Smooth muscle cell-specific matrix metalloproteinase 3 deletion reduces osteogenic transformation and medial artery calcification.

AIMS: Vascular calcification is highly prevalent in atherosclerosis, diabetes, and chronic kidney disease. It is associated with increased morbidity and mortality in patients with cardiovascular disease. Matrix metalloproteinase 3 (MMP-3), also known as stromelysin-1, is part of the large matrix metalloproteinase family. It can degrade extracellular matrix components of the arterial wall including elastin, which plays a central role in medial calcification. In this study, we sought to determine the role of MMP-3 in medial calcification. METHODS AND RESULTS: We found that MMP-3 was increased in rodent models of medial calcification as well as in vascular smooth muscle cells (SMCs) cultured in a phosphate calcification medium. It was also highly expressed in calcified tibial arteries in patients with peripheral arterial disease (PAD). Knockdown and inhibition of MMP-3 suppressed phosphate-induced SMC osteogenic transformation and calcification, whereas the addition of a recombinant MMP-3 protein facilitated SMC calcification. In an ex vivo organ culture model and a rodent model of medial calcification induced by vitamin D3, we found that MMP-3 deficiency significantly suppressed medial calcification in the aorta. We further found that medial calcification and osteogenic transformation were significantly reduced in SMC-specific MMP-3-deficient mice, suggesting that MMP-3 in SMCs is an important factor in this process. CONCLUSION: These findings suggest that MMP-3 expression in vascular SMCs is an important regulator of medial calcification and that targeting MMP-3 could provide a therapeutic strategy to reduce it and address its consequences in patients with PAD.

Perfluorooctanesulfonic Acid and Perfluorooctanoic Acid Promote Migration of Three-Dimensional Colorectal Cancer Spheroids.

Perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) are persistent environmental contaminants that are of increasing public concern worldwide. However, their relationship with colorectal cancer (CRC) is poorly understood. This study aims to comprehensively investigate the effect of PFOS and PFOA on the development and progression of CRC in vitro using a series of biological techniques and metabolic profiling. Herein, the migration of three-dimensional (3D) spheroids of two CRC cell lines, SW48 KRAS wide-type (WT) and SW48 KRAS G12A, were observed after exposure to PFOS and PFOA at 2 µM and 10 µM for 7 days. The time and dose-dependent migration phenotype induced by 10 µM PFOS and PFOA was further confirmed by wound healing and trans-well migration assays. To investigate the mechanism of action, derivatization-mass spectrometry-based metabolic profiles were examined from 3D spheroids of SW48 cell lines exposed to PFOS and PFOA (2 µM and 10 µM). Our findings revealed this exposure altered epithelial-mesenchymal transition related metabolic pathways, including fatty acid ß-oxidation and synthesis of proteins, nucleotides, and lipids. Furthermore, this phenotype was confirmed by the downregulation of E-cadherin and upregulation of N-cadherin and vimentin. These findings show novel insight into the relationship between PFOS, PFOA, and CRC.

Tuning Ligands Ratio Allows for Controlling Gold Nanocluster Conformation and Activating a Nonantimicrobial Thiol Fragrance for Effective Treatment of MRSA-Induced Keratitis.

Bacterial keratitis is a serious ocular disease that affects millions of people worldwide each year, among which ≈25% are caused by Staphylococcus aureus. With the spread of bacterial resistance, refractory keratitis caused by methicillin-resistant S. aureus (MRSA) affects ≈120 000-190 000 people annually and is a significant cause of infectious blindness. Atomically precise gold nanoclusters (GNCs) recently emerged as promising antibacterial agents; although how the GNC structure and capping ligands control the antibacterial properties remains largely unexplored. In this study, by adjusting the ratio of a "bulky" thiol fragrance to a linear zwitterionic ligand, the GNC conformation is transformed from Au25 (SR)18 to Au23 (SR)16 species, simultaneously converting both inactive thiol ligands into potent antibacterial nanomaterials. Surprisingly, mixed-ligand capped Au23 (SR)16 GNCs exhibit superior antibacterial potency compared to their monoligand counterparts. The optimal GNC is highly potent against MRSA, showing >1024-fold lower minimum inhibitory concentration than the corresponding free ligands. Moreover, it displays excellent potency in treating MRSA-induced keratitis in mice with greatly accelerated corneal recovery (by approximately ninefold). Thus, this study establishes a feasible method to synthesize antibacterial GNCs by adjusting the ligand ratio to control GNC conformation and active non-antibacterial ligands, thereby greatly increasing the repertoires for combating multidrug-resistant bacterial infections.

Sex hormones impact early maturation and immune response in the arteriovenous fistula mouse model.

Arteriovenous fistulae (AVF) fail to mature more frequently in female patients compared with male patients, leading to inferior outcomes and decreased utilization. Since our mouse AVF model recapitulates sex differences in human AVF maturation, we hypothesized that sex hormones mediate these differences during AVF maturation. C57BL/6 mice (9-11 wk) were treated with aortocaval AVF surgery and/or gonadectomy. AVF hemodynamics were measured via ultrasound (days 0-21). Blood was collected for FACS and tissue for immunofluorescence and ELISA (days 3 and 7); wall thickness was assessed by histology (day 21). Inferior vena cava shear stress was higher in male mice (P = 0.0028) after gonadectomy, and they had increased wall thickness (22.0 ± 1.8 vs. 12.7 ± 1.2 µm; P < 0.0001). Conversely, female mice had decreased wall thickness (6.8 ± 0.6 vs. 15.3 ± 0.9 µm; P = 0.0002). Intact female mice had higher proportions of circulating CD3+ T cells on day 3 (P = 0.0043), CD4+ (P = 0.0003) and CD8+ T cells (P = 0.005) on day 7, and CD11b+ monocytes on day 3 (P = 0.0046). After gonadectomy, these differences disappeared. In intact female mice, CD3+ T cells (P = 0.025), CD4+ T cells (P = 0.0178), CD8+ T cells (P = 0.0571), and CD68+ macrophages (P = 0.0078) increased in the fistula wall on days 3 and 7. This disappeared after gonadectomy. Furthermore, female mice had higher IL-10 (P = 0.0217) and TNF-α (P = 0.0417) levels in their AVF walls than male mice. Sex hormones mediate AVF maturation, suggesting that hormone receptor signaling may be a target to improve AVF maturation.NEW & NOTEWORTHY After arteriovenous fistula creation, females have lower rates of maturation and higher rates of failure than males. In a mouse model of venous adaptation that recapitulates human fistula maturation, sex hormones may be mechanisms of the sexual dimorphism: testosterone is associated with reduced shear stress, whereas estrogen is associated with increased immune cell recruitment. Modulating sex hormones or downstream effectors suggests sex-specific therapies and could address disparities in sex differences in clinical outcomes.

Correction: Screening on-chip fabricated nanoparticles for penetrating the blood-brain barrier.

Correction for 'Screening on-chip fabricated nanoparticles for penetrating the blood-brain barrier' by Qinghong Hou et al., Nanoscale, 2022, DOI: 10.1039/d1nr05825h.

Modulating the catalytic activity of gold nanoparticles using amine-terminated ligands.

Nanozymes have broad applications in theranostics and point-of-care tests. To enhance the catalytic activity of nanozymes, the conventional strategy is doping metals to form highly active nanoalloys. However, high-quality and stable nanoalloys are hard to synthesize. Ligand modification is a powerful strategy to achieve chemoselectivity or bioactivity by changing the surface chemistry. Here, we explore different ligands to enhance the catalytic activity of nanozymes, e.g., gold nanoparticles (AuNPs). We systematically studied the impacts on the enzymatic activity of AuNPs by ligand engineering of surface chemistry (charge, group, and surface distance). Our work established critical guidelines for surface modification of nanozymes. The amine group favors higher activity of AuNPs than other groups. The flexible amine-rich ligand enhances the catalytic activity of AuNPs in contrast to other ligands and unmodified AuNPs. Using a proof-of-concept model, we screened many candidate ligands to obtain polyamine-AuNPs, which have strongly enhanced peroxidase-like activity and 100 times enhanced sensitivity compared to unmodified AuNPs. The strategy of enhancing the catalytic activity of AuNPs using ligands will facilitate the catalysis-related applications of nanozymes in biology and diagnostics.

Screening on-chip fabricated nanoparticles for penetrating the blood-brain barrier.

The inability of drugs to cross the blood-brain barrier (BBB) makes it difficult to treat diseases in the central nervous system. It is known that peptides with or without specific receptors on the BBB showed different or even controversial neuron targeting capability in different reports. So, it is necessary to clarify how these peptides work as targeting molecules in the central nervous system. Herein, we evaluate and compare the performance of 6 kinds of peptides with (T7, D-T7, and GSH) or without (TGN, CGN, and TAT) BBB-specific receptors by conjugating these peptides on lipids to serve as a shell to encapsulate a core of PLGA and lamotrigine to form nanoparticles for targeted epilepsy therapy. In vitro assay shows that the TAT-modified nanoparticles show the highest internalization efficacy in the BBB model cell line bEnd·3 cells and hippocampal neurons. By contrast, experiments in mice show that the D-T7-modified nanoparticles have the highest brain targeting and epilepsy therapeutic efficiency. Thus, our experiments uncover the different performances of the 6 peptides at different levels (in vitro and in vivo), which is insightful for developing novel delivery systems for treating diseases in the central nervous system.

Small Molecule-Capped Gold Nanoclusters for Curing Skin Infections.

With the long-term and extensive abuse of antibiotics, bacteria can mutate into multidrug-resistant (MDR) strains, resist the existing antibiotics, and escape the danger of being killed. MDR bacteria-caused skin infections are intractable and chronic, becoming one of the most significant and global public-health issues. Thus, the development of novel antimicrobial materials is urgently needed. Non-antibiotic small molecule-modified gold nanoclusters (AuNCs) have great potential as a substitute for commercial antibiotics. Still, their narrow antibacterial spectrum hinders their wide clinical applications. Herein, we report that 4,6-diamino-2-pyrimidinethiol (DAPT)-modified AuNCs (DAPT-AuNCs) can fight against Gram-negative and Gram-positive bacterial strains as well as their MDR counterparts. By modifying DAPT-AuNCs on nanofibrous films, we develop an antibiotic film as innovative dressings for curing incised wounds, which exhibits excellent therapeutic effects on wounds infected by MDR bacteria. Compared to the narrow-spectral one, the broad-spectral antibacterial activity of the DAPT-AuNCs-modified film is more suitable for preventing and treating skin infections caused by various kinds of unknown bacteria. Moreover, the antibacterial films display excellent biocompatibility, implying the great potential for clinical applications.

Reversing Bacterial Resistance to Gold Nanoparticles by Size Modulation.

One major frustration in developing antibiotics is that bacteria can quickly develop resistance that would require an entirely new cycle of research and clinical testing to overcome. Although plenty of bactericidal nanomaterials have been developed against increasingly severe superbugs, few reports have studied the resistance to these nanomaterials. Herein, we show that antibacterial 4,6-diamino-2-pyrimidine thiol (DAPT)-capped gold nanoparticles (AuDAPTs) can induce a 16-fold increased minimum inhibitory concentration (MIC) of E. coli only after very long term exposure (183 days), without developing cross-resistance to commercialized antibiotics. Strikingly, we recovered the bactericidal activities of AuDAPTs to the resistant strain by tuning the sizes of AuDAPTs without employing new chemicals. Such slow, easy-to-handle resistance induced by AuDAPTs is unprecedented compared to traditional antibiotics or other nanomaterials. In addition to the novel antibacterial activities and biocompatibilities, our approach will accelerate the development of gold nanomaterial-based therapeutics against multi-drug-resistant (MDR) bacterial infections.

A Soft, Conductive External Stent Inhibits Intimal Hyperplasia in Vein Grafts by Electroporation and Mechanical Restriction.

Intimal hyperplasia (IH) in vein grafts (VGs) is a major issue in coronary artery bypass grafting (CABG) surgery. Although external stents can attenuate IH of VGs to some extent, none of the existing external stents have shown satisfactory clinical outcomes. Here we develop a flexible, biodegradable, and conductive external metal-polymer conductor stent (MPCS) that can electroporate the vessel wall and produce a protein that prevents IH. We designed the plasmid DNA encoding the tissue inhibitor of metalloproteinases-3 (TIMP-3) and lyophilized it on the inner surface of the MPCS to deliver into the adventitia and the middle layer of VGs for gene therapy. Coupled with its continuous mechanical support to prevent dilation after implanting, the MPCS can inhibit the IH of VGs significantly in the rabbit model. This proof-of-concept demonstration may aid the development of other implantable bioelectronics for electroporation gene therapy.

Activating the Antibacterial Effect of 4,6-Diamino-2-pyrimidinethiol-Modified Gold Nanoparticles by Reducing their Sizes.

Adequately decorated gold nanoparticles (GNPs) have excellent antibiotic activities against multidrug-resistant (MDR) bacteria. Nanoparticles exhibiting Gram selective antibacterial actions are beneficial to precise therapy. Here, we present a strategy to tune the antibacterial spectrum of a small molecule (4,6-diamino-2-pyrimidinethiol, DAPT)-modified GNPs (DAPT-GNPs, DGNPs) by adjusting their sizes. Compared to large (ca. 14 nm diameter) DGNPs (lDGNPs) and medium-sized (3-4 nm diameter) DGNPs (mDGNPs), which have no antibacterial effect or only target Gram-negative (G-) bacteria, ultrasmall DGNPs (uDGNPs, <2 nm) have a broad antibacterial spectrum, especially showing an over 60-fold increase in antibacterial efficacy against Gram-positive (G+) bacteria. Moreover, the uDGNPs-functionalized scaffolds (agarose gel) can serve as general wound dressings for healing burnt infections. Our strategy is insightful for exploring properties of the nanomaterials and their applications.

Biological Safe Gold Nanoparticle-Modified Dental Aligner Prevents the Porphyromonas gingivalis Biofilm Formation.

Oral microbiology could directly influence overall health. Porphyromonas gingivalis (P. gingivalis) is a highly pathogenic bacterium that causes periodontitis and other related systematic diseases, including Alzheimer's disease. Orthodontic devices (e.g., invisalign aligner) is commonly used in populations with periodontitis who are also at a high risk of systematic diseases. In this study, newly explored antibacterial 4,6-diamino-2-pyrimidinethiol-modified gold nanoparticles (AuDAPT) were coated onto aligners. The coated aligners showed favorable antibacterial activity against P. gingivalis. In the presence of the coated aligner, the number of planktonic cells was decreased, and biofilm formation was prevented. This material also showed favorable biocompatibility in vivo and in vitro. This study reveals a new method for treating oral P. gingivalis by coating aligners with AuDAPT, which has typical advantages compared to other treatments for both periodontitis and related systematic diseases.

Near-Infrared Light-Activated Phototherapy by Gold Nanoclusters for Dispersing Biofilms.

A bacterial biofilm is strongly associated with chronic infections and is difficult to be eradicated, posing serious threats to public health. Development of effective therapeutic strategies to prevent and control hospital-acquired infections via eradication of bacteria shielded by biofilms is challenging. Herein, we developed deoxyribonuclease (DNase)-functionalized gold nanoclusters (AuNCs) (DNase-AuNCs), which are capable of killing Gram-positive and Gram-negative bacteria, especially dispersing the surrounding biofilms. The DNase can break down the extracellular polymeric substance matrix to expose the defenseless bacteria to photothermal therapy (PTT) and photodynamic therapy (PDT) by DNase-AuNCs under 808 nm laser irradiation. The combination of enzymolysis, PDT, and PTT can effectively remove biofilms with a dispersion rate of up to 80% and kill ∼90% of the shielded bacteria. DNase-AuNCs exhibit an outstanding therapeutic effect in treating bacterial biofilm-coated orthodontic devices (Invisalign aligners), suggesting their potential applications in medical devices.

Gold Nanoclusters-Coated Orthodontic Devices Can Inhibit the Formation of Streptococcus mutans Biofilm.

Oral health is an issue that has attracted increasing attention recently. Poor oral hygiene may induce the formation of oral biofilm on orthodontic devices, and cause gingivitis and dental caries. Here, we present a strategy for modifying orthodontic devices (e.g., invisalign aligner) with quaternary ammonium (QA)-modified gold nanoclusters (QA-GNCs) as an antibiotic reagent to prevent bacterial contamination and biofilm formation. The QA-GNCs-coated aligner can efficiently inhibit the adhesion of cariogenic pathogenic Streptococcus mutans and the formation of biofilm. Moreover, the antibacterial activity of the coated QA-GNCs can be maintained for at least 3 months and after repeated usage (>3 cycles). Furthermore, the QA-GNCs coating shows excellent biosafety confirmed by the cell viability test, the hemolysis assay, and animal experiments. Our strategy for antibacterial coating has the advantages of broad applications, low cost, good stability, high antibacterial efficiency, good biocompatibility, and low risk of antibiotic contamination, which could be particularly useful in preventing infections involving implantable medical devices or wearable electronics.

Triple-Targeting Delivery of CRISPR/Cas9 To Reduce the Risk of Cardiovascular Diseases.

A high level of low-density lipoprotein cholesterol (LDL-C) in the blood is a major risk factor for coronary heart disease. Herein, we present a triple-targeting strategy to generate a loss-of-function mutation in Pcsk9, which regulates plasma cholesterol levels, using a nanocarrier-delivered CRISPR/Cas9 system. Nuclear localization signal (NLS)-tagged Cas9 and Pcsk9-targeted single guide RNA (sgPcsk9) were complexed with gold nanoclusters (GNCs) modified with cationic HIV-1-transactivating transcriptor (TAT) peptide and further encapsulated in a galactose-modified lipid layer to target the nanoclusters to the liver. The resulting nanoclusters had an in vitro Pcsk9-editing efficiency of about 60 % and resulting in a decrease in plasma LDL-C in mice of approximately 30%. No off-target mutagenesis was detected in 10 sites with high similarity. This approach may have therapeutic potential for the prevention and treatment of cardiovascular disease without side effects.

Macrolides induce severe cardiotoxicity and developmental toxicity in zebrafish embryos.

Macrolide antibiotics (MALs) are widely used for both human and animal health. Most MALs and their metabolites transfer into aquatic organisms and environment resulting in violent consequences. Previous studies show that MALs cause cardiotoxicity in humans and mammals. However, the potential risk of these chemicals in aquatic organisms remains unclear. Here, we used zebrafish embryos as a model to evaluate the toxicity of MALs. Zebrafish embryos were exposed to four typical MALs including azithromycin (AZM), clarithromycin (CLR), tilmicosin (TMS) and tylosin (TYL) to study their cardiotoxicity. The heart rate of zebrafish embryos showed similar biphasic distribution in the presence of four MALs at 2 days post-fertilization (dpf). The heart rate increased significantly at low levels of MALs while decreased obviously at high levels. Subsequently, TMS was chose to study its acute toxicity and developmental toxicity, which caused pericardial edema and spinal curvature in zebrafish embryos at 4 dpf. Furthermore, we found that TMS triggered oxidative stress, with decreased SOD activities and increased MDA contents. Lastly, apoptosis was observed in zebrafish embryos under TMS treatment, with up-regulation of apoptosis associated genes such as p53, bcl 2, bax, caspase 3 and caspase 9, confirmed by increased protein expression based on Western blot analysis. Taken together, these data indicate that MALs can cause serious toxicity in the development of zebrafish. Great caution should be taken due to the huge consumption of MALs for food animal production and treatments with TMS for infections in aquaculture.

Gold Nanoclusters for Targeting Methicillin-Resistant Staphylococcus†aureus In†Vivo.

Widespread multidrug resistance caused by the abuse of antibiotics calls for novel strategies and materials. Gold nanoclusters (AuNCs) are scarcely explored for combating multidrug-resistant (MDR) bacteria in vivo. We herein synthesized a novel class of AuNCs, namely quaternary ammonium (QA) capped AuNCs (QA-AuNCs) as potent antibiotics selectively targeting MDR Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), in vivo. QA-AuNCs kill bacteria through a combined physicochemical mechanism, and show excellent therapeutic effects in both a skin infection model and a bacteremia model induced by MRSA. In addition, owing to their intense fluorescence, QA-AuNCs can be used for the discrimination of live/dead bacteria and bacteria counting, suggesting their potential for clinical theranostics.

Thermo-triggered Release of CRISPR-Cas9 System by Lipid-Encapsulated Gold Nanoparticles for Tumor Therapy.

CRISPR/Cas9 system is a powerful toolbox for gene editing. However, the low delivery efficiency is still a big hurdle impeding its applications. Herein, we report a strategy to deliver Cas9-sgPlk-1 plasmids (CP) by a multifunctional vehicle for tumor therapy. We condensed CPs on TAT peptide-modified Au nanoparticles (AuNPs/CP, ACP) via electrostatic interactions, and coated lipids (DOTAP, DOPE, cholesterol, PEG2000-DSPE) on the ACP to form lipid-encapsulated, AuNPs-condensed CP (LACP). LACP can enter tumor cells and release CP into the cytosol by laser-triggered thermo-effects of the AuNPs; the CP can enter nuclei by TAT guidance, enabling effective knock-outs of target gene (Plk-1) of tumor (melanoma) and inhibition of the tumor both in vitro and in vivo. This AuNPs-condensed, lipid-encapsulated, and laser-controlled delivery system provides a versatile method for high efficiency CRISPR/Cas9 delivery and targeted gene editing for treatment of a wide spectrum of diseases.

Genome Editing for Cancer Therapy: Delivery of Cas9 Protein/sgRNA Plasmid via a Gold Nanocluster/Lipid Core-Shell Nanocarrier.

The type II bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (CRISPR-associated protein) system (CRISPR-Cas9) is a powerful toolbox for gene-editing, however, the nonviral delivery of CRISPR-Cas9 to cells or tissues remains a key challenge. This paper reports a strategy to deliver Cas9 protein and single guide RNA (sgRNA) plasmid by a nanocarrier with a core of gold nanoclusters (GNs) and a shell of lipids. By modifying the GNs with HIV-1-transactivator of transcription peptide, the cargo (Cas9/sgRNA) can be delivered into cell nuclei. This strategy is utilized to treat melanoma by designing sgRNA targeting Polo-like kinase-1 (Plk1) of the tumor. The nanoparticle (polyethylene glycol-lipid/GNs/Cas9 protein/sgPlk1 plasmid, LGCP) leads to >70% down-regulation of Plk1 protein expression of A375 cells in vitro. Moreover, the LGCP suppresses melanoma progress by 75% on mice. Thus, this strategy can deliver protein-nucleic acid hybrid agents for gene therapy.