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Chondrocyte-derived exosomes have shown efficacy in differentiating osteoarthritis-affected cartilage. Intervertebral disc degeneration (IVDD) and osteoarthritis often affect facet joints of the spine and show common epidemiological and pathophysiological characteristics. However, the potential of chondrocyte-derived exosomes for treating IVDD remains unclear. The present study aimed to confirm the effect of end plate chondrocyte-derived exosomes (EPC-Exo) on IVDD and elucidate the underlying mechanism. EPC-Exos were isolated and identified by ultracentrifugation, Western blotting, electron microscopy, and nanoparticle tracking analysis. In the in vitro, EPC-Exo uptake by nucleus pulposus (NP) cells reduced cell death by blocking the nuclear factor-κB (NF-κB) signaling pathway. In the in vivo study, EPC-Exos injected into rat intervertebral discs mitigated lipopolysaccharide-induced IVDD, as revealed by a decreased loss of disc height and improved magnetic resonance imaging findings and histological scores. Bioinformatics and sequencing analyses indicated that EPC-Exos alleviated IVDD through the miR-133a-3p/MAML1 axis. The present study suggests that EPC-Exos reduced IVDD incidence via the miR-133a-3p/MAML1 axis-mediated suppression of NF-κB signaling, which prevented the pyroptosis of NP cells.

Melanoma, with its steadily rising global incidence, is characterized by high invasiveness, leading to poor prognosis in advanced stages. There remains an unmet clinical need for the development of radiolabeled PET imaging probes for the early diagnosis of melanoma. Integrin VLA-4, a key factor in melanoma metastasis, presents a promising protein target to address the specificity shortcomings of existing probes in melanoma imaging. This study evaluates ⁶⁸Ga-labeled DOTA-LLP2A PET probes for melanoma imaging by modifying different carboxyl sites and employing various polyethylene glycol (PEG) linkers based on the structure of the high-affinity ligand LLP2A for VLA-4. The ligand intermediates LLP2A-NH₂ and LLP2A(tBu)-OH, as well as their conjugates (probe precursors), were synthesized via solid-phase synthesis. The specificity and cytotoxicity of the probes were assessed in VLA-4-positive B16F10 cells and VLA-4-negative A375 cells. Targeting efficacy of the probes in B16F10 and A375 xenograft models was compared through PET imaging and biodistribution studies. VLA-4 expression in tissues was evaluated via immunofluorescence, while H&E staining was employed to assess the safety profile of the probes. The probe ([⁶⁸Ga]Ga-T-CH) modified at the Aminocyclohexane carboxylic acid (Ach) exhibited greater signal accumulation in B16F10 melanoma (3.90 ± 0.43%ID/g at 1 h) compared to the 2-aminoadipic acid (Aad) side-chain-modified probe ([⁶⁸Ga]Ga-T-AD) (1.43 ± 0.23%ID/g at 1 h). PET images of the three PEG conjugates derived from the Ach demonstrated bright tumor signals and low background noise, showing a progressive increase in tumor signal intensity from [⁶⁸Ga]Ga-T6 to [⁶⁸Ga]Ga-T4 and [⁶⁸Ga]Ga-T2. Tumor uptake, tumor-to-muscle ratio, and tumor-to-blood ratio from biodistribution were significantly higher for [⁶⁸Ga]Ga-T2 than for [⁶⁸Ga]Ga-T4 and [⁶⁸Ga]Ga-T6 (tumor: 3.58 ± 0.28 vs 2.90 ± 0.16 vs 1.87 ± 0.22%ID/g at 1 h; tumor/muscle: 13.38 ± 0.43 vs 10.62 ± 0.70 vs 7.19 ± 1.15 at 1 h; tumor/blood: 8.64 ± 1.12 vs 5.32 ± 0.91 vs 4.36 ± 0.59 at 1 h; P < 0.05). These data suggest that the series of PEG derivatives [⁶⁸Ga]Ga-T2, [⁶⁸Ga]Ga-T4, and [⁶⁸Ga]Ga-T6, linked at the Ach site, are excellent ⁶⁸Ga-labeled probes for melanoma and other potential VLA-4-positive tumors. Among them, [⁶⁸Ga]Ga-T2 shows the highest tumor-to-background contrast for melanoma, positioning it as the most promising candidate for clinical translation.

The high content of vitamin E, including tocopherols and tocotrienols (TCF-TTE), in palm oil (Elaeis guineensis) has made it a promising candidate for the alternative treatment of atopic dermatitis (AD). However, the limited solubility of TCF-TTE has restricted its therapeutic efficacy. In this study, pluronic-based micelles (MCs) encapsulating palm oil-derived TCF-TTE were formulated with dissolvable microarray patch-micelles (DMP-MC) using carboxymethyl cellulose (CMC) synthesized from empty fruit bunches of palm to optimize its delivery for AD. The MC was prepared using a direct dissolution method using Pluronic F68 and F127. The results showed that MC increased the solubility of TCF-TTE, which was further confirmed by an in vitro study where 90.23 ± 2.07% TCF and 4.56 ± 1.36% TTE were released compared to the unencapsulated TCF-TTE extract. Furthermore, CMC biopolymers and MC integrated into DMP-MC with polyvinylpyrrolidone (PVP) exhibited favorable physical properties, such as mechanical strength and penetration ability. DMP-MC also exhibited a better platform with lower permeation, indicating higher retention and increased localized effects on AD skin than cream-MC. Additionally, dermatokinetic profile parameters showed significant improvement. The mean residence time (MRT) parameter indicated that TCF-TTE was retained for longer times 19.28 ± 0.02 h and 20.68 ± 0.01 h. Moreover, an in vivo study revealed that DMP-MC could relieve AD symptoms more rapidly than oral doses and cream-MC, indicating that DMP-MC proved to be more efficient. Furthermore, DMP-MC showed no tissue destruction (granulation and fibrosis) in rats treated with DMP-MC on the seventh day. Therefore, this study successfully developed the MC formula in DMP-MC formulation using synthesized CMC, which could potentially improve AD's therapeutic efficacy.

At the end of 2019, SARS-CoV-2 emerged and rapidly spread, having a profound negative impact on human health and socioeconomic conditions. In response to this unprecedented global health crisis, significant advancements were made in the mRNA vaccine technology. In this study, we have compared the difference between two SARS-CoV-2 receptor-binding domain (RBD) mRNA-Lipid nanoparticle (LNP) vaccines prepared from two different ionizable cationic lipids: ALC-0315 and MC3. Characterization of RBD mRNA-LNPs showed that both MC3-LNP and ALC-0315-LNP are highly uniform and stable. Furthermore, we assessed the humoral immune response in mice after immunization; our findings indicated that both vaccine formulations effectively enhanced the formation and differentiation of germinal center (GC). Notably, the mice immunized with the ALC-0315-LNP vaccine elicited higher levels of IgG and its subclasses and significantly enhanced the activation of dendritic cells and T cells in draining lymph nodes (dLNs) compared to those immunized with the MC3-LNP vaccine. Further analysis of the T cell phenotype after splenic restimulation showed that mice injected with both LNP mRNA vaccines had significantly increased activation of the splenic T cells and Th1-type cytokine production. In addition, our finding showed that both LNP mRNA vaccines significantly increased the proportions of follicular helper T cells (Tfh) and long-lasting plasma cells in the dLNs of mice on day 14 postimmunization compared to control. In conclusion, both ALC-0315 and MC3 exhibited good stability and immunogenicity as mRNA-LNP recipes, but the ALC-0315-based mRNA-LNP vaccine showed higher efficacy in humoral and cellular immune responses compared to MC3.

Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer term. Here, we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. Using parallelized multitarget TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay on wastewater, soil, and human stool samples, we found 90% agreement on positive calls and 89% agreement on negative calls. Additionally, we show how long-read and short-read sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish subspecies. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.

Gallium, a trace metal not found in its elemental form in nature, has garnered significant interest as a biocide, given its ability to interfere with iron metabolism in bacteria. Consequently, several gallium compounds have been developed and studied for their antimicrobial properties but face challenges of poor solubility and formulation for delivery. Organizing the metal into three-dimensional, hybrid scaffolds, termed metal-organic frameworks (MOFs), is an emerging platform with potential to address many of these limitations. Gallium MOFs show improved solubility and antibacterial potency relative to the free metal due to their ability to coload antibiotics and functional biomolecules. Synthetic strategies are equally versatile, with several rapid, cost-effective, and scalable methods available. In this review, we present the advantages and disadvantages of these various synthetic strategies with respect to their antibacterial efficiency, product purity, and reaction control. The activity of gallium-based MOFs against Gram-positive and Gram-negative pathogens in mono- and combinatorial therapeutic settings is discussed in the context of their mechanisms of action and structure-function-performance relationships collated from recent studies. While gallium MOF development as antibacterials is still in its nascent stages, the examples discussed here highlight their potential as a novel class of therapeutics poised to impact the fight against pan-drug-resistant bacterial pathogens.

Although glycine is the simplest of the amino acids, its solution and solid-state properties are far from straightforward. The aqueous solubility of glycine plays an important role in various applications, including nutrition, food products, biodegradable plastics, and drug development. There is evidence that glycine in subsaturated pH 3-8 solutions forms a dimer, as suggested by several techniques. However, what takes place below pH 3 and above pH 8 in saturated solutions has been sparsely explored and is thought to exhibit complex properties. Although the solubility measurements in the pH 0-13 range have been reported by several groups, the interlaboratory variance between the data below pH 3 and above pH 8 has been high. In a couple of cases, there appears to be no pH dependence on solubility across the wide pH range, even though the reported glycine pK_a values are 2.34 and 9.61. The solubility of the salt forms of glycine is largely uncharacterized. The solubility products of the simplest salts, glycine hydrochloride and sodium glycinate, appear not to have been published. In this study, five series of precision solubility measurements of glycine and its salts were performed at 25 °C, covering the range of pH -0.4 to 12.4, where in each case, just enough glycine was added to reach saturation. We have developed an equilibrium model to rationalize the complicated salt regions. Elemental analysis of isolated solids from saturated solutions supports the speciation model. At least three different salt forms have been indicated in acidic solutions and one salt form in alkaline solutions. Solubility products are reported here. The presence of a water-soluble cationic dimer is also proposed. Data analysis was performed with the aid of the pDISOL-X computer program. Activity corrections based on the Stokes-Robinson hydration theory have been implemented in saturated solutions with ionic strength in some cases exceeding 5 M. Although salt solubility is not a constant, since it depends on two independently controlled reactant concentrations, the salt solubility product is commonly expected to be a constant. However, in the glycine salt region below pH 3, our solubility measurements demonstrate that the solubility products depend on the total amount of added glycine in a saturated solution. We view this as an "uncommon" common-ion effect.

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB). Tuberculous granuloma is the central and key pathological structure of tuberculosis and is characterized by tissue hypoxia and ineffective drug delivery. To address these issues, this study fabricated a composite nanoparticle loaded with catalase (CAT) and levofloxacin (LEV) (CAT@LEV-NPs) and then combined it with ultrasound (US) to investigate the bactericidal effect and underlying mechanisms using TB spheroids. The TB spheroids were constructed using attenuated Bacillus Calmette-Guérin (BCG) instead of MTB to facilitate operation under general experimental conditions. This study examined the physical properties and oxygen production efficiency of CAT@LEV-NPs. Subsequently, we treated TB spheroids with nanoparticles alone or in combination with US and found that ultrasound significantly increased drug permeability and activated CAT@LEV-NPs to produce a large number of reactive oxygen species (ROS). The combined treatment showed excellent antibacterial effects, resulting in more severe damage to the bacterial structure than other treatments. Additionally, the combined treatment induced a higher M1 polarization of macrophages, increased the apoptosis rate, and improved the anoxic microenvironment in TB spheroids. These factors may be closely related to the enhanced bactericidal effects of combined treatment. In conclusion, our study suggests that US combined with CAT@LEV-NPs could serve as a novel, noninvasive, safe, and effective treatment modality for intractable MTB infections.

Traditional chemotherapy often encounters failure attributed to drug resistance mediated by tumor-repopulating cells (TRCs) and chemotherapy-triggered immune suppression. The effective inhibition of TRCs and the mitigation of drug-induced immune suppression are pivotal for the successful chemotherapy. Here, TRC-derived microparticles (3D-MPs), characterized by excellent tumor-targeting and high TRC uptake properties, are utilized to deliver metformin and the chemotherapeutic drug doxorubicin ((DOX+Met)@3D-MPs). (DOX+Met)@3D-MPs efficiently enhance tumor accumulation and are highly internalized in tumor cells and TRCs. Additionally, (DOX+Met)@3D-MPs significantly decrease the chemotherapy-triggered upregulation in P-glycoprotein expression to enhance intracellular doxorubicin retention, resulting in increased chemotherapy sensitivity and immunogenic cell death in tumor cells and TRCs for improved antitumor immunity. Importantly, (DOX+Met)@3D-MPs also remarkably reduce chemotherapy-induced PD-L1 expression, efficiently alleviating immune suppression facilitated by the PD-L1/PD-1 axis to further enhance immunological response against malignancy. These results underscore the (DOX+Met)@3D-MPs' potential as a viable platform for augmenting the efficacy of antitumor therapies.