Nanosuspension-Loaded Dissolving Microneedle Patches for Enhanced Transdermal Delivery of a Highly Lipophilic Cannabidiol.

Purpose: Transdermal Drug Delivery System (TDDS) offers a promising alternative for delivering poorly soluble drugs, challenged by the stratum corneum's barrier effect, which restricts the pool of drug candidates suitable for TDDS. This study aims to establish a delivery platform specifically for highly lipophilic drugs requiring high doses (log P > 5, dose > 10 mg/kg/d), to improve their intradermal delivery and enhance solubility. Methods: Cannabidiol (CBD, log P = 5.91) served as the model drug. A CBD nanosuspension (CBD-NS) was prepared using a bottom-up method. The particle size, polydispersity index (PDI), zeta potential, and concentration of the CBD-NS were characterized. Subsequently, CBD-NS was incorporated into dissolving microneedles (DMNs) through a one-step manufacturing process. The intradermal dissolution abilities, physicochemical properties, mechanical strength, insertion depth, and release behavior of the DMNs were evaluated. Sprague-Dawley (SD) rats were utilized to assess the efficacy of the DMN patch in treating knee synovitis and to analyze its skin permeation kinetics and pharmacokinetic performance. Results: The CBD-NS, stabilized with Tween 80, exhibited a particle size of 166.83 ± 3.33 nm, a PDI of 0.21 ± 0.07, and a concentration of 46.11 ± 0.52 mg/mL. The DMN loaded with CBD-NS demonstrated favorable intradermal dissolution and mechanical properties. It effectively increased the delivery of CBD into the skin, extended the action's duration in vivo, and enhanced bioavailability. CBD-NS DMN exhibited superior therapeutic efficacy and safety in a rat model of knee synovitis, significantly inhibiting TNF-α and IL-1ß compared with the methotrexate subcutaneous injection method. Conclusion: NS technology effectively enhances the solubility of the poorly soluble drug CBD, while DMN facilitates penetration, extends the duration of action in vivo, and improves bioavailability. Furthermore, CBD has shown promising therapeutic outcomes in treating knee synovitis. This innovative drug delivery system is expected to offer a more efficient solution for the administration of highly lipophilic drugs akin to CBD, thereby facilitating high-dose administration.

Recent progress of polymeric microneedle-assisted long-acting transdermal drug delivery.

Microneedle (MN)-assisted drug delivery technology has gained increasing attention over the past two decades. Its advantages of self-management and being minimally invasive could allow this technology to be an alternative to hypodermic needles. MNs can penetrate the stratum corneum and deliver active ingredients to the body through the dermal tissue in a controlled and sustained release. Long-acting polymeric MNs can reduce administration frequency to improve patient compliance and therapeutic outcomes, especially in the management of chronic diseases. In addition, long-acting MNs could avoid gastrointestinal reactions and reduce side effects, which has potential value for clinical application. In this paper, advances in design strategies and applications of long-acting polymeric MNs are reviewed. We also discuss the challenges in scale manufacture and regulations of polymeric MN systems. These two aspects will accelerate the effective clinical translation of MN products.

Development of Novel Nanocarriers by Ultrasound and Ethanol-Assisted Soy Protein Isolate: Enhancing the Resistance of Lutein to Environmental Stress.

The aim of this work was to investigate the effects of the processing sequence of ultrasound and ethanol on the physicochemical properties of soy protein isolate (SPI), which were further evaluated for the morphology and stability of SPI-lutein coassembled nanoparticles. The results showed that the sequence of ultrasound followed by ethanol treatment was the optimal one. The samples were subjected to ultrasonication followed by subunit disassembly and reassembly induced by 40% (v/v) ethanol, with the resulting molecular unfolding and subsequent aggregation being attributed to intramolecular hydrogen bonds. The recombined nanoparticles had smaller particle size (142.43 ± 2.91 nm) and turbidity (0.16 ± 0.01), and the exposure of more hydrophobic groups (H0 = 6221.00 ± 130.20) induced a shift of SPI structure toward a more ordered direction. The homogeneous and stable particle provided excellent stability for the loading of lutein. The bioaccessibility (from 25.48 ± 2.35 to 65.85 ± 1.78%) and release rate of lutein were modulated in gastrointestinal digestion experiments. Our discoveries provide a new perspective for the development of combined physicochemical modification of proteins as nanocarriers in functional foods.

Wheat gliadin hydrolysates based nano-micelles for hydrophobic naringin: Structure characterization, interaction, and in vivo digestion.

In this study, enzymatic hydrolysis was used to fabricate wheat gliadin hydrolysates (WGHs) for the encapsulation and protection of naringin. The exposure of hydrophilic amino acids decreased the critical micelle concentration (from 0.53 ± 0.02 mg/mL to 0.35 ± 0.03 mg/mL) and improved solubility, which provided amphiphilic conditions for the delivery of naringin. The hydrolysates with a degree of hydrolysis (DH) of 9 % had the strongest binding affinity with naringin, and exhibited the smallest particle size (113.7 ± 1.1 nm) and the highest encapsulation rate (83.2 ± 1.3 %). The storage, heat and photochemical stability of naringin were improved via the encapsulation of micelles. Furthermore, the micelles made up of hydrolysates with a DH of 12 % significantly enhanced the bioavailability of naringin (from 19.4 ± 4.3 % to 46.8 ± 1.4 %). Our experiment provides theoretical support for the utilization of delivery systems based on water-insoluble proteins.

Effects of Halogenation on Quinoline-Malononitrile-based AIEgens: Photophysical Properties Investigation and Wash-Free Imaging.

Developing halogen-functionalized fluorescent dyes with intriguing photophysical properties, including enhanced photostability, is particularly important for bioimaging. In this work, we synthesized two new halogen-functionalized aggregation-induced emission (AIE)-active molecules, DQMF-OH and DQMCl-OH, based on the quinoline-malononitrile chromophore. The halogen effect on the photophysical characteristics was detailedly studied by absorption and fluorescence spectroscopy, density functional theory calculations, and crystal structures. Compared with non-halogen substituted AIE luminogen (AIEgen) DQM-OH, the halogen substituted DQMF-OH and DQMCl-OH exhibited red-shifted absorptions and emissions in the solution and solid state. In addition, DQMF-OH and DQMCl-OH also possessed enhanced fluorescence toward viscosity changes. These AIEgens served as remarkable imaging tools for cell tracking in a wash-free manner. Furthermore, DQMF-OH and DQMCl-OH showed much more excellent photobleaching resistance than DQM-OH. Our work sheds new light on developing fluorescent halogenated dyes with enhanced photophysical performances for biological applications.

Thermal stability of exenatide encapsulated in stratified dissolving microneedles during storage.

As low-temperature storage and transportation of peptides require high costs, improving the dosage form of peptides can reduce costs. We developed a thermostable and fast-releasing stratified dissolving microneedle (SDMN) system for delivering exenatide (EXT) to patients with type 2 diabetes. Among the tested polymers, dextran and polyvinyl alcohol (PVA) were the best at stabilizing EXT under high-temperature storage for 9 weeks. The two polymers possess a relatively high glass transition temperature (Tg) and weak hydrogen bonding between PVA and EXT. Additionally, zinc sulfate (ZnSO4) had a stabilizing effect on EXT among the selected stabilizers, suggesting that EXT formed a dimer after coordination with zinc ions (Zn2+). In addition, the denaturation temperature (Tm) of EXT was increased by adding ZnSO4, thus stabilizing EXT. Accordingly, SDMNs consisting of a tip layer (dextran encapsulating the Zn2+-EXT complex) and a base layer (PVA) were fabricated. Within 2 min of implantation, the EXT loaded on the patch was quickly released into the skin. Transdermal pharmacokinetics studies showed that manufactured SDMNs generated comparable efficacy to subcutaneous injection. Significantly, the remaining EXT amount was not significantly different under storage at 40 °C and -20 °C for 3 months, supporting that the SDMN system had excellent delivery efficiency and stability, thus reducing the dependence on the cold chain.

Design and Evaluation of Complex Polypeptide-Loaded Dissolving Microneedles for Improving Facial Wrinkles in Different Areas.

Wrinkles are one of the most intuitive manifestations of skin aging. Complex polypeptide-loaded dissolving microneedles (CP-DMNs) for facial wrinkles in different areas have been developed and evaluated for the first time. In optimizing formulations, we compared the differences in CP-DMNs heights on skin insertion depth and skin repair and healing. Furthermore, systemic safety experiments were carried out to provide a reference for clinical application. On this basis, an 84-day efficacy assessment based on the improvement of facial wrinkles in different areas and a comparison between CP-DMNs vs. placebo was performed on 30 healthy subjects. As a result, DMNs with a height of 300 µm presented sufficient strength to pierce the stratum corneum with minimized skin damage. In addition, CP-DMNs possessed excellent biological safety and skin compatibility for clinical application. Compared with placebo, CP-DMNs exhibited obvious improvements in wrinkles distributed in the corners of eyes, under-eyes, and nasolabial folds. Furthermore, after using CP-DMNs for 84 days, facial wrinkles in five different areas were smoothed. In short, the complex polypeptides showed apparent anti-wrinkle efficacy with the aid of DMNs technology, and CP-DMNs seemed to work better on deeper wrinkles, such as frown lines and nasolabial folds.

Progress on Multifunction Enzyme-Activated Organic Fluorescent Probes for Bioimaging.

Bioimaging techniques are of increasing importance in clinical and related fields, which also have been successfully applied in the in vivo/in vitro imaging system. Due to the vital factor of enzymes in biological systems, enzyme-activated fluorophores, which could turn "on" the fluorescence signal from an "off" state, offer non-invasive and effective potential for the accurate bioimaging of particular cells, tissues, or bacteria. Comparing with the traditional imaging probes, enzyme-activated organic small fluorophores can visualize living cells within small animals with high sensitivity, high imaging resolution, non-invasiveness, and real-time feedback. In this mini review, well-designed enzyme-activated organic fluorescent probes with multiple functions are exclusively reviewed through the latest development and progress, focusing on probe design strategy, fluorescence property, enzyme activation process, and bioimaging applications. It is worth noting that multi-enzyme-activated strategies, which could avoid the production of "false-positive" signals in complex biological systems, effectively provide high selective and real-time bioimaging, indicating the exciting potential of intraoperative fluorescence imaging and diagnosis tools.

Structure Rigidification Promoted Ultrabright Solvatochromic Fluorescent Probes for Super-Resolution Imaging of Cytosolic and Nuclear Lipid Droplets.

Lipid droplets (LDs) containing cytosolic and nuclear LDs have recently received increasing attention because of their diverse biological roles in living systems. However, developing fluorescent probes for super-resolution visualization of these subcellular LDs still remains challenging due to insufficient fluorescence brightness and poor nuclear membrane permeability. Herein, we rationally synthesized a series of ultrabright solvatochromic fluorescent probes based on benzoboranils (BBAs) for LD-specific super-resolution imaging using structured illumination microscopy (SIM). The rigidly structured probes exhibit ultrahigh fluorescence quantum yields of up to 99.9% in low-polar solvents. They also show more significant fluorescence enhancements in lipid environments than commercial LD probes. Owing to these excellent merits, our lipophilic fluorescent probes can specifically light up subcellular LDs at ultralow concentrations down to 10 nM. Further use of BBA-CF3 for super-resolution SIM imaging of cytosolic and nuclear LDs and their fusion process was successfully achieved. The unprecedented spatial resolution for nuclear LDs with an FWHM value of 142 nm was also acquired. Collectively, our ultrabright fluorescent probes hold tremendous potential to unveil the mysterious roles of cytosolic and nuclear LDs in biological research using SIM.

One-Shot Full-Range Quantification of Multi-Biomarkers With Different Abundance by a Tandem Giant Magnetoresistance Assay.

In this study, we reported a tandem giant magnetoresistance (GMR) assay that realized the one-shot quantification of multi-biomarkers of infection, C-reactive protein (CRP) with procalcitonin (PCT), and neutrophil gelatinase-associated lipocalin (NGAL), all of which could cover their clinically relevant concentration ranges under a different principle. In the presence of co-determined assay, we quantified these three biomarkers in undiluted human blood serum in a single test. The tandem principle, based on which quantification of CRP occurs, combines a sandwich assay and an indirect competitive assay, which allows for the discrimination of the concentration values resulting from the multivalued dose-response curve ('Hook' effect), which characterizes the one-step sandwich assay at high CRP concentrations. However, the entire diagnostically dynamic range, in the quantification of PCT and NGAL, was achieved by differential coating of two identical GMR sensors operated in tandem and by combining two standard curves. The sensor quantified low detection limits and a broader dynamic range for the detection of infection biomarkers. The noticeable features of the assay are its dynamic range and small sample volume requirement (50 µL), and the need for a short measurement time of 15 min. These figures of merit render it a prospective candidate for practical use in point-of-care analysis.

Protocells: Milestones and Recent Advances.

The origin of life is still one of humankind's great mysteries. At the transition between nonliving and living matter, protocells, initially featureless aggregates of abiotic matter, gain the structure and functions necessary to fulfill the criteria of life. Research addressing protocells as a central element in this transition is diverse and increasingly interdisciplinary. The authors review current protocell concepts and research directions, address milestones, challenges and existing hypotheses in the context of conditions on the early Earth, and provide a concise overview of current protocell research methods.

Dynamic Range Expansion of the C-Reactive Protein Quantification with a Tandem Giant Magnetoresistance Biosensor.

In this study, we report a convenient analytical method for a full-range quantification of the C-reactive protein (CRP), a blood biomarker of infection and cardiovascular events. We determine CRP over the entire diagnostically relevant concentration range in undiluted human blood serum in a single test, using a tandem giant magnetoresistance (GMR) sensor. The tandem principle combines a sandwich assay and a competitive assay, which allows for the discrimination of the concentration values resulting from the multivalued dose-response curve ("Hook" effect), which characterizes the one-step sandwich assay at high CRP concentrations. The sensor covers a linear detection range for CRP concentration from 3 ng/mL to 350 µg/mL, the detection limit (s/n = 3) is 1 ng/mL. The prominent features of the chip-based method are its expanded dynamic range and low sample volume (50 µL), and the need for a short measurement time of 15 min. These figures of merit, in addition to the low detection limit equal to the established assay instrumentation, make it a viable candidate for use in point-of-care diagnostics.

Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution.

Droplet-based microfluidics has been widely applied in enzyme directed evolution (DE), in either cell or cell-free system, due to its low cost and high throughput. As the isolation principles are based on the labeled or label-free characteristics in the droplets, sorting method contributes mostly to the efficiency of the whole system. Fluorescence-activated droplet sorting (FADS) is the mostly applied labeled method but faces challenges of target enzyme scope. Label-free sorting methods show potential to greatly broaden the microfluidic application range. Here, we review the developments of droplet sorting methods through a comprehensive literature survey, including labeled detections [FADS and absorbance-activated droplet sorting (AADS)] and label-free detections [electrochemical-based droplet sorting (ECDS), mass-activated droplet sorting (MADS), Raman-activated droplet sorting (RADS), and nuclear magnetic resonance-based droplet sorting (NMR-DS)]. We highlight recent cases in the last 5 years in which novel enzymes or highly efficient variants are generated by microfluidic DE. In addition, the advantages and challenges of different sorting methods are briefly discussed to provide an outlook for future applications in enzyme DE.

A tandem giant magnetoresistance assay for one-shot quantification of clinically relevant concentrations of N-terminal pro-B-type natriuretic peptide in human blood.

We report a microfluidic sandwich immunoassay constructed around a dual-giant magnetoresistance (GMR) sensor array to quantify the heart failure biomarker NT-proBNP in human plasma at the clinically relevant concentration levels between 15 pg/mL and 40 ng/mL. The broad dynamic range was achieved by differential coating of two identical GMR sensors operated in tandem, and combining two standard curves. The detection limit was determined as 5 pg/mL. The assay, involving 53 plasma samples from patients with different cardiovascular diseases, was validated against the Roche Cobas e411 analyzer. The salient features of this system are its wide concentration range, low detection limit, small sample volume requirement (50 µL), and the need for a short measurement time of 15 min, making it a prospective candidate for practical use in point of care analysis.

A highly selective "turn-on" water-soluble fluorescent sensor for gallium ion detection.

In this work, a novel sensor, (E)-N'-(3-(tert-butyl)-2-hydroxybenzylidene)thiophene-2-carbohydrazide (1), based on salicylaldehyde and thiophene hydrazide moieties was designed and synthesized. The single-crystal structure of 1 was achieved and studied for understanding its functional properties. The interaction and recognition abilities of 1 with different metal ions were investigated. Sensor 1 showed excellent "turn-on" fluorescence with highly selective and specific recognition ability in the presence of gallium ions (Ga3+) in an aqueous solution. The sensing behavior of 1 with Ga3+ was also studied by photophysical experiments, ESI-MS analysis, and 1H NMR titration. The limit of detection (LOD) and limit of quantification (LOQ) of 1 for the detection of Ga3+ in an aqueous solution were calculated as 58 nM, and 192 nM, respectively. DFT calculations were carried out to optimize the configuration of 1 and 1-Ga3+ complexes and rationalize the photophysical experimental data. Highly selective test strips based on sensor 1 were developed for Ga3+ detection. Sensor 1 was also used to detect Ga3+ in actual water samples, and a considerable recovery rate was obtained.

"Turn-On" Fluorescent Biosensors for High Selective and Sensitive Detection of Al3+ Ion.

A series of new compounds (1-4) based on pyrrole hydrazone Schiff bases were designed and synthesized. The interactions of these new compounds with metal ions and their fluorescent recognition were investigated. All compounds showed "turn-on" fluorescence in the presence of Al3+ in aqueous solution. Their sensing behaviors with Al3+ were studied using photophysical experiments, ESI-MS spectrometry analysis, 1H NMR titration, and DFT calculation. The detection limits of 1-4 for the analysis of Al3+ were found to reach a 10-8 M level in aqueous solution, which are far lower than the WHO guidelines for drinking water (7.41 mM for Al3+). A high selectivity test paper has been fabricated for Al3+ detection based on sensor 3. Theoretical calculations (DFT) have been carried out to elucidate the configuration of 1-4 and their Al complexes and rationalize experimental absorption data.

A novel chrysin thiazole derivative polarizes macrophages to an M1 phenotype via targeting TLR4.

Tumor-associated macrophages (TAMs) are an important cause of tumorigenesis and tumor development. M2 macrophages can promote tumor growth while M1 macrophages kill tumor cells, therefore, polarizing macrophages to achieve a functional M1 phenotype could effectively play its anti-tumor role. In the current study, we synthesized a novel chrysin derivative which is termed as ChR-TD. And we found ChR-TD might be a ligand of TLR4 that polarized the TAMs towards M1 phenotype and played its anti-tumor role. Further study indicated that ChR-TD reprogrammed the macrophages into an M1 phenotype via TLR4 activation. Moreover, ChR-TD activated TLR4/NF-κB signaling pathway and promoted the NF-κB/p65 translocated into the nuclear, leading to the activation of NF-κB and proinflammatory cytokines release. In addition, type I interferon signaling was also activated by ChR-TD, leading to the expressions of IFN-α and IFN-ß and its targeted genes NOS2, MCP-1 and IP-10 were significantly increased in macrophages. Importantly, these effects were disturbed in TLR4-/- macrophages, which are constructed by using CRISPR/Cas9 system. And the molecule docking simulation further indicated that ChR-TD could bind to TLR4 and might be a ligand of TLR4. Hence, these findings suggested that ChR-TD might be a ligand of TLR4 and can be used as a potential lead compound for tumors treatment.

Recent Developments in Fluorescent Materials for Heavy Metal Ions Analysis From the Perspective of Forensic Chemistry.

Forensic chemistry deals with the analysis of various types of physical evidences related to crime, corresponding to the detection of target substances or elements in complex matrices. There is a vital need for highly selective, rapid, and sensitive biosensing technologies in heavy metal ions analysis especially those from living persons, autopsy, food, water, soil, and other identified substances at very preliminary stages. Fluorescent materials-based method for heavy metal ions detection is one of the most important analytical methods, resulting in the ability to measure analytes in complex matrices with unsurpassed selectivity and sensitivity. In this mini review, different fluorescent materials-based analytical methods aiming at several heavy metal ions detection are exclusively reviewed through a comprehensive literature survey. In addition, current challenges to achieve integrated evidence analysis process are briefly discussed to provide an outlook for heavy metal ions detection based on fluorescent analytical methods in the forensic chemistry field.

Recent Trends in Fluorescent Organic Materials for Latent Fingerprint Imaging.

Fingerprints are an important kind of material evidence with the key function in personal identification, which are unique and life-long to everyone. Latent (invisible) fingerprints are common at the crime scene, needing to be visualized with proper methods in order to identify sources of the fingerprints in routine forensic practice. Fluorescent imaging of latent fingerprints has the advantage of high contrast, sensitivity, selectivity, and less dependency on instruments. Taking the environment and users' safety into consideration, organic materials for fluorescent imaging of latent fingerprints are reviewed mainly in recent 5 years. New strategies of fingerprint reagents and improved performances established for fingerprint development based on fluorescent organic materials are discussed in the view of forensic practice. In addition, we briefly highlight current challenges of recent fluorescent imaging works based on organic materials for the latent fingerprints development in forensic practice.