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Inhibition of tumor growth and normalization of immune responses in the tumor microenvironment (TME) are critical issues for improving cancer therapy. However, in the treatment of glioma, effective nanomedicine has limited access to the brain because of the blood-brain barrier (BBB). Previously, we demonstrated nano-sized ginseng-derived exosome-like nanoparticles (GENs) consisting of phospholipids including various bioactive components, and evaluated anti-tumor immune responses in T cells and Tregs to inhibit tumor progression. It was found that the enhanced targeting ability of GENs to the BBB and glioma induced a significant therapeutic effect and exhibited strong efficacy in recruiting M1 macrophage expression in the TME. GENs were demonstrated to be successful candidates in glioma therapeutics both in vitro and in vivo, suggesting excellent potential for inhibiting glioma progression and regulating tumor-associated macrophages (TAMs).
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Exosomas , Glioma , Nanopartículas , Panax , Humanos , Barrera Hematoencefálica/metabolismo , Microambiente Tumoral , Exosomas/metabolismo , Glioma/patología , Línea Celular TumoralRESUMEN
In this paper, we investigate a motion-tracking system for robotic computer-assisted implant surgery. Failure of the accurate implant positioning may result in significant problems, thus an accurate real-time motion-tracking system is crucial for avoiding these issues in computer-assisted implant surgery. Essential features of the motion-tracking system are analyzed and classified into four categories: workspace, sampling rate, accuracy, and back-drivability. Based on this analysis, requirements for each category have been derived to ensure that the motion-tracking system meets the desired performance criteria. A novel 6-DOF motion-tracking system is proposed which demonstrates high accuracy and back-drivability, making it suitable for use in computer-assisted implant surgery. The results of the experiments confirm the effectiveness of the proposed system in achieving the essential features required for a motion-tracking system in robotic computer-assisted implant surgery.
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Procedimientos Quirúrgicos Robotizados , Robótica , Cirugía Asistida por Computador , Procedimientos Quirúrgicos Robotizados/métodos , Robótica/métodos , Cirugía Asistida por Computador/métodos , Movimiento (Física) , ComputadoresRESUMEN
Typically, the actual volume of the residual limb changes over time. This causes the prosthesis to not fit, and then pain and skin disease. In this study, a prosthetic socket was developed to compensate for the volume change of the residual limb. Using an inflatable air bladder, the proposed socket monitors the pressure in the socket and keeps the pressure distribution uniform and constant while walking. The socket has three air bladders on anterior and posterior tibia areas, a latching type 3-way pneumatic valve and a portable control device. In the paper, the mechanical properties of the air bladder were investigated, and the electromagnetic analysis was performed to design the pneumatic valve. The controller is based on a hysteresis control algorithm with a closed loop, which keeps the pressure in the socket close to the initial set point over a long period of time. In experiments, the proposed prosthesis was tested through the gait simulator that can imitate a human's gait cycle. The active volume compensation of the socket was successfully verified during repetitive gait cycle using the weight loads of 50, 70, and 90 kg and the residual limb model with a variety of volumes. It was confirmed that the pressure of the residual limb recovered to the initial state through the active control. The pressure inside the socket had a steady state error of less than 0.75% even if the volume of the residual limb was changed from -7% to +7%.
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Muñones de Amputación , Miembros Artificiales , Humanos , Extremidad Inferior , Diseño de Prótesis , TibiaRESUMEN
As agonists of TLR7/8, single-stranded RNAs (ssRNAs) are safe and promising adjuvants that do not cause off-target effects or innate immune overactivation. However, low stability prevents them from mounting sufficient immune responses. This study evaluates the adjuvant effects of ssRNA derived from the cricket paralysis virus intergenic region internal ribosome entry site, formulated as nanoparticles with a coordinative amphiphile, containing a zinc/dipicolylamine complex moiety as a coordinative phosphate binder, as a stabilizer for RNA-based adjuvants. The nanoformulated ssRNA adjuvant was resistant to enzymatic degradation in vitro and in vivo, and that with a coordinative amphiphile bearing an oleyl group (CA-O) was approximately 100â nm, promoted effective recognition, and improved activation of antigen-presenting cells, leading to better induction of neutralizing antibodies following single immunization. Hence, CA-O may increase the efficacy of ssRNA-based adjuvants, proving useful to meet the urgent need for vaccines during pathogen outbreaks.
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Adyuvantes Inmunológicos/farmacología , Células Presentadoras de Antígenos/inmunología , Composición de Medicamentos , Inmunidad Humoral/efectos de los fármacos , Nanotecnología , ARN/química , Adyuvantes Inmunológicos/química , Animales , HumanosRESUMEN
A body pressure relief system was newly developed with optical pressure sensors for pressure ulcer prevention. Unlike a conventional alternating pressure air mattress (APAM), this system automatically regulates air flow into a body supporting mattress with adaptive inflation (or deflation) duration in response to the pressure level in order to reduce skin stress due to prolonged high pressures. The system continuously quantifies the body pressure distribution using time-of-flight (ToF) optical sensors. The proposed pressure sensor, a ToF optical sensor in the air-filled cell, measures changes in surface height of mattress when pressed under body weight, thereby indirectly indicating the interface pressure. Non-contact measurement of optical sensor usually improves the durability and repeatability of the system. The pressure sensor was successfully identified the 4 different-predefined postures, and quantitatively measured the body pressure distribution of them. Duty cycle of switches in solenoid valves was adjusted to 0-50% for pressure relief, which shows that the interface pressure was lower than 32 mmHg for pressure ulcer prevention.
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Enzymatic synthesis of RNA nanostructures is achieved by isothermal rolling circle transcription (RCT). Each arm of RNA nanostructures provides a functional role of Dicer substrate RNA inducing sequence specific RNA interference (RNAi). Three different RNAi sequences (GFP, RFP, and BFP) are incorporated within the three-arm junction RNA nanostructures (Y-RNA). The template and helper DNA strands are designed for the large-scale in vitro synthesis of RNA strands to prepare self-assembled Y-RNA. Interestingly, Dicer processing of Y-RNA is highly influenced by its physical structure and different gene silencing activity is achieved depending on its arm length and overhang. In addition, enzymatic synthesis allows the preparation of various Y-RNA structures using a single DNA template offering on demand regulation of multiple target genes.
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ARN Helicasas DEAD-box/genética , Nanoestructuras/química , ARN/biosíntesis , Ribonucleasa III/genética , Transcripción Genética , ARN Helicasas DEAD-box/química , ADN/química , Silenciador del Gen , Humanos , Conformación de Ácido Nucleico , ARN/química , ARN/genética , Interferencia de ARN , Ribonucleasa III/químicaRESUMEN
A novel mechanochemical method was firstly developed to synthesize carbon nanodots (CNDs) or carbon nano-onions (CNOs) through high-pressure homogenization of cellulose powders as naturally abundant resource depending on the treatment times. While CNDs (less than 5 nm in size) showed spherical and amorphous morphology, CNOs (10-50 nm in size) presented polyhedral shape, and onion-like outer lattice structure, graphene-like interlattice spacing of 0.36 nm. CNOs showed blue emissions, moderate dispersibility in aqueous media, and high cell viability, which enables efficient fluorescence imaging of cellular media.
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Biological ligands such as aptamer, antibody, glucose, and peptide have been widely used to bind specific surface molecules or receptors in tumor cells or subcellular structures to improve tumor-targeting efficiency of nanoparticles. However, this active-targeting strategy has limitations for tumor targeting due to inter- and intraheterogeneity of tumors. In this study, we demonstrated an alternative active-targeting strategy using metabolic engineering and bioorthogonal click reaction to improve tumor-targeting efficiency of nanoparticles. We observed that azide-containing chemical reporters were successfully generated onto surface glycans of various tumor cells such as lung cancer (A549), brain cancer (U87), and breast cancer (BT-474, MDA-MB231, MCF-7) via metabolic engineering in vitro. In addition, we compared tumor targeting of artificial azide reporter with bicyclononyne (BCN)-conjugated glycol chitosan nanoparticles (BCN-CNPs) and integrin αvß3 with cyclic RGD-conjugated CNPs (cRGD-CNPs) in vitro and in vivo. Fluorescence intensity of azide-reporter-targeted BCN-CNPs in tumor tissues was 1.6-fold higher and with a more uniform distribution compared to that of cRGD-CNPs. Moreover, even in the isolated heterogeneous U87 cells, BCN-CNPs could bind artificial azide reporters on tumor cells more uniformly (â¼92.9%) compared to cRGD-CNPs. Therefore, the artificial azide-reporter-targeting strategy can be utilized for targeting heterogeneous tumor cells via bioorthogonal click reaction and may provide an alternative method of tumor targeting for further investigation in cancer therapy.
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Química Clic/métodos , Nanopartículas/química , Azidas/química , Neoplasias Encefálicas/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Quitosano/química , Femenino , Humanos , Neoplasias Pulmonares/metabolismoRESUMEN
Labeling of stem cells aims to distinguish transplanted cells from host cells, understand in vivo fate of transplanted cells, particularly important in stem cell therapy. Adipose-derived mesenchymal stem cells (ASCs) are considered as an emerging therapeutic option for tissue regeneration, but much remains to be understood regarding the in vivo evidence. In this study, a simple and efficient cell labeling method for labeling and tracking of stem cells was developed based on bio-orthogonal copper-free click chemistry, and it was applied in a mouse hindlimb ischemia model. The human ASCs were treated with tetra-acetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) to generate glycoprotein with unnatural azide groups on the cell surface, and the generated azide groups were fluorescently labeled by specific binding of dibenzylcyclooctyne-conjugated Cy5 (DBCO-Cy5). The safe and long-term labeling of the hASCs by this method was first investigated in vitro. Then the DBCO-Cy5-hASCs were transplanted into the hindlimb ischemia mice model, and we could monitor and track in vivo fate of the cells using optical imaging system. We could clearly observe the migration potent of the hASCs toward the ischemic lesion. This approach to design and tailor new method for labeling of stem cells may be useful to provide better understanding on the therapeutic effects of transplanted stem cells into the target diseases.
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Rastreo Celular/métodos , Isquemia/terapia , Células Madre Mesenquimatosas/citología , Tejido Adiposo/citología , Animales , Azidas/química , Química Clic/métodos , Modelos Animales de Enfermedad , Colorantes Fluorescentes/química , Miembro Posterior , Humanos , Imagenología Tridimensional , Isquemia/patología , Trasplante de Células Madre Mesenquimatosas , RatonesRESUMEN
Establishment of an appropriate cell labeling and tracking method is essential for the development of cell-based therapeutic strategies. Here, we are introducing a new method for cell labeling and tracking by combining metabolic gylcoengineering and bioorthogonal copper-free Click chemistry. First, chondrocytes were treated with tetraacetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) to generate unnatural azide groups (-N3) on the surface of the cells. Subsequently, the unnatural azide groups on the cell surface were specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented strong NIRF signals with relatively low cytotoxicity and the amounts of azide groups and DBCO-650 could be easily controlled by feeding different amounts of Ac4ManNAz and DBCO-650 to the cell culture system. For the in vivo cell tracking, DBCO-650-labeled chondrocytes (1 × 10(6) cells) seeded on the 3D scaffold were subcutaneously implanted into mice and the transplanted DBCO-650-labeled chondrocytes could be effectively tracked in the prolonged time period of 4 weeks using NIRF imaging technology. Furthermore, this new cell labeling and tracking technology had minimal effect on cartilage formation in vivo.
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Cartílago/citología , Condrocitos/citología , Química Clic , Cobre/química , Animales , Citometría de Flujo , Ratones , Ingeniería de TejidosRESUMEN
Oligonucleotide-based biosensors have drawn much attention because of their broad applications in in vitro diagnostics and environmental hazard detection. They are particularly of interest to many researchers because of their high specificity as well as excellent sensitivity. Recently, oligonucleotide-based biosensors have been used to achieve not only genetic detection of targets but also the detection of small molecules, peptides, and proteins. This has further broadened the applications of these sensors in the medical and health care industry. In this review, we highlight various examples of oligonucleotide-based biosensors for the detection of diseases, drugs, and environmentally hazardous chemicals. Each example is provided with detailed schematics of the detection mechanism in addition to the supporting experimental results. Furthermore, future perspectives and new challenges in oligonucleotide-based biosensors are discussed.
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Técnicas Biosensibles , Contaminantes Ambientales/análisis , Oligonucleótidos/química , Enfermedades Transmisibles/diagnóstico , Humanos , Técnicas In Vitro , Neoplasias/diagnóstico , Enfermedades Neurodegenerativas/diagnóstico , Detección de Abuso de Sustancias/métodosRESUMEN
Transient cartilage and a mineralizing microenvironment play pivotal roles in mesenchymal cell ossification during bone formation. In order to recreate these microenvironmental cues, C3H10T1/2 murine mesenchymal stem cells (MSCs) were exposed to chondrocyte-conditioned medium (CM) and seeded onto three-dimensional mineralized scaffolds for bone regeneration. Expansion of C3H10T1/2 cells with CM resulted in enhanced expression levels of chondrogenic markers such as aggrecan, type II collagen, type X collagen, and Sox9, rather than of osteogenic genes. Interestingly, CM expansion led to reduced expression levels of osteogenic genes such as alkaline phosphatase (ALP), type I collagen, osteocalcin, and Runx2. However, CM-expanded C3H10T1/2 cells showed enhanced osteogenic differentiation as indicated by increased ALP and Alizarin Red S staining upon osteogenic factor exposure. In vivo, CM-expanded C3H10T1/2 mesenchymal cells were seeded onto mineralized scaffolds (fabricated with polydopamine and coated with simulated body fluids) and implanted into critical-sized calvarial-defect mouse models. After 8 weeks of implantation, mouse skulls were collected, and bone tissue regeneration was evaluated by micro-computed tumography and Masson's trichrome staining. In accordance with the in vitro analysis, CM-expanded C3H10T1/2 cells gave enhanced bone mineral deposition. Thus, chondrocyte-conditioned factors and a mineralized microenvironment stimulate the bone formation of MSCs.
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Calcificación Fisiológica/fisiología , Condrocitos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/fisiología , Animales , Diferenciación Celular , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos BALB C , Ingeniería de TejidosRESUMEN
Insulin induces the activation of Na,K-ATPase while translationally controlled tumor protein (TCTP) inhibits this enzyme and the associated pump activity. Because binding of insulin with its membrane receptor is known to mediate the phosphorylation of multiple intracellular proteins, phosphorylation of TCTP by insulin might be related to the sodium pump regulation. We therefore examined whether insulin induces TCTP phosphorylation in embryonic kidney 293T cells. Using immunoprecipitation and Western blotting, we found that insulin phosphorylates serine (Ser) residues of TCTP. Following fractionation of the insulin-treated cells into cytosol and membrane fractions, phosphorylated TCTP at its Ser residue (p-Ser-TCTP) was detected exclusively in the cytosolic part and not in the membrane fraction. Phosphorylation of TCTP reached maximum in about 10 min after insulin treatment in 293T cells. In studies of cell-type specificity of insulin-mediated phosphorylation of TCTP, insulin did not phosphorylate TCTP in HeLa cells. Computational prediction and immunoprecipitation using several constructs having Ser to Ala mutation at potential p-Ser sites of TCTP revealed that insulin phosphorylated the serine-9 and -15 residues of TCTP. Elucidations of how insulin-mediated TCTP phosphorylation promotes Na,K-ATPase activation, may offer potential therapeutic approaches to diseases associated with vascular activity and sodium pump dysregulation.
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Biomarcadores de Tumor/metabolismo , Células HEK293/efectos de los fármacos , Insulina/farmacología , Serina/metabolismo , Biomarcadores de Tumor/química , Biomarcadores de Tumor/genética , Membrana Celular/metabolismo , Citosol/metabolismo , Células HEK293/metabolismo , Células HeLa , Humanos , Mutación , Fosforilación , Serina/genética , Proteína Tumoral Controlada Traslacionalmente 1RESUMEN
Programmable molecular self-assembly of siRNA molecules provides precisely controlled generation of dendrimeric siRNA nanostructures. The second-generation dendrimers of siRNA can be effectively complexed with a low-molecular-weight, cationic polymer (poly(ß-amino ester), PBAE) to generate stable nanostructures about 160â nm in diameter via strong electrostatic interactions. Condensation and gene silencing efficiencies increase with the increased generation of siRNA dendrimers due to a high charge density and structural flexibility.
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Dendrímeros/química , Silenciador del Gen , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Nanoestructuras/química , Polímeros/químicaRESUMEN
This study compared the effects of polyethylene glycol (PEG) shielding and mannose-conjugated ligands density on lipid nanoparticles (LNPs) for intracellular uptake to macrophages in vitro and accumulation in spleens in vivo. Fabricated phosphatidyl serine-incorporated LNPs (sLNPs) was physically decorated with mannose-conjugated DSPE-PEG (DPM) at different DPM/LNP molar ratios achieving the DPM density from 0 to 0.6 PEGs/nm2. We demonstrated that low PEG shielding sLNPs with mannose ligands (sLNP-DPMs) displayed superior uptake to macrophages (RAW 264.7 cells) compared with high PEG shielding sLNP-DPMs in vitro. However, high PEG shielding sLNP-DPMs showed significant spleen accumulation compared with low PEG shielding sLNP-DPMs in vivo after intravenous injection. In particular, high PEG shielding sLNPs coated with DSPE-methoxyPEG (DP) and DPM mixture at DP/DPM molar ratios of 5/5 exhibited greater accumulation in red pulp of spleens than naked sLNPs by 2.7-folds in vivo. These results suggested that the optimal PEG shielding and mannose densities per a particle might be different between in vitro cellular uptake to macrophages and in vivo spleen accumulation after systemic administration. Taken together, precision-tailored LNP-surface modifications achieved through optimization of PEG shielding and mannose density can greatly enhance accumulation of LNPs in red pulp of spleens, which could be applied for the delivery of nucleic acid-based drugs and vaccines to spleens in vivo.
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In this study, histidine oligomer (oHis; 10mer)-incorporating LNPs (H10LNPs) are developed as a novel carrier for efficient siRNA delivery. Notably, the unmodified oHis (10mer) is greatly incorporated within LNPs through ionic interaction with siRNAs, which serves as an endosome escape enhancer. H10LNPs with a size of ≈65 nm demonstrate a significantly enhanced extent of endosomal escape, as evidenced by calcein assay and confocal microscopy images of intracellular fluorescence, surpassing conventional LNPs. Furthermore, the half inhibitory concentration (IC50) of the human endogenous globotriaosylceramide synthase (Gb3 synthase) gene in H10LNPs-treated cells exhibits a significant threefold decrease, compared to that in LNP-treated cells. Notably, H10LNPs maintain comparable biocompatibility and biodistribution both in vitro and in vivo. Considering that the fabricated siRNA H10LNPs exhibit excellent biocompatibility and superior gene silencing activity over conventional LNPs, these particles can be harnessed for the safe delivery of therapeutic siRNAs. Additionally, this study introduces promising, feasible, simple, and alternative formulation processes for integrating unmodified functional cationic peptides into LNPs to enhance the delivery efficiency of a wide range of nucleic acid-based drugs.
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Histidina , Nanopartículas , ARN Interferente Pequeño , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacología , Histidina/química , Humanos , Nanopartículas/química , Animales , Lípidos/química , Silenciador del Gen , Ratones , Técnicas de Transferencia de Gen , Distribución Tisular , LiposomasRESUMEN
During the COVID-19 pandemic, mRNA vaccines emerged as a rapid and effective solution for global immunization. The success of COVID-19 mRNA vaccines has increased interest in the use of lipid nanoparticles (LNPs) for the in vivo delivery of mRNA therapeutics. Although mRNA exhibits robust expression profiles, transient protein expression is often observed, raising uncertainty regarding the frequency of its administration. Additionally, various RNA therapeutics may necessitate repeated dosing to achieve optimal therapeutic outcomes. Nevertheless, the impact of repeated administrations of mRNA/LNP on immune responses and protein expression efficacy remains unclear. In this study, we investigated the influence of the formulation parameters, specifically ionizable lipids and polyethylene glycol (PEG) lipids, on the repeat administration of mRNA/LNP. Our findings revealed that ionizable lipids had no discernible impact on the dose-responsive efficacy of repeat administrations, whereas the lipid structure and molar ratio of PEG lipids were primary factors that affected mRNA/LNP performance. The optimization of the LNP formulation with PEG lipid confirmed the sustained dose-responsive efficacy of mRNA after repeated administrations. This study highlights the critical importance of optimizing LNP formulations for mRNA therapeutics requiring repeated administrations.
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Several studies have utilized a lipid nanoparticle delivery system to enhance the effectiveness of mRNA therapeutics and vaccines. However, these nanoparticles are recognized as foreign materials by the body and stimulate innate immunity, which in turn impacts adaptive immunity. Therefore, it is crucial to understand the specific type of innate immune response triggered by lipid nanoparticles. This article provides an overview of the immunological response in the body, explores how lipid nanoparticles activate the innate immune system, and examines the adverse effects and immunogenicity-related development pathways associated with these nanoparticles. Finally, we highlight and explore strategies for regulating the immunogenicity of lipid nanoparticles.
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Nanopartículas , Vacunas , Vacunas de ARNm , LiposomasRESUMEN
RNA therapeutics show a significant breakthrough for the treatment of otherwise incurable diseases and genetic disorders by regulating disease-related gene expression. The successful development of COVID-19 mRNA vaccines further emphasizes the potential of RNA therapeutics in the prevention of infectious diseases as well as in the treatment of chronic diseases. However, the efficient delivery of RNA into cells remains a challenge, and nanoparticle delivery systems such as lipid nanoparticles (LNPs) are necessary to fully realize the potential of RNA therapeutics. While LNPs provide a highly efficient platform for the in vivo delivery of RNA by overcoming various biological barriers, several challenges remain to be resolved for further development and regulatory approval. These include a lack of targeted delivery to extrahepatic organs and a gradual loss of therapeutic potency with repeated doses. In this review, we highlight the fundamental aspects of LNPs and their uses in the development of novel RNA therapeutics. Recent advances in LNP-based therapeutics and preclinical/clinical studies are overviewed. Lastly, we discuss the current limitations of LNPs and introduce breakthrough technologies that might overcome these challenges in future applications.
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COVID-19 , Nanopartículas , Humanos , ARN Interferente Pequeño/genética , Lípidos , LiposomasRESUMEN
In this study, a novel polyhistidine-incorporated lipid nanoparticle (pHis/LNP) is developed for the delivery of therapeutic globotriaosylceramide (Gb3) synthase siRNAs using a microfluidic device with pHis as a biocompatible method of endosome escape. To inhibit the expression of Gb3 synthase, six siRNAs against Gb3 synthase are designed and an optimal siRNA sequence is selected. Selected Gb3 synthase siRNA is incorporated into pHis/LNP to prepare a spherical siRNA pHis/LNP with a size of 62.5 ± 1.9 nm and surface charge of -13.3 ± 4.2 mV. The pHis/LNP successfully protects siRNAs from degradation in 50% serum condition for 72 h. Prepared pHis/LNP exhibits superior stability for 20 days and excellent biocompatibility for A549 cells. After treatment with fluorescence-labeled LNPs, dotted fluorescent signals are co-localized with Lysotracker in cells with LNPs, whereas strong and diffused fluorescence intensity is observed in cells with pHis/LNPs probably due to successful endosomal escape. The extent of Gb3 synthase gene silencing by siRNA pHis/LNP is greatly improved (6.0-fold) compared to that by siRNA/LNP. Taken together, considering that the fabricated siRNA pHis/LNP exhibits excellent biocompatibility and superior gene silencing activity over conventional LNP, these particles can be utilized for the delivery of a wide range of therapeutic siRNAs.