Zhen Huang

A pH/Enzyme-responsive tumor-specific delivery system for doxorubicin

To overcome the cardiotoxicity of doxorubicin, a self-assembled pH/enzyme-responsive system was developed. Cationic gelatin combined polyGC-DOX intercalation tightly to form compact nanoscale complexes (CPX1) which then combined by a pH-sensitive pegylated alginate to form CPX2. CPX2 could be digested and release DOX under the co-digestion of gelatinase (GA) and Dnase I when pH < 6.9. More importantly, tumor homogenate supernatant (THS) could effectively release DOX from CPX2 while the plasma and liver homogenate supernatant (LHS) could not, which was confirmed by an in vivo test. The results indicated that this formulation had the tumor-specific drug-release effect. This effect resulted in an increased drug concentration in tumor tissue and decreased content in heart and liver. The changed bio-distribution of DOX delivered by CPX2 greatly enhanced the anti-cancer activity and reduced the cardiotoxicity of the drug. The anti-cancer efficiency of DOX delivered by CPX2 is more than 2 times of the free doxorubicin, and the mortality caused by the high-dose DOX was completely prevented by CPX2. All results suggested that this easy-manufactured, cost-effective nanocomplex holds great promise to be developed into a formulation of doxorubicin and the other anthracyclines with high anti-cancer activity and low cardiotoxicity.

miR-141 Regulates colonic leukocytic trafficking by targeting CXCL12β during murine colitis and human Crohn's disease

Objective Emerging evidence suggests that microRNA (miRNA)-mediated gene regulation influences a variety of autoimmune disease processes, including Crohns disease (CD), but the biological function of miRNAs in CD remains unclear. We examine miRNA level in colon tissues and study the potential functions of miRNAs that regulate pathological genes during the inflammation process. Design miRNA levels were assayed in the inflamed colon of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced and IL-10 knockout (KO) chronic colitis mice and CD patients by microarray or qRT-PCR. The influence of differently expressed miR-141 on its putative target genes, CXCL12β, and leukocyte migration was investigated in colonic epithelia cells, colitis models and CD patients. The role of miR-141 was further studied in the experimental colitis mice by intracolonic administration of miR-141 precursors or inhibitors. Results An inverse correlation between miR-141 and CXCL12β/total-CXCL12 was observed predominantly in the epithelial cells of the inflamed colons from colitic mice and CD patients. Further study demonstrated that miR-141 directly regulated CXCL12β expression and CXCL12β-mediated leukocyte migration. Upregulation or downregulation of miR-141 in the TNBS-induced or IL-10 KO colitic colon regulated leukocyte infiltration and alleviated or aggravated experimental colitis, respectively. Additionally, colonic overexpression of CXCL12β abolished the therapeutic effect of miR-141 in TNBS-induced colitis. Conclusions This study showed that the pathway of miR-141 targeting CXCL12β is a possible mechanism underlying inflammatory cell trafficking during colonic inflammation process. Inhibiting colonic CXCL12β expression and blocking colonic immune cell recruitment by using miRNA precursors represents a promising approach that may be valuable for CD treatment.

Targeted delivery of oligonucleotides into tumor-associated macrophages for cancer immunotherapy

Tumor-associated macrophages (TAMs) have been proven to be a driving force in the initiation, proliferation, metastasis and angiogenesis of various tumors. Specifically, alterations in the functions of TAMs exhibited inhibitory effects on tumor growth. However, there is currently no research being conducted on the targeting delivery of drugs into TAMs for cell-specific tumor immunotherapy. In the present study, we developed a TAMs targeted delivery system that is triggered by the acidic microenvironment in the tumor to release a TAMs-recognizing nano-complex loaded with oligonucleotides. By using this system, we demonstrated a significant anti-tumor effect of an oligonucleotide combination of CpG oligonucleotide, anti-IL-10 and anti-IL-10 receptor oligonucleotides. These nucleic acid drugs delivered by the delivery system accumulated in the TAMs of an allograft hepatoma murine model by intravenous injection, suppressed the pro-tumor functions and stimulated the anti-tumor activities of TAMs. More importantly, the nucleic acid drug-based immune-regulation was restricted to the tumor microenvironment and did not cause an upregulation of serum inflammatory cytokines. Our present study provides an effective therapeutic strategy for regulating cell-specific functions using nucleic acid drugs.

The Autoregulatory Feedback Loop of MicroRNA-21/Programmed Cell Death Protein 4/Activation Protein-1 (MiR-21/PDCD4/AP-1) as a Driving Force for Hepatic Fibrosis Development

Background: MicroRNA-21 is important in hepatic fibrosis development, but the mechanism is unclear. Results: MicroRNA-21 is predominantly up-regulated in activated hepatic stellate cells and could form a double negative feedback loop that links fibrogenic machinery. Conclusion: The microRNA-21-mediated loop is a main driving force for hepatic fibrosis progression. Significance: It suggests a mechanism for how microRNA-21 contributes to hepatic fibrosis. Sustained activation of hepatic stellate cells (HSCs) leads to hepatic fibrosis, which is characterized by excessive collagen production, and for which there is no available drug clinically. Despite tremendous progress, the cellular activities underlying HSC activation, especially the driving force in the perpetuation stage, are only partially understood. Recently, microRNA-21 (miR-21) has been found to be prevalently up-regulated during fibrogenesis in different tissues, although its detailed role needs to be further elucidated. In the present study, miR-21 expression was examined in human cirrhotic liver samples and in murine fibrotic livers induced by thioacetamide or carbon tetrachloride. A dramatic miR-21 increase was noted in activated HSCs. We further found that miR-21 maintained itself at constant high levels by using a microRNA-21/programmed cell death protein 4/activation protein-1 (miR-21/PDCD4/AP-1) feedback loop. Disrupting this loop with miR-21 antagomir or AP-1 inhibitors significantly suppressed fibrogenic activities in HSCs and ameliorated liver fibrosis. In contrast, reinforcing this loop with small interfering RNA (siRNA) against PDCD4 promoted fibrogenesis in HSCs. Further analysis indicated that the up-regulated miR-21 promoted the central transforming growth factor-β (TGF-β) signaling pathway underlying HSC activation. In summary, we suggest that the miR-21/PDCD4/AP-1 autoregulatory loop is one of the main driving forces for hepatic fibrosis progression. Targeting this aberrantly activated feedback loop may provide a new therapeutic strategy and facilitate drug discovery against hepatic fibrosis.

Anti-tumor immune responses of tumor-associated macrophages via toll-like receptor 4 triggered by cationic polymers.

Agonists of toll-like receptors (TLRs) are potential therapeutic reagents for cancer immunotherapy. Cationic polymers such as polyethyleneimine (PEI) with nucleic acid drug delivery capability are approved for use in clinical trials, and recent reports indicate that these cationic polymers have significant immunological activity mediated by TLRs. In the present study, we demonstrated that cationic polymers such as PEI and cationic dextran could reverse tumor-associated macrophages (TAMs) polarization and promote IL-12 expression both inxa0vitro and inxa0vivo. The stimulatory role of cationic polymers was remarkably attenuated in TAMs pre-treated with TLR-4 blocking antibody or TAMs from TLR-4 knockout mice. Additionally, these cationic polymers exerted direct tumoricidal activity by promoting Th 1 and NK cell infiltration, suppressing tumor angiogenesis and prolonging the survival of sarcoma-bearing wild-type. These phenomena were abrogated in TLR-4 knockout mice, suggesting that the immune stimulation was primarily mediated by TLR-4. In conclusion, these results demonstrated that cationic polymers could transform the immunotolerogenic phenotype of TAMs through TLR-4 signaling, thereby promoting therapeutic anti-tumor immunity. Our present study suggests a new class of drugs as a candidate for future cancer immunotherapy.

Targeting delivery of anti-TNFα oligonucleotide into activated colonic macrophages protects against experimental colitis

Background and aims Tumour necrosis factor α (TNFα) is a focal point of the inflammatory cascade in Crohns disease (CD). As an emerging approach to block cytokines, antisense oligonucleotide (ASO) has developed quickly, but is thwarted by a key obstacle—safe and effective delivery to specified cells. Here a novel nano-complex, based on galactosylated low molecular weight chitosan (gal-LMWC) and an ASO against TNFα, is presented which may be effective for CD treatment. The aim of this study was to investigate the targeting delivery ability of the gal-LMWC/ASO complex into activated macrophages and its potential therapeutic action in experimental colitis. Methods Gal-LMWC was associated with ASO to form a stable nano-complex and the complex was injected into mice by intracolonic administration. Cellular localisation of the gal-LMWC/ASO complex in the colon was determined. The therapeutic effects were further studied in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and CD4+CD45RBhi T cell transfer colitis. Results Intracolonic administration of the gal-LMWC/ASO complex resulted in the successful delivery of ASO into activated colonic macrophages and a significant reduction of colonic TNFα in mice with colitis. A single injection in TNBS colitis or repeated treatment in CD45RBhi transfer colitis both significantly ameliorated the clinical and histopathological severity of the wasting disease, reduced tissue levels of inflammatory cytokines and abrogated body weight loss, diarrhoea and intestinal protein loss. Conclusions It is the first time a non-viral gene vector has been combined with an ASO targeted to activated macrophages in the treatment of CD. The inhibition of TNFα by this strategy represents a promising therapeutic approach for the treatment of CD.

The promotion of type 1 T helper cell responses to cationic polymers in vivo via toll-like receptor-4 mediated IL-12 secretion

Cationic polymers with nucleic acid drug delivery ability are widely used in experimental and clinical studies. However, their interactions with the immune systems are rarely studied. In the present study, cationic polymers including PEI, polylysine, cationic dextran and cationic gelatin exhibited strong stimulation on Th1 response which was characterized by the induction of the proliferation of CD4(+) T cells and the secretion of Th1 related cytokines. Experiments performed on macrophages demonstrated that cationic polymers specifically stimulated the macrophage to secrete IL-12 which is one of the main Th1-inducing cytokines. The result that MyD88 inhibitor remarkably reduced the IL-12 expression induced by cationic polymers suggested that this stimulation was mainly mediated by toll-like receptor (TLR) pathway. Additionally, cationic polymers could strongly inhibit LPS-induced TNF-alpha secretion in macrophages. This result implied that cationic polymers may interact with macrophages through TLR-4 which is the receptor of LPS. The following test of inhibiting IL-12 expression stimulated by cationic polymers using TLR-4 antibody proved that the stimulation was mainly mediated by TLR-4. Data in the present study demonstrated that the stimulation ability of cationic polymer was related with its cationic degree and neutralizing cationic polymer with anionic polymer completely abrogated the stimulation effect. The molecular weight of the polymers also influenced their stimulation ability, larger molecular means stronger stimulation ability. In conclusion, the present study revealed that cationic polymers could promote Th1 responses in vivo via TLR-4 mediated IL-12 secretion and the molecular weight and cationic degree of the polymers determined the stimulation ability.

Identification of a panel of five serum miRNAs as a biomarker for Parkinson's disease

BACKGROUND AND OBJECTIVEnParkinsons disease (PD) is the second most common age-related neurodegenerative disorder after Alzheimers disease. The aim of this work was to determine whether the differences of serum miRNAs profiling could distinguish PD patients from healthy individuals.nnnMETHODSnWe collected serum samples from 106 sporadic PD patients and 91 age/gender-matched healthy controls. Serum miRNAs were analysed by Solexa sequencing followed by a qRT-PCR examination. The qRT-PCR assay, which was divided into two phases, was used to validate the expression of miRNAs screened by Solexa sequencing. Receiver operating characteristic (ROC) curve analysis and clustering analysis were performed to determine the diagnostic usefulness of the selected miRNAs for PD.nnnRESULTSnIn this study, we generated a profile of 5 serum miRNAs: miR-195 was up-regulated, and miR-185, miR-15b, miR-221 and miR-181a were down-regulated.nnnCONCLUSIONnThis group of five miRNAs can precisely distinguish PD patients from health individuals and may be used as a potential serum-based biomarker for the diagnosis of PD.

3,3'-Diindolylmethane alleviates oxazolone-induced colitis through Th2/Th17 suppression and Treg induction.

The T cell is pivotal in orchestrating and promoting an immune response during ulcerative colitis (UC). The aryl hydrocarbon receptor (AhR) is involved in the regulation of T cell responses, and 3,3-diindolylmethane (DIM) is a known ligand of AhR. The aim of this study was to examine the therapeutic effects of DIM in experimental colitis and to investigate the possible mechanisms underlying its effects on mucosal T cell responses. The therapeutic effects of DIM were studied in an oxazolone-induced colitis model. The pathologic markers of colitis were measured, moreover, T-helper cell (Th)- and regulatory T cell (Treg)-related transcription factor expression and associated colonic cytokine production were determined. The impact of DIM on T cell differentiation was further investigated in cultures of naive Th cells that were stimulated with anti-CD3/CD28 monoclonal antibodies (mAbs). The administration of DIM attenuated experimental colitis, as determined by pathological indices. DIM may affect signaling pathways downstream of AhR, leading to decreased Th2/Th17 cells and increased Tregs. Ultimately, this could result in the alleviation of experimental colitis. DIM has shown anti-UC activity in animal models via inhibition of Th2/Th17 cells and promotion of Tregs and may thus offer potential treatments for UC patients.

3,3′-Diindolylmethane decreases VCAM-1 expression and alleviates experimental colitis via a BRCA1-dependent antioxidant pathway

Reactive oxygen species (ROS) exhibit a key role in the pathogenesis of inflammatory bowel disease (IBD). 3,3-Diindolylmethane (DIM) can protect against oxidative stress in a breast cancer susceptibility gene 1 (BRCA1)-dependent manner. The aim of this study was to examine the therapeutic effects of DIM in experimental colitis and investigate the possible mechanisms underlying its effects on intestinal inflammation. The therapeutic effects of DIM were studied in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Pathological markers of colitis severity, antioxidant activity, and ROS generation in colonic tissue were measured. The impact of DIM on ROS-induced endothelial vascular cell adhesion molecule 1 (VCAM-1) expression and leukocyte-endothelial cell interaction was further investigated in cultures of endothelial cells and in the TNBS-induced colitis model. Administration of DIM was demonstrated to attenuate experimental colitis, as judged by pathological indices. DIM could effectively stimulate the expression of BRCA1 in vitro and in vivo and reduce ROS generation, leading to the inhibition of VCAM-1 expression and leukocyte-endothelial cell adhesion, and finally resulted in an alleviation of experimental colitis. DIM has shown anti-IBD activity in animal models by inhibiting ROS-induced VCAM-1 expression and leukocyte recruitment via a BRCA1-dependent antioxidant pathway and thus may offer potential treatments for IBD patients.