Damage sensing through TLR9 Promotes Viral Clearance and Recovery During Influenza Infection.

Host response aimed at eliminating the infecting pathogen, as well as the pathogen itself, can cause tissue injury. Tissue injury leads to the release of a myriad of cellular components including mitochondrial DNA, which the host senses through pattern recognition receptors. How the sensing of tissue injury by the host shapes the anti-pathogen response remains poorly understood. In this study, we utilized mice that are deficient in toll-like receptor-9 (TLR9), which binds to unmethylated CpG DNA sequences such as those present in bacterial and mitochondrial DNA. To avoid direct pathogen sensing by TLR9, we utilized the influenza virus, which lacks ligands for TLR9, to determine how damage sensing by TLR9 contributes to anti-influenza immunity. Our data show that TLR9-mediated sensing of tissue damage promotes an inflammatory response during early infection, driven by the myeloid cells and associated cytokine responses. Along with the diminished inflammatory response, the absence of damage sensing through TLR9 led to impaired viral clearance manifested as a higher and prolonged influenza burden in the lung. The absence of TLR9 led to extensive infection of myeloid cells including monocytes and macrophages rendering them highly inflammatory, despite having a low initial inflammatory response. The persistent inflammation driven by infected myeloid cells led to persistent lung injury and impaired recovery in influenza-infected TLR9-/- mice. Further, we show elevated circulating TLR9 ligands in the plasma samples of patients with influenza, demonstrating its clinical relevance. Overall, over data show an essential role of damage sensing through TLR9 in promoting anti-influenza immunity.

A comparative study on schizophyllan and chitin nanoparticles for ellagic acid delivery in treating breast cancer.

In this study, following encapsulation of ellagic acid (EA), an anti-cancer agent, loaded in schizophyllan (EA/SPG-NP) and chitin (EA/Ch-NP) nanoparticles, its release in 95% ethanol, and different mediums of digestive systems with pH ranging 1.5 to 7.4, were examined before investigating for treatment of breast cancer MCF-7cells. Following synthesis, the EA was characterized by FT-IR, SEM, XRD, DLS and zeta potential analysis. Loading capacity of schizophyllan and chitin were 30.08 and 79.52%, respectively, while SEM images indicated respective size distributions of 217.8 and 39.82 nm, with the corresponding zeta potentials being +27 and -9.14 mV. As EA was loaded in nanoparticles, antioxidant activity, examined by DPPH method, of the free EA was found to be higher than both EA/SPG-NP and EA/Ch-NP, but lower than the latter at 7.4 pH. Interestingly, scavenging activities for EA and EA/SPG-NP reduced for higher pH. The MTT cytotoxicity indicated that EA/SPG-NP and EA/Ch-NP inhibited effectively cell growth of breast cancer cell lines at IC50 of 60 and 115 µg/ml, respectively.

Aptamer functionalized curcumin-loaded human serum albumin (HSA) nanoparticles for targeted delivery to HER-2 positive breast cancer cells.

In this study, an HER2 aptamer-decorated curcumin-loaded human serum albumin nanoparticle (Apt-HSA/CCM NP) was developed and characterized as a new anticancer formulation for targeted delivery to human epithelial growth factor receptor 2 (HER2) overexpressing breast cancer cells. Conjugation of HER2 Apt to the surface of HSA NPs was confirmed by gel electrophoresis and FTIR analysis. The obtained NPs have the hydrodynamic diameter of 281.1 ±â€¯11.1 nm and zeta potential of -33.3 ±â€¯2.5 mV. The data demonstrated that encapsulation of curcumin in HSA NPs by desolvation method has increased water solubility by 400 folds. Fluorescent microscopy image demonstrated remarkable cytoplasmic uptake of Apt-HSA/CCM NPs in HER2-overexpressing SK-BR-3 cells compared to unconjugated counterparts. Cytotoxicity experiments demonstrated no significant difference between cytotoxic effect of free curcumin and non-targeted HSA/CCM NPs in both HER2 positive and HER2 negative cell lines. However, the toxicity of Apt-HSA/CCM NPs was significantly higher and cell viability reached 36% after 72 h in SK-BR3 cell line. These results suggest that this targeted delivery system has the potential to be considered as a promising candidate for the treatment of HER2 positive cancer cells.

Synthesis and characterization of schizophyllan nanogels via inverse emulsion using biobased materials.

Oxidized sucrose cross-linked Schizophyllan nanogel was successfully synthesized via inverse emulsion method for the first time. The synthesis process was conducted in the absence of both toxic cross-linker and organic solvent. The nanogel crosslinking network was prepared using fractionated coconut oil as the continuous phase and oxidized sucrose as the cross-linker. The formation of ether linkage on schizophyllan cross-linked structure was confirmed by Fourier transform infrared microscopy (FTIR) analysis. Size and morphology were evaluated by DLS analysis and SEM. The results showed that increasing the concentration of surfactant causes the decrease in the size. By keeping surfactant amounts constant, the increase in the amount of crosslinker caused increase in the size and swelling degree. Nontoxicity of the nanogels was proved by in vitro MTT analysis. The obtained nanogels, possess special properties such as high water content, colloidal stability, bioactivity, functionality, and interior network for drug loading capacity offer great potential for the utilization of nanogels in biomedical applications.

Redox responsive curcumin-loaded human serum albumin nanoparticles: Preparation, characterization and in vitro evaluation.

The purpose of this study was to fabricate redox-responsive human serum albumin (HSA) nanoparticles (NPs) through self-assembly of HSA molecules for incorporation of curcumin (CCM) as a hydrophobic drug molecule. The structural changes of HSA in self-assembly process of nanoparticle formation were investigated using fluorescence, UV-vis, circular dichroism spectroscopy and X-ray diffraction. Spectroscopy data show changes in secondary and tertiary structures of HSA in the process of nanoparticle formation, which can be indicative of the interaction of the hydrophobic drug with HSA molecules. Unlike free CCM, nanoparticulate curcumin is readily dispersed in aqueous medium. Furthermore, self-assembled HSA-CCM NPs release CCM in an environment imitating the intracellular environment with the trigger of acidic pH and redox potential. The in vitro release studies showed that at pH7.4 only 26% CCM was released from CCM-loaded HSA NPs in 48h. However, the release of CCM was significantly accelerated in the presence of 10mM glutathione (GSH) at pH7.4 or pH5.5, in which 57% and 70% of CCM was released, respectively. The incorporation of CCM in HSA nanoparticles enhanced the cellular uptake of CCM in comparison with free CCM results in higher anticancer efficacy against MCF-7 cells.

Multifunctional nanoparticles for cancer immunotherapy.

During the last decades significant progress has been made in the field of cancer immunotherapy. However, cancer vaccines have not been successful in clinical trials due to poor immunogenicity of antigen, limitations of safety associated with traditional systemic delivery as well as the complex regulation of the immune system in tumor microenvironment. In recent years, nanotechnology-based delivery systems have attracted great interest in the field of immunotherapy since they provide new opportunities to fight the cancer. In particular, for delivery of cancer vaccines, multifunctional nanoparticles present many advantages such as targeted delivery to immune cells, co-delivery of therapeutic agents, reduced adverse outcomes, blocked immune checkpoint molecules, and amplify immune activation via the use of stimuli-responsive or immunostimulatory materials. In this review article, we highlight recent progress and future promise of multifunctional nanoparticles that have been applied to enhance the efficiency of cancer vaccines.

MPG-based nanoparticle: An efficient delivery system for enhancing the potency of DNA vaccine expressing HPV16E7.

DNA vaccines against human papillomavirus (HPV) type 16 have not been successful in clinical trials, due to the lack of an appropriate delivery system. In this study, a peptide-based gene delivery system, MPG, which forms stable non-covalent nanoparticles with nucleic acids, was used for in vitro and in vivo delivery of HPV16 E7 DNA as a model antigen. The results demonstrated that at Nitrogen/Phosphate (N/P) ratio over 10:1, this peptide can effectively condense plasmid DNA into stable nanoparticles with an average size of 180-210nm and a positive surface charge. The transfection efficiency of MPG-based nanoparticles was shown to be comparable with Polyethyleneimine (PEI). The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPG-based nanoparticles as a potent delivery system in DNA vaccine formulations. Immunization with MPG/E7DNA nanoparticles at an N/P ratio of 10:1 induced a stronger Th1 cellular immune response with a predominant interferon-γ (IFN-γ) profile than those induced by E7DNA alone in a murine tumor model. These findings suggest that MPG peptide as a novel gene delivery system could have promising applications in improving HPV therapeutic vaccines.

Evaluation of Cell Penetrating Peptide Delivery System on HPV16E7 Expression in Three Types of Cell Line.

BACKGROUND: The poor permeability of the plasma and nuclear membranes to DNA plasmids are two major barriers for the development of these therapeutic molecules. Therefore, success in gene therapy approaches depends on the development of efficient and safe non-viral delivery systems. OBJECTIVES: The aim of this study was to investigate the in vitro delivery of plasmid DNA encoding HPV16 E7 gene using cell penetrating peptide delivery system to achieve the best conditions for cell transfection and protein expression. For this purpose, we have used a cationic peptide delivery system, MPG which forms stable non-covalent complexes with nucleic acids for delivery of pEGFP-E7 as a model antigen in vitro. MATERIALS AND METHODS: DNA construct encoding HPV16 E7 (pEGFP-E7) was prepared in large scale with high purity. MPG peptide/ DNA complexes were prepared at different N/P (nitrogen/phosphate) ratios and physicochemical characterization and stability of nanoparticles were investigated. In vitro peptide-mediated E7-GFP DNA transfection, and its expression was evaluated in three cell types. To quantify the transfection efficiency of this delivery system, transfected cells were harvested and assessed for GFP-positive cells by flow cytometry. Furthermore, E7-GFP expression was confirmed by western blot analysis. RESULTS: The cellular uptake of MPG based nanoparticles was shown to be comparable with standard reagent PEI. The COS-7 cells transfected by MPG-based nanoparticles at an N/P ratio of 15:1 showed the highest transfection efficiency and gene expression. CONCLUSIONS: The results indicated that the efficient gene expression depends on both cell type and N/P ratio applied, in vitro. The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPGbased nanoparticles as a potent gene delivery system.

Polymeric nanoparticles: potent vectors for vaccine delivery targeting cancer and infectious diseases.

Nanocarriers with various compositions and biological properties have been extensively applied for in vitro/in vivo drug and gene delivery. The family of nanocarriers includes polymeric nanoparticles, lipid-based carriers (liposomes/micelles), dendrimers, carbon nanotubes, and gold nanoparticles (nanoshells/nanocages). Among different delivery systems, polymeric carriers have several properties such as: easy to synthesize, inexpensive, biocompatible, biodegradable, non-immunogenic, non-toxic, and water soluble. In addition, cationic polymers seem to produce more stable complexes led to a more protection during cellular trafficking than cationic lipids. Nanoparticles often show significant adjuvant effects in vaccine delivery since they may be easily taken up by antigen presenting cells (APCs). Natural polymers such as polysaccharides and synthetic polymers have demonstrated great potential to form vaccine nanoparticles. The development of new adjuvants or delivery systems for DNA and protein immunization is an expanding research field. This review describes polymeric carriers especially PLGA, chitosan, and PEI as vaccine delivery systems.