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Stem cell-related therapeutic technologies have garnered significant attention of the research community for their multi-faceted applications. To promote the therapeutic effects of stem cells, the strategies for cell microencapsulation in hydrogel microparticles have been widely explored, as the hydrogel microparticles have the potential to facilitate oxygen diffusion and nutrient transport alongside their ability to promote crucial cell-cell and cell-matrix interactions. Despite their significant promise, there is an acute shortage of automated, standardized, and reproducible platforms to further stem cell-related research. Microfluidics offers an intriguing platform to produce stem cell-laden hydrogel microparticles (SCHMs) owing to its ability to manipulate the fluids at the micrometer scale as well as precisely control the structure and composition of microparticles. In this review, the typical biomaterials and crosslinking methods for microfluidic encapsulation of stem cells as well as the progress in droplet-based microfluidics for the fabrication of SCHMs are outlined. Moreover, the important biomedical applications of SCHMs are highlighted, including regenerative medicine, tissue engineering, scale-up production of stem cells, and microenvironmental simulation for fundamental cell studies. Overall, microfluidics holds tremendous potential for enabling the production of diverse hydrogel microparticles and is worthy for various stem cell-related biomedical applications.
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PURPOSE: Post-induction hypotension (PIH) is an independent risk factor for prolonged postoperative stay and hospital death. Patients undergoing transcatheter aortic valve implantation (TAVI) are prone to develop PIH. This study aimed to develop a predictive model for PIH in patients undergoing TAVI. METHODS: This single-center retrospective observational study included 163 patients who underwent TAVI. PIH was defined as at least one measurement of systolic arterial pressure <90 mmHg or at least one incident of norepinephrine infusion at a rate >6 µg/min from anesthetic induction until 20 min post-induction. Multivariate logistic regression analysis was performed to develop a predictive model for PIH in patients undergoing TAVI. RESULTS: In total, 161 patients were analyzed. The prevalence of PIH was 57.8%. Multivariable logistic regression analysis showed that baseline mean arterial pressure ≥90 mmHg [adjusted odds ratio (aOR): 0.413, 95% confidence interval (95% CI): 0.193-0.887; p=0.023] and higher doses of fentanyl (per 1-µg/kg increase, aOR: 0.619, 95% CI: 0.418-0.915; p=0.016) and ketamine (per 1-mg/kg increase, aOR: 0.163, 95% CI: 0.062-0.430; p=0.002) for induction were significantly associated with lower risk of PIH. A higher dose of propofol (per 1-mg/kg increase, aOR: 3.240, 95% CI: 1.320-7.920; p=0.010) for induction was significantly associated with higher risk of PIH. The area under the curve (AUC) for this model was 0.802. CONCLUSION: The present study developed predictive models for PIH in patients who underwent TAVI. This model may be helpful for anesthesiologists in preventing PIH in patients undergoing TAVI.
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Carrier-free naked mRNA vaccines may reduce the reactogenicity associated with delivery carriers; however, their effectiveness against infectious diseases has been suboptimal. To boost efficacy, we targeted the skin layer rich in antigen-presenting cells (APCs) and utilized a jet injector. The jet injection efficiently introduced naked mRNA into skin cells, including APCs in mice. Further analyses indicated that APCs, after taking up antigen mRNA in the skin, migrated to the lymph nodes (LNs) for antigen presentation. Additionally, the jet injection provoked localized lymphocyte infiltration in the skin, serving as a physical adjuvant for vaccination. Without a delivery carrier, our approach confined mRNA distribution to the injection site, preventing systemic mRNA leakage and associated systemic proinflammatory reactions. In mouse vaccination, the naked mRNA jet injection elicited robust antigen-specific antibody production over 6 months, along with germinal center formation in LNs and the induction of both CD4- and CD8-positive T cells. By targeting the SARS-CoV-2 spike protein, this approach provided protection against viral challenge. Furthermore, our approach generated neutralizing antibodies against SARS-CoV-2 in non-human primates at levels comparable to those observed in mice. In conclusion, our approach offers a safe and effective option for mRNA vaccines targeting infectious diseases.
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Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vacinas de mRNA , Animais , Camundongos , SARS-CoV-2/imunologia , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacinas de mRNA/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Antivirais/imunologia , Feminino , Células Apresentadoras de Antígenos/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Linfócitos T CD8-Positivos/imunologia , Anticorpos Neutralizantes/imunologia , Humanos , Vacinação/métodosRESUMO
Controlling the end-groups of biocompatible polymers is crucial for enabling polymer-based therapeutics and nanomedicine. Typically, end-group diversification is a challenging and time-consuming endeavor, especially for polymers prepared via ionic polymerization mechanisms with limited functional group tolerance. In this study, we present a facile end-group diversification approach for poly(2-oxazoline)s (POx), enabling quick and reliable production of heterotelechelic polymers to facilitate POxylation. The approach relies on the careful tuning of reaction parameters to establish differential reactivity of a pentafluorobenzyl initiator fragment and the living oxazolinium chain-end, allowing the selective introduction of N-, S-, O-nucleophiles via the termination of the polymerization, and a consecutive nucleophilic para-fluoro substitution. The value of this approach for the accelerated development of nanomedicine is demonstrated through the synthesis of well-defined lipid-polymer conjugates and POx-polypeptide block-copolymers, which are well-suited for drug and gene delivery. Furthermore, we investigated the application of a lipid-POx conjugate for the formulation and delivery of mRNA-loaded lipid nanoparticles for immunization against the SARS-COV-2 virus, underscoring the value of POx as a biocompatible polymer platform.
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Nanomedicina , Oxazóis , Oxazóis/química , Nanomedicina/métodos , Humanos , SARS-CoV-2 , Polímeros/química , Polímeros/síntese química , Nanopartículas/química , Polimerização , AnimaisRESUMO
Adjuvanticity and delivery are crucial facets of mRNA vaccine design. In modern mRNA vaccines, adjuvant functions are integrated into mRNA vaccine nanoparticles, allowing the co-delivery of antigen mRNA and adjuvants in a unified, all-in-one formulation. In this formulation, many mRNA vaccines utilize the immunostimulating properties of mRNA and vaccine carrier components, including lipids and polymers, as adjuvants. However, careful design is necessary, as excessive adjuvanticity and activation of improper innate immune signalling can conversely hinder vaccination efficacy and trigger adverse effects. mRNA vaccines also require delivery systems to achieve antigen expression in antigen-presenting cells (APCs) within lymphoid organs. Some vaccines directly target APCs in the lymphoid organs, while others rely on APCs migration to the draining lymph nodes after taking up mRNA vaccines. This review explores the current mechanistic understanding of these processes and the ongoing efforts to improve vaccine safety and efficacy based on this understanding.
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Vacinas , Vacinas de mRNA , Adjuvantes Imunológicos , Antígenos , RNA Mensageiro/genéticaRESUMO
In comparison to monolithic perovskite/perovskite double-junction solar cells, a four-terminal spectrum-splitting system is a simple method to obtain a higher power conversion efficiency (PCE) because it has no constraints of unifying the structures of the top and bottom cells. In this work, utilizing the fact that low-bandgap Sn-Pb bottom cells work the best in p-i-n while Pb-based wide-bandgap top cells work better in an n-i-p architecture, a wide-bandgap (Eg = 1.61 eV) perovskite solar cell with a mesoscopic structure and a narrow-bandgap (Eg = 1.27 eV) perovskite solar cell with an inverted structure were combined to fabricate a double-junction four-terminal spectral splitting solar cell. The double-junction solar cell with the 801 nm spectral splitting with an active area of 0.18 cm2 was found to work with a PCE of 25.3%, which is the highest reported so far for a 4-T all-perovskite double-junction spectral splitting solar cell.
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mRNA-based therapeutics are revolutionizing the landscape of medical interventions. However, the short half-life of mRNA and transient protein expression often limits its therapeutic potential, demanding high treatment doses or repeated administrations. Self-replicating RNA (RepRNA)-based treatments could offer enhanced protein production and reduce the required dosage. Here, we developed polymeric micelles based on flexible poly(ethylene glycol)-poly(glycerol) (PEG-PG) block copolymers modified with phenylalanine (Phe) moieties via biodegradable ester bonds for the efficient delivery of RepRNA. These polymers successfully encapsulated RepRNA into sub-100 nm micelles assisted by the hydrophobicity of the Phe moieties and their ability to π-π stack with the bases in RepRNA. The micelles made from Phe-modified PEG-PG (PEG-PG(Phe)) effectively maintained the integrity of the loaded RepRNA in RNase-rich serum conditions. Once taken up by cells, the micelles triggered a pH-responsive membrane disruption, promoted by the strong protonation of the amino groups at endosomal pH, thereby delivering the RepRNA to the cytosol. The system induced strong protein expression in vitro and outperformed commercial transfecting reagents in vivo, where it resulted in enhanced and long-lasting protein expression.
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Micelas , Fenilalanina , RNA , Linhagem Celular Tumoral , Concentração de Íons de Hidrogênio , Polímeros/química , Polietilenoglicóis/química , RNA Mensageiro , Portadores de Fármacos/químicaRESUMO
Various cationic polymers are used to deliver polyplex-mediated antisense oligonucleotides (ASOs). However, few studies have investigated the structural determinants of polyplex functionalities in polymers. This study focused on the polymer hydrophobicity. A series of amphiphilic polyaspartamide derivatives possessing various hydrophobic (R) moieties together with cationic diethylenetriamine (DET) moieties in the side chain (PAsp(DET/R)s) were synthesized to optimize the R moieties (or hydrophobicity) for locked nucleic acid (LNA) gapmer ASO delivery. The gene knockdown efficiencies of PAsp(DET/R) polyplexes were plotted against a hydrophobicity parameter, logD7.3, of PAsp(DET/R), revealing that the gene knockdown efficiency was substantially improved by PAsp(DET/R) with logD7.3 higher than -2.4. This was explained by the increased polyplex stability and improved cellular uptake of ASO payloads. After intratracheal administration, the polyplex samples with a higher logD7.3 than -2.4 induced a significantly higher gene knockdown in the lung tissue compared with counterparts with lower hydrophobicity and naked ASO. These results demonstrate that the hydrophobicity of PAsp(DET/R) is crucial for efficient ASO delivery in vitro and in vivo.
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Oligonucleotídeos Antissenso , Polímeros , Polímeros/químicaRESUMO
Cell-penetrating peptides (CPPs), for example, arginine (Arg) rich peptides, are used for the intracellular delivery of nucleic acids. In this study, oligosarcosine-conjugated Arg-rich peptides were designed as plasmid DNA (pDNA) carriers, and the physicochemical parameters and transfection efficiency of the peptide/pDNA complexes were evaluated. Oligosarcosine with different lengths were conjugated to a base sequence composed of arginine and α-aminoisobutyric acid (Aib) [(Aib-Arg-Arg)3]. Oligosarcosine conjugation inhibited the aggregation of the complexes after mixing with pDNA, shielded the positive charge of the complexes, and provided efficient pDNA transfection in cultured cells. The efficiency of the pDNA transfection was improved by varying the length of the oligosarcosine moiety (10-15 units were optimal). The cellular uptake efficiency and intracellular distribution of pDNA were the same regardless of oligosarcosine conjugation. These results implied that intracellular processes, including the decondensation of pDNA, contributed to the efficiency of the protein expression from pDNA. This study demonstrated the advantages of oligosarcosine conjugation to Arg-rich CPPs and provided valuable insight into the future design of CPPs.
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Arginina , Peptídeos Penetradores de Células , Arginina/genética , DNA/química , DNA/genética , Plasmídeos/genética , Transfecção , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genéticaRESUMO
Fibrosis is a disease that causes abnormal accumulation of collagen and other extracellular matrix components. It can lead to organ failure and is responsible for one-third of all deaths worldwide. However, there is no cure for this disease, and the development of minimally invasive therapies is urgently needed. We have previously reported techniques for adjusting the shape and flexibility of fibrous tissue by traction while denaturing it with heat. However, studies comparing heat and traction on fibrous tissue are limited, so this paper examined that. Applying heat and traction to bovine Achilles tendon tissue has been shown to cause the denaturation of collagen molecules to accumulate in the tissue in response to these loads. Heat-induced collagen denaturation was nondirectional and omnidirectional, whereas mechanical stress-induced collagen denaturation was concentrated in the direction of traction. When both heat and traction were applied, collagen denaturation increased more than under a single load, indicating a synergistic effect.
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Colágeno , Animais , Bovinos , Colágeno/fisiologia , Estresse MecânicoRESUMO
The influence of chloride integration on perovskite film deposition, encompassing both the structures of intermediate phases and the properties of the final films, was explored. Our methodology involved the fabrication of perovskite intermediate-phase films with varying concentrations of methylammonium chloride (MACl). Subsequently, we conducted an analysis employing X-ray diffraction and Rietveld refinement, incorporating the March-Dollase correction, to gain insights into how chloride-induced intermediate phases impact film morphology. Remarkably, a distinct preferred orientation was observed in the (020) lattice plane perpendicular to the substrate surface, and this orientation was found to be directly correlated to the MACl concentration. This distinctive arrangement of chloride-induced intermediate-phase complexes facilitated controlled crystallization, leading to highly oriented crystals and an improved film morphology. As a consequence, perovskite solar cell devices incorporating chloride-containing methylammonium lead iodide achieved a power conversion efficiency exceeding 20%. These findings suggest a crucial link between the preferred orientation observed in the final chlorine-derived perovskite films and the intermediate-phase structure formed during the initial stages of perovskite formation. These results suggest a profound impact of intermediate phase compositions and crystal structures on perovskite formation, emphasizing the importance of a comprehensive understanding of these factors to enable precise control over ideal structures and the subsequent transformation into high-quality perovskite films.
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The effect of TiO2 interfacial morphology on perovskite crystallinity was investigated by modifying the micro and nanoscale surface roughness of compact TiO2. While surface treatments of the compact TiO2 layer are recognized as effective strategies to enhance the photovoltaic performance of perovskite solar cells, the discussion regarding the crystallinity of perovskite atop TiO2 has been limited. In this study, we explored the impact of micro and nano scale interface morphology on perovskite crystal formation and its subsequent effects on device performance. Surprisingly, despite the absence of noticeable voids at the interface between the compact TiO2 and perovskite layers, the perovskite crystal morphology exhibited significant improvement following either micro or nanoscale interfacial modification. This enhancement ultimately led to improved photoconversion efficiency and reduced I-V hysteresis. These results emphasize the importance of underlayer surface morphology in the perovskite crystallization and suggest that the presence of grain boundaries within the perovskite layer may also contribute to I-V hysteresis in perovskite solar cells.
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This paper presents the molecular dynamics simulation of 1 mol kg-1 LiPF6 in a binary solvent of ethylene carbonate (EC) and dimethylcarbonate, which is a representative electrolyte solution for lithium-ion batteries. The simulation successfully reproduced the diffusion coefficient, ionic conductivity, and shear viscosity as functions of EC content at 300 K, which had been experimentally determined in our previous study. The Yukawa potential was adopted to model intercharge interactions to reduce computational costs, which consequently allowed us to precisely calculate the conductivity and viscosity by directly integrating time-correlation functions without explicitly modeling the molecular polarization. Breaking down microscopic current correlation functions into components revealed that, whereas the cation-anion attractive interaction dominantly impedes the conduction when the EC content is low, it is the cation-cation and anion-anion repulsive interactions that reduce the conductivity at a high EC content. An analysis of the pressure correlations revealed that all components positively contribute to the viscosity in the binary solvent without the electrolyte. On the other hand, negative terms are observed in five out of six cross correlations in the presence of the electrolyte, implying that these correlations negatively contribute to the shear stress and entropy production, both of which are net positive.
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BACKGROUND: Left atrial auricle (LAA) clipping is a common method of preventing cardiogenic thromboembolism. However, acute coronary syndrome (ACS) has been reported as a fatal complication of LAA clipping. We describe a case of ACS 2 months after LAA clipping. CASE PRESENTATION: A 33-year-old male with atrial fibrillation was scheduled LAA clipping during aortic valve replacement for congenital aortic bicuspid valve. The surgery went smoothly with no postoperative complications, but he suddenly went into cardiac arrest 2 months later. Emergency coronary angiography and intravascular ultrasonography revealed that compression by the clip of the left main coronary trunk had caused the ACS. Percutaneous coronary intervention with stents was performed, and the clip was removed under general anesthesia. CONCLUSION: Even in the remote timepoint of LAA clipping, compression of the coronary artery by the clip should be differentiated as a cause of ACS.
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Integrating antigen-encoding mRNA (Messenger RNA) and immunostimulatory adjuvant into a single formulation is a promising approach to potentiating the efficacy of mRNA vaccines. Here, we developed a scheme based on RNA engineering to integrate adjuvancy directly into antigen-encoding mRNA strands without hampering the ability to express antigen proteins. Short double-stranded RNA (dsRNA) was designed to target retinoic acid-inducible gene-I (RIG-I), an innate immune receptor, for effective cancer vaccination and then tethered onto the mRNA strand via hybridization. Tuning the dsRNA structure and microenvironment by changing its length and sequence enabled the determination of the structure of dsRNA-tethered mRNA efficiently stimulating RIG-I. Eventually, the formulation loaded with dsRNA-tethered mRNA of the optimal structure effectively activated mouse and human dendritic cells and drove them to secrete a broad spectrum of proinflammatory cytokines without increasing the secretion of anti-inflammatory cytokines. Notably, the immunostimulating intensity was tunable by modulating the number of dsRNA along the mRNA strand, which prevents excessive immunostimulation. Versatility in the applicable formulation is a practical advantage of the dsRNA-tethered mRNA. Its formulation with three existing systems, i.e., anionic lipoplex, ionizable lipid-based lipid nanoparticles, and polyplex micelles, induced appreciable cellular immunity in the mice model. Of particular interest, dsRNA-tethered mRNA encoding ovalbumin (OVA) formulated in anionic lipoplex used in clinical trials exerted a significant therapeutic effect in the mouse lymphoma (E.G7-OVA) model. In conclusion, the system developed here provides a simple and robust platform to supply the desired intensity of immunostimulation in various formulations of mRNA cancer vaccines.
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Neoplasias , RNA de Cadeia Dupla , Humanos , Animais , Camundongos , RNA de Cadeia Dupla/genética , Adjuvantes Imunológicos/farmacologia , Antígenos , Imunidade Celular , Citocinas/genética , RNA Mensageiro/genética , Camundongos Endogâmicos C57BL , Neoplasias/terapiaRESUMO
Nanoparticle-based delivery systems have contributed to the recent clinical success of RNA therapeutics, including siRNA and mRNA. RNA delivery using polymers has several distinct properties, such as enabling RNA delivery into extra-hepatic organs, modulation of immune responses to RNA, and regulation of intracellular RNA release. However, delivery systems should overcome safety and stability issues to achieve widespread therapeutic applications. Safety concerns include direct damage to cellular components, innate and adaptive immune responses, complement activation, and interaction with surrounding molecules and cells in the blood circulation. The stability of the delivery systems should balance extracellular RNA protection and controlled intracellular RNA release, which requires optimization for each RNA species. Further, polymer designs for improving safety and stability often conflict with each other. This review covers advances in polymer-based approaches to address these issues over several years, focusing on biological understanding and design concepts for delivery systems rather than material chemistry.
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Nanopartículas , Polímeros , Humanos , RNA Interferente Pequeno/uso terapêutico , Polímeros/químicaRESUMO
Starting with the clinical application of two vaccines in 2020, mRNA therapeutics are currently being investigated for a variety of applications. Removing immunogenic uncapped mRNA from transcribed mRNA is critical in mRNA research and clinical applications. Commonly used capping methods provide maximum capping efficiency of around 80-90% for widely used Cap-0- and Cap-1-type mRNAs. However, uncapped and capped mRNA possesses almost identical physicochemical properties, posing challenges to their physical separation. In this work, we develop hydrophobic photocaged tag-modified cap analogs, which separate capped mRNA from uncapped mRNA by reversed-phase high-performance liquid chromatography. Subsequent photo-irradiation recovers footprint-free native capped mRNA. This approach provides 100% capping efficiency even in Cap-2-type mRNA with versatility applicable to 650 nt and 4,247 nt mRNA. We find that the Cap-2-type mRNA shows up to 3- to 4-fold higher translation activity in cultured cells and animals than the Cap-1-type mRNA prepared by the standard capping method.
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Biossíntese de Proteínas , Capuzes de RNA , Animais , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Cultivadas , Capuzes de RNA/metabolismoRESUMO
Naturally occurring peptides with high membrane permeability often have ester bonds on their backbones. However, the impact of amide-to-ester substitutions on the membrane permeability of peptides has not been directly evaluated. Here we report the effect of amide-to-ester substitutions on the membrane permeability and conformational ensemble of cyclic peptides related to membrane permeation. Amide-to-ester substitutions are shown to improve the membrane permeability of dipeptides and a model cyclic hexapeptide. NMR-based conformational analysis and enhanced sampling molecular dynamics simulations suggest that the conformational transition of the cyclic hexapeptide upon membrane permeation is differently influenced by an amide-to-ester substitution and an amide N-methylation. The effect of amide-to-ester substitution on membrane permeability of other cyclic hexapeptides, cyclic octapeptides, and a cyclic nonapeptide is also investigated to examine the scope of the substitution. Appropriate utilization of amide-to-ester substitution based on our results will facilitate the development of membrane-permeable peptides.
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Amidas , Peptídeos Cíclicos , Peptídeos Cíclicos/química , Metilação , Ésteres , Permeabilidade da Membrana Celular , Peptídeos/química , PermeabilidadeRESUMO
Amphipathic peptides composed of cationic amino acids and hydrophobic amino acids have cell-penetrating ability and are often used as a delivery tool for membrane-impermeable compounds. Small interfering RNA (siRNAs) are one of the delivery targets for such cell-penetrating peptides (CPPs). Cationic CPPs can associate with anionic siRNAs by electrostatic interactions resulting in the formation of nano-sized complexes, which can deliver siRNAs intracellularly. CPPs containing unnatural amino acids offer promising tools to siRNA delivery. However, the detailed structure-activity relationship in siRNA delivery has been rarely studied. In the current study, we designed peptides containing dipropylglycine (Dpg) and explored the cellular uptake and cytotoxicity of peptide/siRNA complexes. The amphipathic structure of the peptides played a key role in complexation with siRNAs and intracellular siRNA delivery. In the amphipathic peptides, cellular uptake of siRNA increased with increasing peptide length, but cytotoxicity was reduced. A peptide containing four Dpg exhibited an effective gene-silencing effect with small amounts of peptides without cytotoxicity in medium containing serum. These findings will be helpful for the design of novel CPPs for siRNA delivery.