A novel p55PIK signaling peptide inhibitor alleviates neuroinflammation via the STAT3/NF-kB signaling pathway in experimental stroke.

BACKGROUND: Ischemic stroke remains the predominant contributor to mortality and disability globally. Microglia undergo rapid activation and initiate inflammatory cascade reactions by phenotypic polarization, participating in the regulation of inflammatory injury and tissue repair post-ischemic stroke. Regulating microglia-mediated neuroinflammation is a promising therapeutic strategy for ischemic stroke. Previously, we designed and synthesized a novel p55PIK inhibitor, TAT-N15 polypeptide, which presents inhibitive activity on NF-κB signaling-mediated inflammation in acute conjunctivitis and allergic rhinitis. The present study aimed to explore the therapeutic effect and mechanism of TAT-N15 on ischemia stroke. METHODS: The mouse model of transient cerebral ischemia was made using the intraluminal filament method. After being treated with daily intraperitoneal injections of TAT-N15 (10 mg/kg) for 7 d, the neurological outcomes and the cerebral infarction volume were evaluated. Histopathology of the ischemia cerebral hemisphere was observed by H&E and Nissl staining. Neuronal survival, astrogliosis, and co-labeling of CD86/Iba1 and CD206/Iba1 were detected by immunofluorescence. The cell apoptosis was estimated by TUNEL staining. The expression levels of apoptosis-associated proteins, proinflammatory cytokines, protein markers of M1 and M2 microglia, and the phosphorylation of NF-κB and STAT3 proteins in the ischemic penumbra were detected by Western blot. RESULTS: TAT-N15 treatment significantly decreased the infarct volume and alleviated neurological functional impairment, neuronal injury, and neuron apoptosis. Meanwhile, TAT-N15 treatment restrained the activation of microglia and astrocytes as well as the protein expression of proinflammatory cytokine in ischemic penumbra. Additionally, the administration of TAT-N15 treatment resulted in a significant reduction in the density of M1 phenotype microglia while concurrently increasing the density of M2 phenotype microglia within the ischemic penumbra. Finally, mechanical analysis unveiled that TAT-N15 exerted a substantial inhibitory effect on the protein expression of phosphorylated STAT3 and NF-κB. CONCLUSION: TAT-N15 may inhibit neuroinflammation via regulating microglia activation and polarization through the STAT3/NF-κB pathway, which exhibits the neuroprotection effect in ischemic stroke.

Chiral aldehyde-nickel dual catalysis enables asymmetric α-propargylation of amino acids and stereodivergent synthesis of NP25302.

The combined catalytic systems derived from organocatalysts and transition metals exhibit powerful activation and stereoselective-control abilities in asymmetric catalysis. This work describes a highly efficient chiral aldehyde-nickel dual catalytic system and its application for the direct asymmetric α-propargylation reaction of amino acid esters with propargylic alcohol derivatives. Various structural diversity α,α-disubstituted non-proteinogenic α-amino acid esters are produced in good-to-excellent yields and enantioselectivities. Furthermore, a stereodivergent synthesis of natural product NP25302 is achieved, and a reasonable reaction mechanism is proposed to illustrate the observed stereoselectivity based on the results of control experiments, nonlinear effect investigation, and HRMS detection.

P55PIK Regulates P53-Dependent Apoptosis in Cancer Cells by Interacting with P53 DNA-Specific Domain.

PURPOSE: Phosphatidylinositol 3-kinase (PI3K) plays an important role in tumorigenesis by cross-talking with several signaling pathways. p55PIK is a unique regulatory subunit of PI3K and contains an extra 24-residue N-terminal domain (N24). This study aimed to explore the interaction of p55PIK with p53 and the role of p55PIK in regulating p53-dependent apoptosis in cancer cells. MATERIALS AND METHODS: The expression of p55PIK was detected in cancer cells, and the interaction of p55PIK with p53 was examined by immunoprecipitation and pull-down assay. The expression of p53-dependent apoptosis-related genes was detected by PCR. RESULTS: N24 domain of p55PIK interacted with DNA-specific binding domain (DBD) of p53. The increase or decrease of p55PIK expression led to the change of the expression of p53 and p53-regulated genes in cancer cells. Moreover, N24 peptide led to the change of the expression of p53-regulated genes. Moreover, a membrane-permeable N24 peptide enhanced p53-dependent apoptosis induced by methyl methanesulfonate. CONCLUSION: Our results reveal a novel mechanism that regulates p53-dependent apoptosis in cancer cells via p55PIK-p53 interaction.

In situ real-time tracing of hierarchical targeting nanostructures in drug resistant tumors using diffuse fluorescence tomography.

Nanoparticles that respond to specific endogenous or exogenous stimuli in tumor tissues are actively being developed to address multidrug resistance owing to multiple advantages, including a prolonged circulation time, enhanced permeability and retention effect, and superior cellular uptake. Although some exciting results have been obtained, existing nanoparticles have limited routes to overcome the drug resistance of tumor cells; this limitation results in a failure to ablate resistant tumors via intravenous administration. To resolve this dilemma, we developed a smart theranostic nanoplatform with programmable particle size, activatable target ligands and in vivo multimodal imaging. This nanoplatform, which includes stealth zwitterionic coating, was shown to be quickly trapped in tumor tissue from the blood circulation within 5 min. Subsequently, the targeting moieties were activated in response to the acidic tumor microenvironment by triggering the zwitterionic shell detachment, driving the peeled nanoparticles to penetrate into tumor cells. These smart nanoparticles completely inhibited drug-resistant tumor growth and did not cause any damage to normal organ tissues in live animals. The designed nanoplatforms simultaneously acted as a nanoprobe for fluorescence imaging. Moreover, we also used noninvasive pharmacokinetic diffuse fluorescence tomography (DFT) to dynamically monitor and in situ real-time trace the nanoplatforms' behavior throughout the entire tumor in live animals. The nanoplatforms enabled rapid drug accumulation and deep penetration throughout the entire tumor. The rate of drug accumulation after the administration of nanoplatforms was five-fold higher compared with that after the administration of the free drug, which resulted in increased drug delivery efficiency and improved antitumor efficacy. Collectively, this hierarchical vehicle design provides promising insights for the development of theragnosis for multidrug resistant tumors.

Pathways of topological rank analysis (PoTRA): a novel method to detect pathways involved in hepatocellular carcinoma.

Complex diseases such as cancer are usually the result of a combination of environmental factors and one or several biological pathways consisting of sets of genes. Each biological pathway exerts its function by delivering signaling through the gene network. Theoretically, a pathway is supposed to have a robust topological structure under normal physiological conditions. However, the pathway's topological structure could be altered under some pathological condition. It is well known that a normal biological network includes a small number of well-connected hub nodes and a large number of nodes that are non-hubs. In addition, it is reported that the loss of connectivity is a common topological trait of cancer networks, which is an assumption of our method. Hence, from normal to cancer, the process of the network losing connectivity might be the process of disrupting the structure of the network, namely, the number of hub genes might be altered in cancer compared to that in normal or the distribution of topological ranks of genes might be altered. Based on this, we propose a new PageRank-based method called Pathways of Topological Rank Analysis (PoTRA) to detect pathways involved in cancer. We use PageRank to measure the relative topological ranks of genes in each biological pathway, then select hub genes for each pathway, and use Fisher's exact test to test if the number of hub genes in each pathway is altered from normal to cancer. Alternatively, if the distribution of topological ranks of gene in a pathway is altered between normal and cancer, this pathway might also be involved in cancer. Hence, we use the Kolmogorov-Smirnov test to detect pathways that have an altered distribution of topological ranks of genes between two phenotypes. We apply PoTRA to study hepatocellular carcinoma (HCC) and several subtypes of HCC. Very interestingly, we discover that all significant pathways in HCC are cancer-associated generally, while several significant pathways in subtypes of HCC are HCC subtype-associated specifically. In conclusion, PoTRA is a new approach to explore and discover pathways involved in cancer. PoTRA can be used as a complement to other existing methods to broaden our understanding of the biological mechanisms behind cancer at the system-level.

miR2Pathway: A novel analytical method to discover MicroRNA-mediated dysregulated pathways involved in hepatocellular carcinoma.

MicroRNAs (miRNAs) are small, non-coding RNAs involved in the regulation of gene expression at a post-transcriptional level. Recent studies have shown miRNAs as key regulators of a variety of biological processes, such as proliferation, differentiation, apoptosis, metabolism, etc. Aberrantly expressed miRNAs influence individual gene expression level, but rewired miRNA-mRNA connections can influence the activity of biological pathways. Here, we define rewired miRNA-mRNA connections as the differential (rewiring) effects on the activity of biological pathways between hepatocellular carcinoma (HCC) and normal phenotypes. Our work presented here uses a PageRank-based approach to measure the degree of miRNA-mediated dysregulation of biological pathways between HCC and normal samples based on rewired miRNA-mRNA connections. In our study, we regard the degree of miRNA-mediated dysregulation of biological pathways as disease risk of biological pathways. Therefore, we propose a new method, miR2Pathway, to measure and rank the degree of miRNA-mediated dysregulation of biological pathways by measuring the total differential influence of miRNAs on the activity of pathways between HCC and normal states. miR2Pathway proposed here systematically shows the first evidence for a mechanism of biological pathways being dysregulated by rewired miRNA-mRNA connections, and provides new insight into exploring mechanisms behind HCC. Thus, miR2Pathway is a novel method to identify and rank miRNA-dysregulated pathways in HCC.

Hierarchical design of a polymeric nanovehicle for efficient tumor regression and imaging.

Effective delivery of therapeutics to disease sites significantly contributes to drug efficacy, toxicity and clearance. Here we designed a hierarchical polymeric nanoparticle structure for anti-cancer chemotherapy delivery by utilizing state-of-the-art polymer chemistry and co-assembly techniques. This novel structural design combines the most desired merits for drug delivery in a single particle, including a long in vivo circulation time, inhibited non-specific cell uptake, enhanced tumor cell internalization, pH-controlled drug release and simultaneous imaging. This co-assembled nanoparticle showed exceptional stability in complex biological media. Benefiting from the synergistic effects of zwitterionic and multivalent galactose polymers, drug-loaded nanoparticles were selectively internalized by cancer cells rather than normal tissue cells. In addition, the pH-responsive core retained their cargo within their polymeric coating through hydrophobic interaction and released it under slightly acidic conditions. In vivo pharmacokinetic studies in mice showed minimal uptake of nanoparticles by the mononuclear phagocyte system and excellent blood circulation half-lives of 14.4 h. As a result, tumor growth was completely inhibited and no damage was observed for normal organ tissues. This newly developed drug nanovehicle has great potential in cancer therapy, and the hierarchical design principle should provide valuable information for the development of the next generation of drug delivery systems.

An Acid-Triggered Degradable and Fluorescent Nanoscale Drug Delivery System with Enhanced Cytotoxicity to Cancer Cells.

To reduce side-effects of anticancer drugs, development of nanocarriers with precise biological functions is a critical requirement. In this study, the multifunctional nanoparticles combining imaging and therapy for tumor-targeted delivery of hydrophobic anticancer drugs were prepared via self-assembly of amphiphilic copolymers obtained using RAFT polymerization, specifically, acid-labile ortho ester and galactose. First, boron-dipyrromethene dye-conjugated chain transfer agent provides fluorescent imaging capability for diagnostic application. Second, nanoparticles were stable under physiological conditions but degraded in acidic tumor microenvironment, leading to enhanced anticancer efficacy. Third, the application of biocompatible glycopolymers efficiently increased the target-to-background ratio through carbohydrate-protein interactions. Data from cell viability, cellular internalization, flow cytometry, biodistribution and anticancer efficacy tests showed that the drug-loaded nanoparticles were capable of inhibiting cancer cell proliferation with significantly enhanced capacity. Our newly developed multifunctional nanoparticles may thus facilitate the development of effective drug delivery systems for application in diagnosis and therapy of cancer.

A pH gated, glucose-sensitive nanoparticle based on worm-like mesoporous silica for controlled insulin release.

This study prepares a kind of core-shell hybrid nanoparticles, which is worm-like, pH gated, and glucose-sensitive. It has a mesoporous silica nanoparticle (MSN) core and polymer shell (cross-linked and non-cross-linked), bearing 3-acrylamidophenylboronic acid (AAPBA) and N-isopropylacrylamide (NIPAM) as sensor moieties. The shell of the nanoparticles has presented a distinct transition from swollen state to collapsed state as the temperature increases, which offers easy access to drug loading. Here, insulin is applied as a model drug and the behaviors of its loading/release are investigated. Insulin loading is up to 15% via mesoporous silica core. In vitro experiment shows that the cumulative release of insulin is dependent on glucose concentration, and the glucose sensitivity could be adjusted simply by different pH values. Simultaneously, compared with the non-cross-linked shell, the cross-linked shell, using dextran-maleic acid (Dex-Ma) as a macromolecule cross-link, enables insulin to release more persistently. Also, cell viability assay indicates that these nanoparticles have good biocompatibility. Consequently, the novel, pH gated, glucose-sensitive core-shell nanoparticles may have potential applications as a vehicle of self-regulated insulin delivery system.

Hydrotropic polymeric mixed micelles based on functional hyperbranched polyglycerol copolymers as hepatoma-targeting drug delivery system.

Mixed copolymer nanoparticles (NPs) self-assembled from ß-cyclodextrin-grafted hyperbranched polyglycerol (HPG-g-CD) and lactobionic acid (LA)-grafted hyperbranched polyglycerol (HPG-g-LA) were applied as carriers for a hydrophobic antitumor drug, paclitaxel (PTX), achieving hepatocellular carcinoma-targeted delivery. The resulting NPs exhibited high drug loading capacity and substantial stability in aqueous solution. In vitro drug release studies demonstrated a controlled drug release profile with increased release at acidic pH. Remarkably, tumor proliferation assays showed that PTX-loaded mixed copolymer NPs inhibited asialoglycoprotein (ASGP) receptor positive HepG2 cell proliferation in a concentration-dependent manner in comparison with ASGP receptor negative BGC-823 cells. Moreover, the competition assay demonstrated that the small molecular LA inhibited the cellular uptake of the PTX-loaded mixed copolymer NPs, indicating the ASGP receptor-mediated endocytosis in HepG2 cells. In addition, the intracellular uptake tests by confocal laser scanning microscopy showed that the mixed copolymer NPs were more efficiently taken up by HepG2 cells compared with HPG-g-CD NPs. These results suggest a feasible application of the mixed copolymer NPs as nanocarriers for hepatoma-targeted delivery of potent antitumor drugs.

Glycopolymer micelles with reducible ionic cores for hepatocytes-targeting delivery of DOX.

A novel galactose-decorated cross-linked micelles (cl-micelles) with ionic cores using cystamine (Cys) as a biodegradable cross-linker was prepared by using block ionomer complexes of poly(ethylene glycol)-b-poly(2-acryloxyethyl-galactose)-b-poly(acrylic acid) (PEG-b-PAEG-b-PAA) and Ca(2+) (PEG-b-PAEG-b-PAA cl-micelles/Cys). Doxorubicin (DOX) was successfully incorporated into the ionic cores of such micelles via electrostatic interactions. Proton nuclear magnetic resonance spectrum and Fourier transform infrared spectrometer indicated galactose ligands were exposed at the micellar surface. The micelles were spherical in shape, with an average size of 100nm. The in vitro release studies confirmed that DOX-loaded PEG-b-PAEG-b-PAA cl-micelles/Cys accomplished rapid drug release under reducing condition. Remarkably, PEG-b-PAEG-b-PAA cl-micelles/Cys efficiently delivered and released DOX into the cell nucleus of HepG2 cells, and the intensity of fluorescence observed in HepG2 cells was stronger than that incubated with the micelles without galactose ligands. In contrast, little fluorescence was observed in NIH3T3 cells after incubation with PEG-b-PAEG-b-PAA cl-micelles/Cys. Interestingly, cytotoxicity assays showed that DOX-loaded PEG-b-PAEG-b-PAA cl-micelles/Cys retained higher cell inhibition efficiency in HepG2 cells as compared with NIH3T3 cells, and were more potent than the micelles without galactose ligands and the micelles with non degradable cross-links. These results indicate that PEG-b-PAEG-b-PAA cl-micelles/Cys have great potential in liver tumor-targeted chemotherapy.

A hydrotropic ß-cyclodextrin grafted hyperbranched polyglycerol co-polymer for hydrophobic drug delivery.

The development of successful formulations for poorly water soluble drugs remains a longstanding, critical, and challenging issue in cancer therapy. A ß-cyclodextrin (CD) functionalized hyperbranched polyglycerol (HPG) has been prepared as a potential water insoluble drug carrier. The HPG-g-CD molecules could self-assemble into multimolecular spherical micelles in water, the size of which ranged from 200 to 300 nm, with good dispersity. A high loading capacity and high encapsulation efficiency of paclitaxel, as a model, were obtained. The release profiles of different co-polymer compositions showed a burst release followed by continuous extended release. Furthermore, MTT analysis showed that HPG-g-CD had good biocompatibility, indicating that HPG-g-CD may be considered a promising hydrophobic drug delivery system.

Gradient cross-linked biodegradable polyelectrolyte nanocapsules for intracellular protein drug delivery.

Gradient shell cross-linked hollow polyelectrolyte nanocapsules composed of cysteamine conjugated chitosan and dextran sulfate were prepared by layer-by-layer adsorption on beta-cyclodextrin (beta-CD) functionalized silica spheres followed by cross-linking thiols and removal of silica core. This disulfide bond gradient cross-linked nanocapsules combined reduction and pH sensitive. Gradually increased from the inside to the outside of the cross-linking degree, one purpose is to ensure that cross-linking disulfide bond after reduction cleavage still has pH sensitive, on the other hand is to avoid cross-linked contraction of internal damage the crystal and bioactivity of protein drugs. Disulfide cross-linked nanocapsules were used to enhance the physical stability against acidic pH conditions compared to the un-cross-linked ones. Bovine serum albumin, as a model protein drug, was loaded inside nanocapsules. The disulfide bond cross-linked nanocapsules are intended to remain more stable in physiological pH and decrease the loss of protein drugs caused by the gastric cavity, and can release the drugs in the intracellular environment after glutathione reduction.

Polyelectrolyte nanoparticles based on water-soluble chitosan-poly(L-aspartic acid)-polyethylene glycol for controlled protein release.

Water-soluble chitosan (WSC)-poly(L-aspartic acid) (PASP)-polyethylene glycol (PEG) nanoparticles (CPP nanoparticles) were prepared spontaneously under quite mild conditions by polyelectrolyte complexation. These nanoparticles were well dispersed and stable in aqueous solution, and their physicochemical properties were characterized by turbidity, FTIR spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), and zeta potential. PEG was chosen to modify WSC-PASP nanoparticles to make a protein-protective agent. Investigation on the encapsulation efficiency and loading capacity of the bovine serum albumin (BSA)-loaded CPP nanoparticles was also conducted. Encapsulation efficiency was obviously decreased with the increase of initial BSA concentration. Furthermore, its in vitro release characteristics were evaluated at pH 1.2, 2.5, and 7.4. In vitro release showed that these nanoparticles provided an initial burst release, followed by a slowly sustained release for more than 24 h. The BSA released from CPP nanoparticles showed no significant conformational change compared with native BSA, which is superior to the BSA released from nanoparticles without PEG. A cell viability study suggested that the nanoparticles had good biocompatibility. This nanoparticle system was considered promising as an advanced drug delivery system for the peptide and protein drug delivery.

Chitosan-NAC nanoparticles as a vehicle for nasal absorption enhancement of insulin.

The purpose of this work was to investigate chitosan-N-acetyl-L-cysteine (chitosan-NAC) nanoparticles as a potential carrier system for the nasal delivery of insulin. For the study, we used insulin-loaded chitosan-NAC nanoparticles (140-210 nm in diameter) prepared by in situ gelation with tripolyphosphate (TPP), with positive zeta potential values of +19.5-31.7 mV and insulin loading capacities of 13-42%. The physicochemical properties of the nanoparticles were affected by the number of thiol groups present. Mucoadhesive properties, which were evaluated by measuring the in vitro absorbed mass of mucin, of chitosan-NAC nanoparticles were >1.8-fold that of unmodified chitosan nanoparticles. In aqueous solution, chitosan-NAC nanoparticles exhibited fast swelling behavior. Insulin was released from chitosan-NAC nanoparticles in vitro in an initial burst followed by slow release. Intranasal administration of chitosan-NAC nanoparticles in rats enhanced the absorption of insulin by the nasal mucosa compared with unmodified chitosan nanoparticles and control insulin solution. In light of these observations, the novel thiolated chitosan nanoparticles represent a promising vehicle for nasal insulin administration.

Glucosamine-carrying temperature- and pH-sensitive microgels: preparation, characterization, and in vitro drug release studies.

Glucosamine-carrying temperature- and pH-sensitive microgels with an average diameter of about 100 nm were successfully prepared by free radical precipitation polymerization. The thermo- and pH-responsive properties of the microgels were designed by the incorporation of N-isopropylacrylamide (NIPAM) and acrylic acid (AAc) to copolymerize with acrylamido-2-deoxyglucose (AADG). The stimuli sensitivity of the microgels was studied by the measurement of their sizes and volume phase transition temperature (VPTT) under different surrounding conditions. The results showed that the microgels were responsive to temperature, pH, and ionic strength, and could have a desired VPTT by modifying AADG and AAc contents. The effect of temperature and pH on insulin release from the microgels was also investigated. The release of drug at the tumor-surrounding environment is faster than that under normal physiological conditions. A preliminary in vitro cell study showed that the glucosamine-carrying microgels are more biocompatible to mouse fibroblast cells, compared to the microgels without glucosamine. These glucosamine-carrying dual-sensitive microgels may be promising carriers for targeted drug delivery to tumors.