Pulmonary Delivery of Specialized Pro-Resolving Mediators-Based Nanotherapeutics Attenuates Pulmonary Fibrosis in Preclinical Animal Models.

Pulmonary fibrosis (PF) is a chronic lung disease characterized by excess extracellular matrix deposition and prolonged inflammation that fails to resolve and is druggable. Using resolvins and their precursors for inflammation resolution, we demonstrate a nano-enabled approach for accomplishing robust antifibrotic effects in bleomycin- or engineered nanomaterial-induced mouse and rat PF models. Targeting the lipid peroxidation-triggered NLRP3 inflammasome and NF-κB pathway in macrophages and the ROS-mediated TGF-ß/Smad and S1P signaling in epithelial cells results in these potent protective effects at the ng/mL dosimetry. We further develop an inhalable biocompatible nanoparticle that encapsulates fish oil, a chosen resolvin precursor, with phosphatidylcholine and polyethylene glycol to enhance drug permeability and facilitate crossing the mucosal barrier, forming "fish-oilsome" (FOS). Oropharyngeal aspiration and inhalation of FOS improved the anti-inflammatory status, histological characteristics, and pulmonary function in fibrotic lungs, which was mechanistically supported by transcriptomic and proteomic analyses. Further, scale-up engineered FOS samples with the desired physicochemical properties, anti-PF efficacy, and in vivo biocompatibility were validated in different batch sizes (up to 0.2 L/batch). This study provides a practical and translatable approach to promoting inflammation resolution and PF treatment.

Effective Treatment of Knee Osteoarthritis Using a Nano-Enabled Drug Acupuncture Technology in Mice.

A nano-enabled drug delivery acupuncture technology (nd-Acu) is developed that is based on traditional acupuncture needles where the stainless-steel surface is designed to deliver various payload molecules. To create the nd-Acu platform, an electrochemistry procedure is used to attach methyl salicylate-modified cyclodextrin in which the sugar rings allow the encapsulation of structurally defined single or multiple payload molecules via an inclusion complexation process. Drug loading and release profile are first studied using fluorescent dyes abiotically and at intact animal level. nd-Acu allows more efficient dye loading and time-dependent release compared to pristine needles without cyclodextrin modification. Subsequently, a proof-of-principle efficacy study is conducted using the platform to load a local anesthetic, lidocaine, for the treatment of knee osteoarthritis (KOA) in mice. It is demonstrated that lidocaine-laden nd-Acu can effectively alleviate pain, reduce inflammation, and slow down KOA development biochemically and histologically. Hypothesis-driven and proteomic approaches are utilized to investigate the working mechanisms of lidocaine nd-Acu, indicating that the therapeutic outcome is attributed to the in vivo modulation of the HMGB1/TLR4 signaling pathway. The study also obtained preliminary evidence suggesting the involvement of mitochondria as well as small GTPase such as cdc42 during the treatment by lidocaine nd-Acu.

A Totipotent "All-In-One" Peptide Sequentially Blocks Immune Checkpoint and Reverses the Immunosuppressive Tumor Microenvironment.

Immune checkpoint blockade combined with reversal of the immunosuppressive tumor microenvironment (TME) can dramatically enhance anti-tumor immunity, which can be achieved by using multiple-agent therapy. However, the optimal dose and order of administration of different agents remain elusive. To address this dilemma, multiple agents are often grafted together to construct "all-in-one" totipotent drugs, but this usually comes at the cost of a lack of synergy between the agents. Herein, by comprehensively analyzing the conserved sites of the immune checkpoint and TME drug targets, peptide secondary structures, assembly properties, and other physicochemical properties, a high-content peptide library is designed. By using the "3D-molecular-evolution" screening strategy, an efficient and totipotent "all-in-one" peptide (TAP) is obtained, which possesses the abilities of self-assembling, blocking the PD-1/PD-L1 axis, inhibiting Rbm38-eIF4E complex formation, and activating p53. It is shown that in mice treated with TAP, with either subcutaneous tumors or patient-derived xenografts, PD-L1 is blocked, with increased activation of both T and NK cells whilst reversing the immunosuppressive TME. Moreover, TAP can mitigate tumor activity and suppress tumor growth, showing superior therapeutic effect over antibody-based drugs.

Preparation and MRI performance of a composite contrast agent based on palygorskite pores and channels binding effect to prolong the residence time of water molecules on gadolinium ions.

Herein, gadolinium tannate was simply and conveniently coated on the surface of palygorskite by in situ reaction of a coordination polymer formed between tannic acid and Gd3+. The palygorskite-tannate gadolinium-polyvinyl alcohol integrated composite (PAL@Gd@PVA) is successfully prepared after the introduction of polyvinyl alcohol onto the palygorskite-tannate gadolinium. The structure is characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray diffraction spectroscopy, X-ray photoelectron spectroscopy, and transmission electron microscopy analysis. The results show that TA-Gd and PVA are successfully loaded on the surface of palygorskite, and the rod crystal structure of palygorskite in the composite remains intact. Palygorskite fibres constitute the framework of the composite and play a key role in supporting and crosslinking the composite. The prepared compounds showed negligible cytotoxicity and low haemolysis rate, showing good biocompatibility. In vitro MRI results showed that the longitudinal and transverse relaxation rates of the composite are 59.56 and 340.81 mm-1 s-1, respectively.

Tannin-Mn coordination polymer coated carbon quantum dots nanocomposite for fluorescence and magnetic resonance bimodal imaging.

The MR/FI bimodal imaging has attracted widely studied due to combining the advantages of MRI and FI can bridge gaps in sensitivity and depth between these two modalities. Herein, a novel MR/FI bimodal imaging probe is facile fabricated by coating the Mn-phenolic coordination polymer on the surface of the carbon quantum dots. The structure of the as-prepared nanocomposite probe is carefully validated via SEM, TEM, and XPS. The content of Mn2+ is calculated through the EDS and TGA. The quantum yield (QY) and emission wavelength of the probe are about 7.24% and 490 nm, respectively. The longitudinal r1 value (2.43 mM-1 s-1) with low r2/r1 (4.45) of the probe is obtained. Subsequently, fluorescence and MR imaging are performed. The metabolic pathways in vivo are inferred by studying the bio-distribution of the probe in major organs. Thus, these results indicate that probe would be an excellent dual-modal imaging probe for enhanced MR imaging and fluorescence imaging. MR/FI bimodal imaging probe is built via in-situ coated Mn-phenolic coordination polymer on the surface of the carbon quantum dots. The in vitro and vivo image property of the probe is evaluated.

Gold nanoparticles enhance antibody effect through direct cancer cell cytotoxicity by differential regulation of phagocytosis.

Ramucirumab is the first FDA-approved monotherapy for advanced gastric cancer. In this study, Ramucirumab (Ab) is attached to gold nanoparticles to enhance uptake efficiency. Gold nanoparticles can induce direct cytotoxic effects to cancer cells in the presence of Ab, while individual Ab or gold nanoparticles don't have such an effective anticancer effect even at extremely high concentrations. Proteomic and transcriptomic analyses reveal this direct cytotoxicity is derived predominantly from Ab-mediated phagocytosis. High affinity immunoglobulin gamma Fc receptor I shows differential up-regulation in gastric cancer cells treated by these nanodrugs compared with Ab, especially for Ab with gold nanorods. Simplified and powerful designs of smart nanoparticles are highly desired for clinical application. The enhancement of Ab accumulation with a simple composition, combined with direct cytotoxic effects specific to cancer cells brought improved therapeutic effects in vivo compared with Ab, which can promote further clinical application of gold nanomaterials in the diagnosis and therapeutics of gastric cancer.

Red Fluorescent Emissive Gd-Phenolic Nanoparticles for In Vivo Fluorescence and Magnetic Resonance Bimodal Imaging.

Due to the combination of the high resolution of fluorescence imaging and the no limitation in penetration depth of magnetic resonance imaging, dual-mode imaging of magnetic resonance and fluorescence (MR/FI) have attracted extensive research in recent years. Herein, a novel MR/FI bimodal imaging probe is facile fabricated by attaching the rhodamine fluorophore covalently to the surface of the Gd-phenolic coordination polymer nanoparticles. The contents of Gd3+ and RB of the as prepared probe are calculated to be 8.2% and 12.5%. The quantum yield of the probe is about 8.84% as well as red fluorescent emissive. The longitudinal r1 value is 6.94 mM-1 s-1 and the ratio r2/r1 is very low and about 1.22. Subsequently, the and MR imaging and fluorescence both in vitro and In Vivo are performed. The metabolic pathways In Vivo are inferred by studying the bio-distribution of the probe in major organs. The as-prepared probe exhibits excellent imaging performance and biocompatibility, which is conducive to its further application.

Ultrasensitive Gastric Cancer Circulating Tumor Cellular CLDN18.2 RNA Detection Based on a Molecular Beacon.

Gastric cancer (GC) is a major global cancer burden, and only HER2-targeted therapies have been approved in first line clinical therapy. CLDN18.2 has been regarded as a potential therapeutic target for gastrointestinal tumors, and global clinical trials have been in process. Hence, the precise, efficient, and noninvasive detection of CLDN18.2 expression is important for the effective application of this attractive target. A high similarity of protein sequence between CLDN18.1 and -18.2 made RNA become more suitable for the detection of CLDN18.2 expression. In this study, CLDN18.2 molecular beacon (MB) with a stem-loop hairpin structure was optimized by phosphorothioate and 2'-O-methyl for stability and efficiency. The MB could recognize CLDN18.2 RNA rapidly. Its resolution and selectivity has been verified in several model cells, demonstrating that MB can distinguish CLDN18.2 expression in several model cells. Furthermore, it was applied successfully to the circulating tumor cell (CTC) assay. The concordance in the expression of CLDN18.2 between CTCs and tissue biopsy is 100% (negative: 3 vs 3; positive: 7 vs 7), indicating that CLDN18.2 RNA detection in CTCs based on a MB will be a promising approach for searching potential patients to CLDN 18.2 targeted drug.

Analysis of salivary exosomal proteins in young adults with severe periodontitis.

OBJECTIVES: Salivary exosomes harbour numerous constituents associated with oral and systemic diseases. However, no reports addressed components of salivary exosomes in patients with periodontitis. Our study aims to explore salivary exosomal proteins in young adults with severe periodontitis (SP) and to analyse the relationships between different proteins. MATERIALS AND METHODS: We collected saliva from 11 young adults with SP and 11 periodontally healthy subjects. After isolation of salivary exosomes, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyse proteins. Gene ontology analysis was performed based on GeneCodis, and interaction network analysis for unique salivary exosomal proteins was performed by STRING. RESULTS: Twenty-six proteins were identified only in the SP group, and 58 proteins were identified only in the healthy group. Gene ontology analysis revealed that innate immune response, cytolysis and complement activation were highly enriched in the SP group. Interaction network analysis showed that the correlations among immune-related proteins (e.g. complement components and chemokine (C-C motif) ligand 28) were significant in the SP group. C6 proteins expressed only in the SP group were evaluated by Western blotting. CONCLUSIONS: Salivary exosomes from periodontitis patients are enriched immune-related proteins that might participate in the immune response during the development of periodontitis.

A Novel Peptide Probe for Identification of PLS3-Expressed Cancer Cells.

The T-plastin (PLS3) has a significant implication in epithelial-mesenchymal transition (EMT) and breast cancer prognosis. Using one-bead-one-compound library strategy, a novel peptide TP1 (KVKSDRVC) toward PLS3 was screened and exhibited the specificity for identifying PLS3-expressed cancer cells. Moreover, we found Fluorescein isothiocyanate-labeled TP1 (FITC-TP1) could act as a novel probe for EMT-induced cancer cells, preferentially in the leading edge. It also has satisfactory specificity for PLS3-expressed cancer cells spiked in the blood. FITC-TP1 was expected to become a diagnostic tool to identify PLS3-expressed circulating tumor cells and predict prognosis for patients with breast cancer in the future.

Plastin 3 down-regulation augments the sensitivity of MDA-MB-231 cells to paclitaxel via the p38 MAPK signalling pathway.

Plastin 3 (PLS3) overexpression may serve as a marker for predicting chemotherapeutic outcomes in drug-resistant cancer cells, but the mechanism is unclear. Herein, we show that the down-regulation of PLS3 by PLS3 gene silencing augments the sensitivity of MDA-MB-231 triple-negative breast cancer cells to paclitaxel. Interestingly, a low concentration of paclitaxel was able to induce strong apoptosis in the PLS3-silenced cells. Further study revealed that p38 MAPK signalling was responsible for the increased sensitivity to paclitaxel in these cells, as the p38 MAPK inhibitor SB203580 impaired the changes mediated by PLS3 down-regulation in response to paclitaxel. Therefore, our study identifies PLS3 as a potential target for enhancing the p38 MAPK-mediated apoptosis induced by paclitaxel. Unlike paclitaxel, Abraxane was unable to induce strong apoptosis in the PLS3-silenced cells. As PLS3 was found to be involved in the process of endocytosis in breast cancer cells, the reliance of cellular Abraxane uptake on this process may render it not as efficient as paclitaxel in PLS3-depleted tumour cells. The finding that PLS3 could be a critical regulator of paclitaxel sensitivity may have important implications for breast cancer chemotherapy.

Quantitative proteomic analysis to the first commercialized liposomal paclitaxel nano-platform Lipusu revealed the molecular mechanism of the enhanced anti-tumor effect.

The first nano-platform commercialized as a drug delivery system was a liposomal formulation. The application of liposome technology resolved the issues of paclitaxel (PTX) insolubility and eliminated the use of solvents causing toxic side-effects, which enabled to apply higher drug doses leading to an enhanced drug efficacy. The growth-inhibitory activity of liposome-encapsulated PTX was retained in vitro against a variety of tumor cell. To investigate the drug efficacy in the system biological level, quantitative proteomic analysis was employed to study the molecular mechanism of the anti-tumor effect of Lipusu® (lip) compared with PTX on lung cancer cell A549. The functions of the differential expressed proteins were correlated to the negative effect to cell proliferation due to regulation of hippo pathway and prolonged cell cycle, as well as inhibitory cell exocytosis, which would cause the aggregation of free PTX. This investigation focused on the direct biological effect of lip to cancer cells. It was different from pharmaceutical issues about drug exposure, delivery and distribution which were widely investigated in other traditional studies. It was the first study about the drug effect of lip from the global molecular biological aspect.

Quantitative Proteomic Analysis of Cell Responses to Electroporation, a Classical Gene Delivery Approach.

Electroporation, as an established nonviral technology for breaching cell membrane, has been accepted for the delivery of nucleic acids. Despite satisfactory delivery efficiencies have been achieved on multiple cell kinds by simply exhausting all possible electrical parameters, electroporation is still inefficient, or even invalid, for various kinds of cells. This is largely due to the lack of comprehensive understanding of cell responses to electrical stimulation at biological aspect. Moreover, a systematically investigation of protein variation of electroporated cells is also required for biosafety evaluation before clinically applying electroporation. By employing quantitative proteomic analysis, the biological mechanism of electroporation is explored from the molecular level. The results reveal that electrical stimulations widely influence many biological processes including nucleic acid stabilization, protein synthesis, cytoskeleton dynamic, inflammation, and cell apoptosis. It is found that several antivirus-related processes appeared in the enrichment results. Moreover, SAMD9, a broad spectrum antiviral and antitumor factor, is dramatically downregulated on easy-to-transfect cells while electroporation can not alter SAMD9 expression on hard-to-transfect cells, hinting that electroporation, a pure physical treatment, can induce antivirus-like defensive responses and the altering of SAMD9 can be used to predict the effectiveness of electroporation on transfecting specific kinds of cells.

Device To Study the Cell Invasion Behavior and Phenotypic Profile at Single Cell Level.

Multiple methods for investigating cell invasion behavior in vitro have proven useful in exploring the mechanisms behind the epithelial-mesenchymal transition (EMT) and EMT-related tumor cell invasion, for example, by revealing that cell heterogeneity existed in EMT. However, several hypotheses and predictions regarding EMT heterogeneity have remained unproven because of the inability to quantitatively profile cell invasion at the single cell level. Here, we present a microfluidic chip that provides the capability of simultaneously investigating single cell invasion behavior, phenotypic diversity, and responsiveness to anti-invasion drugs. By assessing single cell invasion behavior in separate wells, cell-cell contacts and their corresponding interference in the invasion process could be excluded. The chip allowed for both precise quantitation of cell invasion and in situ phenotyping, such that any single cell heterogeneity could be detected and accurately quantified. This study has proven that the proposed hybrid epithelial/mesenchymal cell phenotype exists and is important in the EMT process. The invasion abilities of two cell lines were also assessed, either with or without EMT-promoting or EMT-inhibiting agents, proving that the chip can also be used to assess the effectiveness of antimetastatic agents. This study has demonstrated that the strategy of isolating single cells before studying their invasive properties is correct and that it provides an in vitro method for understanding cell heterogeneity during EMT. This approach also provides a mean of screening for anti-invasion agents that are focused on single cell invasion, a process known to be important for blood-borne metastasis to occur.

Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1).

Abraxane (Abr), a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX). To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1), showed significant differential expression (P<0.05) in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes.

Response of Human Osteoblast to n-HA/PEEK--Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite.

Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn't increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca(2+) concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.

Quantitative Proteomic Analysis of Cellular Resistance to the Nanoparticle Abraxane.

Abraxane, an FDA-approved albumin-bound nanoparticle (NP) form of paclitaxel (PTX) to treat breast cancer and nonsmall cell lung cancer (NSCLC), has been demonstrated to be more effective than the original Taxol, the single molecule form. We have established a cell line from NSCLC A549 cells to be resistant to Abraxane. To further understand the molecular mechanisms involved in the NP drug resistance, global protein expression profiles of Abraxane sensitive (A549) and resistant cells (A549/Abr), along with the treatment of Abraxane, have been obtained by a quantitative proteomic approach. The most significantly differentially expressed proteins are associated with lipid metabolism, cell cycle, cytoskeleton, apoptosis pathways and processes, suggesting several mechanisms are working synergistically in A549 Abraxane-resistant cells. Overexpression of proteins in the lipid metabolism processes, such as E3 ubiquitin-protein ligase RNF139 (RNF139) and Hydroxymethylglutaryl-CoA synthase (HMGCS1), have not been reported previously in the study of paclitaxel resistance, suggesting possibly different mechanism between nanoparticle and single molecular drug resistance. In particular, RNF139 is one of the most up-regulated proteins in A549 Abraxane-resistant cell line, but remains no change when the resistant cells were further treated with Abraxane and down-regulated in the sensitive cells after 4 h treatment of Abraxane. This study shows the use of a proteomic strategy to understand the unique response of drug resistant cells to a nanoparticle therapeutic.

Abraxane, the Nanoparticle Formulation of Paclitaxel Can Induce Drug Resistance by Up-Regulation of P-gp.

P-glycoprotein (P-gp) can actively pump paclitaxel (PTX) out of cells and induces drug resistance. Abraxane, a nanoparticle (NP) formulation of PTX, has multiple clinical advantages over the single molecule form. However, it is still unclear whether Abraxane overcomes the common small molecule drug resistance problem mediated by P-gp. Here we were able to establish an Abraxane-resistant cell line from the lung adenocarcinoma cell line A549. We compared the transcriptome of A549/Abr resistant cell line to that of its parental cell line using RNA-Seq technology. Several pathways were found to be up or down regulated. Specifically, the most significantly up-regulated gene was ABCB1, which translates into P-glycoprotein. We verified the overexpression of P-glycoprotein and confirmed its function by reversing the drug resistance with P-gp inhibitor Verapamil. The results suggest that efflux pathway plays an important role in the Abraxane-resistant cell line we established. However, the relevance of this P-gp mediated Abraxane resistance in tumors of lung cancer patients remains unknown.

Secretome analyses of Aß(1-42) stimulated hippocampal astrocytes reveal that CXCL10 is involved in astrocyte migration.

Amyloid-beta (Aß) aggregation plays an important role in the development of Alzheimer's disease (AD). In the AD brain, amyloid plaques are surrounded by reactive astrocytes, and many essential functions of astrocytes have been reported to be mediated by protein secretion. However, the roles of activated astrocytes in AD progression are under intense debate. To provide an in-depth view of the secretomes of activated astrocytes, we present in this study a quantitative profile of rat hippocampal astrocyte secretomes at multiple time points after both brief and sustained Aß(1-42) stimulation. Using SILAC labeling and LC-MS/MS analyses, we identified 19 up-regulated secreted proteins after Aß(1-42) treatment. These differentially expressed proteins have been suggested to be involved in key aspects of biological processes, such as cell recruitment, Aß clearance, and regulation of neurogenesis. Particularly, we validated the role played by CXCL10 in promoting astrocyte aggregation around amyloid plagues through in vitro cell migration analysis. This research provides global, quantitative profiling of astrocyte secretomes produced on Aß stimulation and hence provides a detailed molecular basis for the relationship between amyloid plaques and astrocyte aggregation; the findings thus have important implications for further investigations into AD development and therapy.

In vitro bioactivity and biocompatibility evaluation of bulk nanostructured titanium in osteoblast-like cells by quantitative proteomic analysis.

Nanostructured titanium prepared by the equal-channel angular pressing route (ECAPed Ti) has shown great promise as an implant material over conventional pure titanium. The aim of this report is to investigate its biological properties, surface performance, and comprehensive biological effects at a molecular level when in contact with cells. Protein expression changes of human osteoblast-like MG-63 in response to polished ECAPed Ti had been profiled by employing stable isotope labelling with amino acids in cell culture (SILAC), using cpTi as control after the same polishing process. It was found that ubiquitin proteasome related processes were predominantly enriched in the over-expressed proteins. Superoxide dismutase 2 (SOD2) was apparently up-regulated on the ECAPed Ti surface, which could have contributed to the increase in SOD activity and the decrease in the reactive oxygen species (ROS) level. These expression changes have relationships with protein degradation, bone formation and resistance to oxidative injury, and they suggest that ECAPed Ti has the potential to further promote osteoblast differentiation. On the other hand, the down-regulated proteins exhibited resistance to platelet adhesion on the ECAPed Ti surface. This study reveals the differential expression of proteins in human osteoblasts induced by nanostructured titanium substrates for the first time.