Highly Robust Multilamellar Lipid Vesicles Generated through Intervesicular Self-Assembly Mediated by Hydrolyzed Collagen Peptides.

Despite the well-known advantages of lipid vesicles for drug and gene delivery, structural instability limits their practical applications and requires strictly regulated conditions for transport and storage. Chemical crosslinking and in situ polymerization have been suggested to increase the membrane rigidity and dispersion stability of lipid vesicles. However, such chemically modified lipids sacrifice the dynamic nature of lipid vesicles and obfuscate their in vivo metabolic fates. Here, we present highly robust multilamellar lipid vesicles through the self-assembly of preformed, cationic large unilamellar vesicles (LUVs) with hydrolyzed collagen peptides (HCPs). The cationic LUVs undergo vesicle-to-vesicle attachment and structural reorganization through polyionic complexation with HCPs, resulting in the formation of multilamellar collagen-lipid vesicles (MCLVs). The resulting MCLVs exhibit excellent structural stability against variations in pH and ionic strength and the addition of surfactants. Particularly, the MCLVs maintain their structural stability against repeated freeze-thaw stresses, proving the unprecedented stabilization effect of biological macromolecules on lipid lamellar structures. This work provides a practically attractive technique for the simple and quick fabrication of structurally robust lipid nanovesicles without covalent crosslinkers, organic solvents, and specialized instruments.

Biomimetic Multilayered Lipid Nanovesicles for Potent Protein Vaccination.

Lipid vesicles are widely used for drug and gene delivery, but their structural instability reduces in vivo efficacy and requires specialized handling. To address these limitations, strategies like lipid cross-linking and polymer-lipid conjugation are suggested to enhance stability and biological efficacy. However, the in vivo metabolism of these altered lipids remains unclear, necessitating further studies. A new stabilization technique without chemical modification is urgently needed. Here, a bio-mimetic approach for fabricating robust multilamellar lipid vesicles to enhance in vivo delivery and stabilization of protein antigens is presented. This method leverages 1-O-acylceramide, a natural skin lipid, to facilitate the self-assembly of lipid nanovesicles. Incorporating 1-O-acylceramide, anchoring lipid bilayers akin to its role in the stratum corneum, provides excellent stability under environmental stresses, including freeze-thaw cycles. Encapsulating ovalbumin as a model antigen and the adjuvant monophosphoryl lipid A demonstrates the vesicle's potential as a nanovaccine platform. In vitro studies show enhanced immune responses with both unilamellar and multilamellar vesicles, but in vivo analyses highlight the superior efficiency of multilamellar vesicles in inducing higher antibody and cytokine levels. This work suggests ceramide-induced multilamellar lipid vesicles as an effective nanovaccine platform for enhanced antigen delivery and stability.

Live-Cell Imaging of MicroRNA Expression via Photoinduced Electron Transfer Controlled by Catalytic Hairpin Assembly.

MicroRNAs (miRNAs) serve as emerging biomarkers for a range of diseases, and their quantitative analysis draws increasing attention. Yet, current invasive methods limit continuous tracking within living cells. To overcome this, a nonenzymatic DNA-based nanoprobe is developed for dynamic, noninvasive miRNA tracking via live-cell imaging. This probe features a unique hairpin DNA structure with five guanines that act as internal quenchers, suppressing fluorescence from an attached fluorophore via photoinduced electron transfer. Target miRNA initiates toehold-mediated strand displacement, restoring, and amplifying the fluorescence signal. Additionally, by introducing a single mismatch to the hairpin DNA, the nanoprobe's sensitivity is significantly enhanced, lowering the detection limit to about 60 pM without compromising specificity. To optimize intracellular delivery for prolonged monitoring, the nanoprobe is encapsulated within multilamellar lipid nanovesicles, fluorescently labeled for dual-wavelength ratiometric analysis. The proposed nanoprobe demonstrates a significant advance in live-cell miRNA detection, promising enhanced in situ analysis for a better understanding of miRNAs' pathophysiological function.

Multilayered collagen-lipid hybrid nanovesicles for retinol stabilization and efficient skin delivery.

Lipid-based nanocarriers have been extensively utilized for the solubilization and cutaneous delivery of water-insoluble active ingredients in skincare formulations. However, their practical application is often limited by structural instability, leading to premature release and degradation of actives. Here we present highly robust multilamellar nanovesicles, prepared by the polyionic self-assembly of unilamellar vesicles with hydrolyzed collagen peptides, to stabilize all-trans-retinol and enhance its cutaneous delivery. Our results reveal that the reinforced multilayer structure substantially enhances dispersion stability under extremely harsh conditions, like freeze-thaw cycles, and stabilizes the encapsulated retinol. Interestingly, these multilamellar vesicles exhibit significantly lower cytotoxicity to human dermal fibroblasts than their unilamellar counterparts, likely due to their smaller particle number per weight, minimizing potential disruptions to cellular membranes. In artificial skin models, retinol-loaded multilamellar vesicles effectively upregulate collagen-related gene expression while suppressing the synthesis of metalloproteinases. These findings suggest that the robust multilamellar vesicles can serve as effective nanocarriers for the efficient delivery and stabilization of bioactive compounds in cutaneous applications.

Conjugation-Free Multilamellar Protein-Lipid Hybrid Vesicles for Multifaceted Immune Responses.

Various lipid-based nanocarriers have been developed for the co-delivery of protein antigens with immunological adjuvants. However, their in vivo potency in vaccine delivery is limited by structural instability, which causes off-target delivery and low cross-presentation efficacies. Recent works employ covalent cross-linking to stabilize the lipid nanostructures, though the immunogenicity and side effects of chemically modified protein antigens and lipids can cause a long-lasting safety issue. Here robust "conjugation-free" multilamellar protein antigen-lipid hybrid nanovesicles (MPLVs) are introduced through the antigen-mediated self-assembly of unilamellar lipid vesicles for the co-delivery of protein antigens and immunologic adjuvants. The nanocarriers coated with monophosphoryl lipid A and hyaluronic acids elicit highly increase antigen-specific immune responses in vitro and in vivo. The MPLVs increase the generation of immunological surface markers and cytokines in mouse-derived bone-marrow dendritic cells compared to soluble antigens with adjuvants. Besides, the vaccination of mice with the MPLVs significantly increase the production of anti-antigen antibody and interferon-gamma via the activation of CD4+ and CD8+ T cells, respectively. These findings suggest that MPLVs can serve as a promising nanovaccine delivery platform for efficient antigen cross-presentation through the efficient co-delivery of protein antigens with adjuvants.

Protein-induced metamorphosis of unilamellar lipid vesicles to multilamellar hybrid vesicles.

Protein encapsulation into nanocarriers has been extensively studied to improve the efficacy and stability of therapeutic proteins. However, the chemical modification of proteins or new synthetic carrier materials are essential to achieve a high encapsulation efficiency and structural stability of proteins, which hinders their clinical applications. New strategies to physically incorporate proteins into nanocarriers feasible for clinical uses are required to overcome the current limitation. Here we report the spontaneous protein-induced reorganization of 'pre-formed' unilamellar lipid vesicles to efficiently incorporate proteins within multilamellar protein-lipid hybrid vesicles without chemical modification. Epidermal growth factor (EGF) binds to the surface of cationic unilamellar lipid vesicles and induces layer-by-layer self-assembly of the vesicles. The protein is spontaneously entrapped in the interstitial layers of a multilamellar structure with extremely high loading efficiency, ~99%, through polyionic interactions as predicted by molecular dynamics simulation. The loaded protein exhibits much higher structural, chemical, and biological stability compared to free protein. The method is also successfully applied to several other proteins. This work provides a promising method for the highly efficient encapsulation of therapeutic proteins into multilamellar lipid vesicles without the use of specialized instruments, high energy, coupling agents, or organic solvents.

Artificial Taste Buds: Bioorthogonally Ligated Gustatory-Neuronal Multicellular Hybrids Enabling Intercellular Taste Signal Transmission.

Heterogeneous tissue models require the assembly and co-culture of multiple types of cells. Our recent work demonstrated taste signal transmission from gustatory cells to neurons by grafting single-stranded DNA into the cell membrane to construct multicellular assemblies. However, the weak DNA linkage and low grafting density allowed the formation of large gustatory cell self-aggregates that cannot communicate with neurons efficiently. This article presents the construction of artificial taste buds exhibiting active intercellular taste signal transmission through the hybridization of gustatory-neuronal multicellular interfaces using bioorthogonal click chemistry. Hybrid cell clusters were formed by the self-assembly of neonatal gustatory cells displaying tetrazine with a precultured embryonic hippocampal neuronal network displaying trans-cyclooctene. A bitter taste signal transduction was provoked in gustatory cells using denatonium benzoate and transmitted to neurons as monitored by intracellular calcium ion sensing. In the multicellular hybrids, the average number of signal transmissions was five to six peaks per cell, and the signal transmission lasted for ∼5 min with a signal-to-signal gap time of 10-40 s. The frequent and extended intercellular signal transmission suggests that the cell surface modification by the bioorthogonal click chemistry is a promising approach to fabricating functional multicellular hybrid clusters potentially useful for cell-based biosensors, toxicity assays, and tissue regeneration.

Hydrogel Skin-Covered Neurons Self-Assembled with Gustatory Cells for Selective Taste Stimulation.

Many technical challenges exist in the co-culture of multiple types of cells, including medium optimization, cell-to-cell connection, and selective data acquisition of cellular responses. Particularly, mixed cellular responses limit the precise interpretation of intercellular signal transduction. Here, we report the formation of an agarose gel skin on neurons closely assembled with gustatory cells to selectively stimulate gustatory cells by retarding the diffusion of tastants to neurons. The signal transmission, triggered by denatonium benzoate, from gustatory cells to neurons was monitored using intracellular calcium ion concentrations. The agarose gel skin efficiently suppressed the direct transfer of tastants to neurons, decreasing the number of responsive neurons from 56 to 13% and the number of calcium ion signals per neuron from multiple to single. The assembly of neurons with gustatory cells induced the high level of neuronal responses through taste signal transduction from gustatory cells to neurons. However, the calcium ion signal peaks of free neurons coated with agarose gel were much shorter and weaker than those of neurons closely assembled with gustatory cells. This work demonstrated that agarose gel skin is a simple, fast, and effective means to increase the signal selectivity of cellular responses in the co-culture of multiple types of cells.

Cancer-targeted reactive oxygen species-degradable polymer nanoparticles for near infrared light-induced drug release.

Nanocarriers can be translocated to the peripheral region of tumor tissues through the well-known enhanced permeability and retention effects. However, a high dose of nanocarriers need to be injected due to the low delivery efficiency of nanocarriers, which can also increase the side effects of off-targeted nanocarriers in normal tissues. It was demonstrated that on-demand drug release from tumor-targeted nanocarriers can reduce the effective dosage of anti-cancer drugs by rapidly increasing the local drug concentration in the tumor tissue. Here we report a near-infrared (NIR) photodynamic method to trigger drug release from tumor-targeted polymer nanoparticles via reactive oxygen species (ROS)-mediated polymer degradation. Paclitaxel and silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) were co-encapsulated as an anti-cancer drug and photosensitizer, respectively, within biotin-decorated poly(ethylene glycol)-polythioketal micelles. Upon NIR light illumination under the maximum permissible exposure level, the photoexcited naphthalocyanine generated ROS cleaved the thioketal groups in the micelles to release the encapsulated paclitaxel. The photodynamically-induced release of paclitaxel dramatically reduced the half maximal inhibitory concentration of paclitaxel by 39.9-fold and eliminated lung adenocarcinoma at a concentration an order of magnitude smaller than its maximum tolerated dosage. Even under a simulated deep tissue condition with a tissue-like phantom, the NIR light-illuminated micelles exhibited a high level of cytotoxicity against the tumor cells and efficiently suppressed tumor growth. Our study demonstrates that photodynamic polymer degradation is an effective means to improve the anticancer drug efficacy of tumor-targeted micelles.

Motor and Sensory Function as a Predictor of Respiratory Function Associated With Ventilator Weaning After High Cervical Cord Injury.

OBJECTIVE: To analyze the respiratory function of high cervical cord injury according to ventilator dependence and to examine the correlations between diaphragm movement found on fluoroscopy and sensory and motor functions. METHODS: A total of 67 patients with high cervical spinal cord injury (SCI), admitted to our hospital were enrolled in the study. One rehabilitation physician performed sensory and motor examinations on all patients while each patient was in the supine position on the American Spinal Injury Association (ASIA) standard. In addition, fluoroscopic diaphragm movement studies and bedside spirometry were performed. RESULTS: Bedside spirometry and diaphragm fluoroscopic tests were analyzed according to ventilator dependence. Forced vital capacity and maximal inspiratory pressure were significantly higher in the ventilator weaned group. Natural breathing during the fluoroscopic diaphragm examinations and ventilator weaning showed statistical significance with the movement on the right, while deep breathing showed statistical significance with the movement on both sides. Deep breathing movement has correlation with the C5 key muscle. Diaphragm movement has correlation with right C3 and bilateral C4 sensory functions. CONCLUSION: The present expansion study showed that, through simple bedside physical examinations, rehabilitation physicians could relatively easily predict diaphragm movement and respiratory function recovery, which showed significance with ventilator weaning in patients with high cervical SCI.

Tannin-Titanium Oxide Multilayer as a Photochemically Suppressed Ultraviolet Filter.

UV filters can initiate redox reactions of oxygen and water when exposed to sunlight, generating reactive oxygen species (ROS) that deteriorate the products containing them and cause biological damages. This photochemical reactivity originates from the high chemical potential of UV filters, which also determines the optical properties desirable for sunscreen applications. We hypothesize that this dilemma can be alleviated if the photochemical pathway of UV filters is altered to coupling with redox active molecules. Here, we employ tannic acid (TA) as a key molecule for controlling the photochemical properties of titanium dioxide nanoparticles (TiO2 NPs). TA provides an unusual way for layer-by-layer assembly of TiO2 NPs by the formation of a ligand-to-metal charge transfer complex that alters the nature of UV absorption of TiO2 NPs. The galloyl moieties of TA efficiently scavenge ROS due to the stabilization of ROS by intramolecular hydrogen bonding while facilitating UV screening through direct charge injection from TA to the conduction band of TiO2. The TiO2-TA multilayers assembled in open porous polymer microspheres substantially increased sun protection while dramatically reducing ROS under UV exposure. The assembled structure exhibits excellent in vivo anti-UV skin protection against epidermal hyperplasia, inflammation, and keratinocyte apoptosis without long-term toxicity.

Anorectal Manometric and Urodynamic Parameters According to the Spinal Cord Injury Lesion.

OBJECTIVE: To assess the correlation between the anorectal function and bladder detrusor function in patients with complete spinal cord injury (SCI) according to the type of lesion. METHODS: Medical records of twenty-eight patients with SCI were included in this study. We compared the anorectal manometric and urodynamic (UD) parameters in total subjects. We analyzed the anorectal manometric and UD parameters between the two groups: upper motor neuron (UMN) lesion and lower motor neuron (LMN) lesion. In addition, we reclassified the total subjects into two groups according to the bladder detrusor function: overactive and non-overactive. RESULTS: In the group with LMN lesion, the mean value of maximal anal squeeze pressure (MSP) was slightly higher than that in the group with UMN lesion, and the ratio of MSP to maximal anal resting pressure (MRP) was statistically significant different between the two groups. In addition, although the mean value of MSP was slightly higher in the group with non-overactive detrusor function, there was no statistical correlation of anorectal manometric parameters between the groups with overactive and non-overactive detrusor function. CONCLUSION: The MSP and the ratio of MSP to MRP were higher in the group with LMN lesion. In this study, we could not identify the correlation between bladder and bowel function in total subjects. We conclude that the results of UD study alone cannot predict the outcome of anorectal manometry in patients with SCI. Therefore, it is recommended to perform assessment of anorectal function with anorectal manometry in patients with SCI.

Effect of constraint-induced movement therapy and mirror therapy for patients with subacute stroke.

OBJECTIVE: To evaluate the effectiveness of constraint-induced movement therapy (CIMT) and combined mirror therapy for inpatient rehabilitation of the patients with subacute stroke. METHODS: Twenty-six patients with subacute stroke were enrolled and randomly divided into three groups: CIMT combined with mirror therapy group, CIMT only group, and control group. Two weeks of CIMT for 6 hours a day with or without mirror therapy for 30 minutes a day were performed under supervision. All groups received conventional occupational therapy for 40 minutes a day for the same period. The CIMT only group and control group also received additional self-exercise to substitute for mirror therapy. The box and block test, 9-hole Pegboard test, grip strength, Brunnstrom stage, Wolf motor function test, Fugl-Meyer assessment, and the Korean version of Modified Barthel Index were performed prior to and two weeks after the treatment. RESULTS: After two weeks of treatment, the CIMT groups with and without mirror therapy showed higher improvement (p<0.05) than the control group, in most of functional assessments for hemiplegic upper extremity. The CIMT combined with mirror therapy group showed higher improvement than CIMT only group in box and block test, 9-hole Pegboard test, and grip strength, which represent fine motor functions of the upper extremity. CONCLUSION: The short-term CIMT combined with mirror therapy group showed more improvement compared to CIMT only group and control group, in the fine motor functions of hemiplegic upper extremity for the patients with subacute stroke.

Differences in urodynamic variables for vesicoureteral reflux depending on the neurogenic bladder type.

OBJECTIVE: To compare the urodynamic study variables at the onset of vesicoureteral reflux (VUR) between the overactive and underactive bladders in patients with spinal cord injury who presented with VUR. METHODS: A total of 28 (13 cases of detrusor overactivity and 15 detrusor underactivity) men were enrolled. We compared the urodynamic variables between the two groups; detrusor pressure and bladder compliance, the infused volume at the onset of VUR measured on a voiding cystourethrography and cystometric capacity, maximum detrusor pressure, and bladder compliance during filling cystometry were recorded. RESULTS: At the onset of VUR, the bladder volume and compliance, except for the detrusor pressure, showed a significant difference between the two groups. The detrusor pressure, bladder volume, and bladder compliance relative to the cystometric capacity showed a significant difference between the two groups. The detrusor pressure, bladder volume, and bladder compliance at the onset of VUR relative to the cystometric bladder capacity did not show any significant difference between the two groups. CONCLUSION: There were differences in some variables at the onset of VUR depending on the type of neurogenic bladder. The VUR occurred at a lower capacity in neurogenic bladder with detrusor overactivity than in neurogenic bladder with detrusor underactivity at the same pressure. VUR occurred at a lower intravesical pressure compared to that known as the critical detrusor pressure (≥40 cm H2O) required for the development of VUR. The results of our study demonstrate that the detrusor pressure should be maintained lower than the well known effective critical detrusor pressure for the prevention and treatment of VUR.