, and ζ-potential. The amount of siRNA electrostatically incorporated into the nanoparticle can be tuned by the number of polymeric shells, which in turn influences the cellular uptake and gene silencing effect. In a bioreductive environment, the interlayer disulfide bond breaks to release the siRNA from the degraded polymeric shells. Intravenously injected c-Myc siRNA-incorporated particles accumulate in the tumor site of a murine lung carcinoma model and significantly suppress the tumor growth. Therefore, the combination of a size-tunable AuNP core and an ATRP-functionalized shell offers control and versatility in the effective delivery of siRNA.

Electrospun Nanofibrous Sheets for Selective Cell Capturing in Continuous Flow in Microchannels.

Electrospun nanofibrous meshes were surface-modified for selective capturing of specific cells from a continuous flow in PDMS microchannels. We electrospun nanofibrous mats composed of poly(ε-carprolactone) (PCL) and amine-functionalized block copolymers composed of PCL and poly(ethylenimine) (PEI). A mixture of biotinylated PEG and blunt PEG was chemically tethered to the nanofibrous mats via the surface-exposed amines on the mat. The degree of biotinylation was fluorescently and quantitatively assayed for confirming the surface-biotinylation levels for avidin-specific binding. The incorporation level of avidin gradually increased when the blend ratio of biotinylated PEG on the mat increased, confirming the manipulated surfaces with various degree of biotinylation. Biotinylated cells were incubated with avidin-coated biotinylated mats and the specific binding of biotinylated cells was monitored in a microfluidic channel with a continuous flow of culture medium, which suggests efficient and selective capturing of the biotinylated cells on the nanofibrous mat.

Nanoporous Electrospun Fibrous Meshes: Size-Controlled Reverse Micelles Strategy.

A simple and efficient method to fabricate size-controlled nanoporous-nanofibrous meshes has been demonstrated by introducing and removing novel size-controllable porogens, reverse micelles, on electrospun polymeric nanofibers. Poly(D,L-lactide) and reverse micelles composed of amphiphilic diblock copolymer, poly(ethylene glycol) methyl ether-block-poly(ε-caprolactone), were first dissolved in an acetone/chloroform (3:1, v/v) mixture and then electrospun into nanofibers, followed by 70% EtOH post-treatment. During the post-treatment, the reverse micelles were dissolved in 70% EtOH at room temperature thus separated from the poly(D,L-lactide) nanofibers backbone, resulting in a nanoporous nanofibrous structure. The pores on the nanofibers are size-controllable because the sizes of the reverse micelles can be adjusted by varying the water content inside them. The sizes of reverse micelles, which ranged from 100 nm to 700 nm, are investigated by dynamic light scattering. The pores of various sizes on the poly(D,L-lactide) nanofibers have areas ranging from 20 µm2 to 80 µm2 were observed by field-emission scanning electron microscopy.

Nucleic acid-scavenging electrospun nanofibrous meshes for suppressing inflammatory responses.

Fragmented nucleic acids are potent stimulators for inflammatory responses provoking pathological outcomes by activating adaptive immunity. In this study, highly cationic surfaces were prepared on electrospun nanofibrous meshes to scavenge nucleic acids to the surfaces. Poly(ε-caprolactone) [PCL]-poly(ethylenimine) [PEI] block copolymers were synthesized by coupling the carboxyl-terminated PCL to the primary amines of branched PEI. Polymeric solutions composed of PCL-PEI and PCL were electrospun to nanofibrous mats, and the surfaces were further methylated to prepare highly cationic surfaces on the mats. Raman spectroscopy revealed that the presence of increased methylated amines on the surfaces of the mats compared to unmodified mats. The methylated surfaces showed significant increases of wettability after methylation, suggesting highly charged surfaces caused by methylation of the primary amines. When the blend ratio of PCL-PEI was increased, the scavenged DNA was also increased, and the methylation further strengthened the scavenging ability of the mats. Fluorescently labeled oligodeoxynucleic acids were significantly adsorbed on the surface of the mats depending on the amounts of PCL-PEI and the degree of methylation. In the presence of the methylated nanofibrous mats, inflammatory responses induced by CpG oligonucleotides in murine macrophages were significantly reduced, which was confirmed by measuring inflammatory cytokine levels including TNF-α and IFN-γ.

Optimization of the surface immobilized folate on the magnetic nanoparticles.

Cancer cells overexpressing folate receptors have been targeted using a folate decorated carriers for anti-cancer drugs in aims to overcome the tissue non-specificity of anti-cancer agents. We here prepared magnetic nanoparticles and surface-decorated them with different amounts of folate to optimize the number of the immobilized folate on the carriers for superior targeting effects. Magnetic nanoparticles were prepared by oxidizing ferric or ferrous chloride solution to iron oxide in the presence of poly(vinyl alcohol). The magnetic nanoparticles were functionalized with primary amines for subsequent reactions with the different feed ratios of the activated folate. The magnetization degree of the folate magnetic magnetization were slightly decreased when the folate on the particles were increased. Intracellular uptakes of the nanoparticles were shown to be increased and become saturated dependent on the amounts of the surface-immobilized folate. The folate-decorated magnetic nanoparticles showed negligible cytotoxicity against KB cells from 5 µg to 35 µg of the nanoparticle weights.

Inorganic Nanoparticle Functionalization Strategies in Immunotherapeutic Applications.

Nanotechnology has been increasingly utilized in anticancer treatment owing to its ability of engineering functional nanocarriers that enhance therapeutic effectiveness while minimizing adverse effects. Inorganic nanoparticles (INPs) are prevalent nanocarriers to be customized for a wide range of anticancer applications, including theranostics, imaging, targeted drug delivery, and therapeutics, because they are advantageous for their superior biocompatibility, unique optical properties, and capacity of being modified via versatile surface functionalization strategies. In the past decades, the high adaptation of INPs in this emerging immunotherapeutic field makes them good carrier options for tumor immunotherapy and combination immunotherapy. Tumor immunotherapy requires targeted delivery of immunomodulating therapeutics to tumor locations or immunological organs to provoke immune cells and induce tumor-specific immune response while regulating immune homeostasis, particularly switching the tumor immunosuppressive microenvironment. This review explores various INP designs and formulations, and their employment in tumor immunotherapy and combination immunotherapy. We also introduce detailed demonstrations of utilizing surface engineering tactics to create multifunctional INPs. The generated INPs demonstrate the abilities of stimulating and enhancing the immune response, specific targeting, and regulating cancer cells, immune cells, and their resident microenvironment, sometimes along with imaging and tracking capabilities, implying their potential in multitasking immunotherapy. Furthermore, we discuss the promises of INP-based combination immunotherapy in tumor treatments.

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 .

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.

Milk exosome-infused fibrous matrix for treatment of acute wound.

To provide an advanced therapy for wound recovery, in this study, pasteurized bovine milk-derived exosomes (mEXO) are immobilized onto a polydopamine (PDA)-coated hyaluronic acid (HA)-based electrospun nanofibrous matrix (mEXO@PMAT) via a simple dip-coating method to formulate an mEXO-immobilized mesh as a wound-healing biomaterial. Purified mEXOs (~82 nm) contain various anti-inflammatory, cell proliferation, and collagen synthesis-related microRNAs (miRNAs), including let-7b, miR-184, and miR-181a, which elicit elevated mRNA expression of keratin5, keratin14, and collagen1 in human keratinocytes (HaCaT) and fibroblasts (HDF). The mEXOs immobilized onto the PDA-coated meshes are gradually released from the meshes over 14 days without burst-out effect. After treatment with HaCaT and HDF, the degree of in vitro cell migration increases significantly in the mEXO@PMAT-treated HaCaT and HDF cells compared to the unmodified or PDA-coated meshes-treated cells. Additionally, the mEXO@PMAT provides significantly faster wound closure in vivo without notable toxicity. Thus, the sustained liberation of bioactive mEXO from the meshes can effectively enhance cell proliferation in vitro and accelerate wound closure in vivo, which could be harnessed mEXO@PMAT as a promising wound-healing biomaterial.

Dual-responsive breakdown of nanostructures with high doxorubicin payload for apoptotic anticancer therapy.

Self-assembled nanoaggregates co-encapsulating doxorubicin (DOX) and oligonucleotide are prepared for dual-responsive breakdown of the nanostructure with complete disappearance characteristics. Four-arm poly(ethylene glycol) is co-conjugated with DOX and anti-bcl-2 oligonucleotide with reducible linkers and acid-cleavable linkers, respectively. The conjugate is hydrophobically self-assembled into nanoaggregates in aqueous solution. Elemental scanning of the nanoaggregates reveals their core-shell structure with DOX and oligonucleotide located at the core and the shell, respectively. The tracking of size modulation suggests the complete disappearance of the particles under reducing conditions and the liberation of oligonucleotide at low pH, which is confirmed by dynamic light scattering and electron microscopy. The release of DOX and oligonucleotide is controlled by the pH and the reducing potential of the medium, and most of the drug and DNA are released in 24 h. The released fractions are analyzed by reversed-phase chromatography, which indicates facile cleavage of DOX and oligonucleotide from the carriers. The nanoaggregates with both DOX and oligonucleotide show the lowest IC(50) value when a cytotoxicity assay is performed against A549 cells. Apoptosis assay also confirms that cells treated with the nanoaggregates having both DOX and oligonucleotide show higher fluorescence intensity of antiapoptotic antibody than native DOX.

Electrospun chitosan microspheres for complete encapsulation of anionic proteins: controlling particle size and encapsulation efficiency.

Electrospinning was employed to fabricate chitosan microspheres by a single-step encapsulation of proteins without organic solvents. Chitosan in acetic acid was electrospun toward a grounded sodium carbonate solution at various electric potential and feeding rates. Electrospun microspheres became insoluble and solidified in the sodium carbonate solution by neutralization of chitosan acetate. When the freeze-dried microspheres were examined by scanning electron microscopy, the small particle size was obtained at higher voltages. This is explained by the chitosan droplet size at the electrospinning needle was clearly controllable by the electric potential. The recovery yield of chitosan microspheres was dependent on the concentration of chitosan solution due to the viscosity is the major factor affecting formation of chitosan droplet during curling of the electrospinning jets. For protein encapsulation, fluorescently labeled bovine serum albumin (BSA) was codissolved with chitosan in the solution and electrospun. At higher concentration of sodium carbonate solution and longer solidification time in the solution, the encapsulation efficiency of the protein was confirmed to be significantly high. The high encapsulation efficiency was achievable by instant solidification of microspheres and electrostatic interactions between chitosan and BSA. Release profiles of BSA from the microspheres showed that the protein release was faster in acidic solution due to dissolution of chitosan. Reversed-phase chromatography of the released fractions confirmed that exposure of BSA to acidic solution during the electrospinning did not result in structural changes of the encapsulated protein.

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.

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.

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.

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.

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.

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.

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.