Tumor-Targeted Codelivery of CpG and siRNA by a Dual-Ligand-Functionalized Curdlan Nanoparticle.

The simultaneous delivery of CpG oligonucleotide along with short interfering RNA (siRNA) has the potential to significantly boost the anticancer impact of siRNA medications. Our previous research demonstrated that Curdlan nanoparticles functionalized with adenosine are capable of selectively delivering therapeutic siRNA to cancerous cells through endocytosis mediated by adenosine receptors. Herein, we synthesized a dual-ligand-functionalized Curdlan polymer (denoted by CuMAN) to simultaneously target tumor cells and tumor-associated macrophages (TAMs). CuMAN nanoparticles containing CpG and siRNA demonstrated enhanced uptake by B16F10 tumor cells and bone marrow-derived macrophages, which are facilitated by AR on tumor cells and mannose receptor on macrophages. This led to increased release of pro-inflammatory cytokines in both in vitro and in vivo settings. The synergistic effect of CpG on TAMs and RNAi on tumor cells mediated by the CuMAN nanoparticle not only suppressed the tumor growth but also strongly inhibited the lung metastasis. Our findings indicate that the CuMAN nanoparticle has potential as an effective dual-targeting delivery system for nucleic acid therapeutics.

Tumor targeted siRNA delivery by adenosine receptor-specific curdlan nanoparticles.

Aminated curdlan derivatives are highly effective nucleic acid carriers. Previously, we proved that the ligand-functionalized curdlan derivatives have greatly enhanced cell type specificity induced by receptor-mediated internalization in vitro. In this study, to improve biocompatibility and enhance tumor-targeting efficacy of the curdlan derivative, we pegylated the adenosine functionalized amino curdlan derivative (denoted by pAVC polymer). We confirmed that the uptake of pAVC polymer carrying siRNA by tumor cells was adenosine receptor (AR)-dependent and was specifically inhibited by AMP but not by GMP. The pAVC polymers not only preserved the receptor recognition and exhibited significantly decreased cytotoxicity but also showed remarkable tumor targeting efficiency in vivo. The nanoparticles formulated from siRNA (against STAT3) and pAVC4 polymer, which bears the highest degree of PEG substitution, delivered siRNA highly specifically to tumor tissue, knocked down STAT3, and inhibited tumor growth. The pAVC polymers may be a promising carrier for tumor specific delivery of nucleic acid drugs.

Analysis and identification of key anti-inflammatory molecules in Eerdun Wurile and exploration of their mechanism of action in microglia.

Ethnomedicine Eerdun Wurile (EW) can significantly promote poststroke neuro-recovery through modulation of microglia polarization. Fraction 4-6 (F4-6) isolated from EW via serial fractionation inhibits the expression of pro-inflammatory genes in LPS stimulated microglia. However, the key active molecules of F4-6 have not been identified. Herein, we identified alantolactone (Ala) and dehydrodiisoeugenol (Deh) as the active anti-inflammatory components of F4-6 by UPLC-qTof MS analysis. We confirmed that, F4-6, Ala, Deh and mixture of Ala and Deh (Mix) downregulate the expression of several pro-inflammatory genes including Ccl2, Cox2 and Il6 in LPS-treated microglia in a similar pattern. At the same time upregulate the expression of anti-inflammatory genes including Hmox1, Tgfß, Igf1 and Creb1. Moreover, the conditioned culture media obtained from F4-6 treated microglia significantly enhanced proliferation of N2a cells, and promoted neurite outgrowth possibly through upregulation of Nefh and Dlg4. Mechanistically, F4-6 strongly downregulated the expression of NF-κB p65, while also inhibiting the nuclear translocation of p65, leading to the suppression of transcription of pro-inflammatory genes initiated by NF-κB. Collectively, our data identified and quantified the key chemicals of EW and provide insights into the optimization of the herbal composition for neuroprotection.

The study of ethanol extract of Epilobium angustifolium L. on blood sugar level in type II diabetic rats.

Epilobium angustifolium (EA) is well known as a traditional medicinal plant in many countries with multiple health effects. However, the chemical composition and anti-diabetic effect of EA has not been reported. In our study, the composition and anti-diabetic effects of ethanol extracts from EA in vivo and in streptozotocin (STZ)-induced type II diabetic rats were investigated. EA ethanol extracts exhibited protection effect on H2O2 induced oxidative stress damage INS-1 cells, reduce the body weight loss, blood glucose level and increase insulin level when compared with those of diabetic rats. Following 21 days of EA treatment at 9.2 and 18.4mg/kg, BW increased by 15.85% and 15.53%, respectively, which were extremely higher than diabetic group (9.50%). The fasting blood glucose level of EA 9.2mg/kg group rats significantly decreased by 60.43% and insulin level increased by 2.78 times, respectively. Corresponding to that, the fasting blood glucose level of EA 18.4mg/kg group rats decreased by 52.61% and insulin level increased by 2 times, respectively. Collectively our data suggest that ethanol extract of EA has remarkably hypoglycemic effect in type 2 diabetes and EA might be a promising functional food or medicine for T2DM treatment.

Synthesis of PEGylated cationic curdlan derivatives with enhanced biocompatibility.

Cationic polysaccharides have shown excellent ability of nucleic acids delivery. However, cationic curdlan derivatives with high degree of amination cause damage to the cell membrane and induce considerable cytotoxicity, limiting their in vivo application. Herein, we synthesized PEGylated 6-amino-6-deoxy-curdlan derivatives containing cleavable disulfide bonds. The resulting polymers (denote 6AC-2S PEGx) not only showed high affinity to siRNA but also exhibited significantly decreased cytotoxicity and hemolysis effect, while showing remarkable in vitro transfection efficiency. In vivo study demonstrated that 6AC-2S PEG40, which had a lower LD50 value than that of 6AC-100, did not cause liver damage, as the i.v. injection of 6AC-2S PEG40 to mouse did not increase serum level of ALT/AST. Furthermore, tissue distribution results showed that 6AC-2S PEG40 successfully delivered siRNA to liver, lung and spleen. Collectively, our data confirmed that PEGylation can increase the biocompatibility of cationic curdlan derivatives, which is a promising carrier for nucleic acid therapeutics.

AMP functionalized curdlan nanoparticles as a siRNA carrier: Synthesis, characterization and targeted delivery via adenosine A2B receptor.

Receptor-mediated endocytosis has been used for tissue targeted delivery of short interfering RNA (siRNA) drugs. Herein, we investigated adenosine receptor (AR) as a candidate for receptor-mediated siRNA internalization. We synthesized adenosine functionalized cationic curdlan derivatives (denote CuAMP polymers). One of these polymers, CuAMP4, efficiently delivered siRNA to breast cancer cells expressing high level of A2B receptor. The internalization of siRNA loaded CuAMP4 by cancer cells was inhibited by free AMP as well as endocytosis inhibitors. Moreover, knockdown of A2BR by siRNA, or pre-treatment of the cells with anti-A2BR antibody, strongly inhibited the cellular uptake of CuAMP4. Our findings confirmed that A2BR can be utilized for cell type specific siRNA delivery, and CuAMP4 NP may be a promising delivery system for cancer cell targeted delivery of therapeutic siRNAs.

Oxidized curdlan activates dendritic cells and enhances antitumor immunity.

Curdlan activates dendritic cells (DCs) and enhances DC-based antitumor immunity. However, hydrophobicity and heterogeneity of curdlan particulates hinder perfect binding of curdlan to dectin-1 receptor, resulting in the reduced activation of antigen presenting cells and limited antitumor effects. Herein, we synthesized partially oxidized curdlan derivative (ß-1,3-polyglucuronic acid, denote PGA). PGA-45 polymer, the reaction product prepared from curdlan by oxidation with 4-acetamido-TEMPO/NaClO/NaClO2 systems under acid conditions for 45 min, activated DCs, induced the expression of co-stimulatory molecules and cytokines, and promoted allogenic T cell proliferation as well as the expression of IL-2. Mechanistically, PGA-45 polymer strongly enhanced phosphorylation of IKK-ß and reduced the expression of phosphorylated Akt, suggesting that PGA-45 may activate multiple cell surface receptors such as TLR4 and dectin-1. Administration of tumor lysate pulsed DCs pre-treated with PGA-45 particles induced strong antitumor activity in B16F10 melanoma model. Our data suggest that PGA-45 have strong adjuvant effects for anti-cancer immunity and the design of PGA polymers may provide insights in the development of novel adjuvants for cancer immunotherapy.

Silencing of STAT3 via Peptidomimetic LNP-Mediated Systemic Delivery of RNAi Downregulates PD-L1 and Inhibits Melanoma Growth.

Cutaneous melanoma is the most aggressive skin cancer with notorious drug resistance. Inhibition of immune checkpoint molecules is one of the most promising approaches for cancer therapy. Herein, we show that RNAi mediated silencing of STAT3 expression in the tumor tissue robustly inhibit tumor growth in B16F10 mouse model of melanoma. We designed a peptidomimetic-based lipid nanoparticles (LNPs) for the delivery of siRNA in mouse model of melanoma. When systemically administered, the novel formulation (denote DoCh) preferentially delivered siRNA to the tumor tissue. Remarkably, sequential intravenous injections of siRNA against STAT3 induced profound silencing of STAT3 expression in tumor tissue, which resulted in significant downregulation of PD-L1, leading to significant inhibition of tumor growth through inhibition of tumor immune checkpoint. Moreover, DoCh-mediated siRNA delivery did not show noticeable damage to the major organs. Collectively, our data demonstrated that DoCh LNP is a promising tumor-targeted siRNA delivery system.

The complete mitochondrial genome of Schizothorax pseudaksaiensis (Cypriniformes: Cyprinidae).

The complete mitochondrial genome of Schizothorax pseudaksaiensis was cloned and sequenced in the present study. The genome was 16,582 bp in size, which had a mostly conserved structural organization in comparison with that of other teleost fish. It consisted of 37 genes (13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes), and 2 main non-coding regions (the control region and the origin of the light strand replication). All protein-coding genes started with ATG except for COX1, which began with GTG. However, the termination codons of 13 protein-coding genes varied with TAA, TA, T or TAG. The overall base composition of S. pseudaksaiensis in descending order was A 30.18%, C 27.08%, T 25.37% and G 17.37%, with a slight A + T bias. The complete mitochondrial genome sequence may provide useful information for phylogenetic analysis and studies of population genetics of S. pseudaksaiensis.