Efficacy of orally administered porcine epidemic diarrhea vaccine-loaded hydroxypropyl methylcellulose phthalate microspheres and RANKL-secreting L. lactis.

Here, we examined the efficacy of are combinant subunit antigen-based oral vaccine for preventing porcine epidemic diarrhea virus (PEDV). First, we generated a soluble recombinant partial spike S1 protein (aP2) from PEDV in E. coli and then evaluated the utility of aP2 subunit vaccine-loaded hydroxypropyl methylcellulose phthalate microspheres (HPMCP) and RANKL-secreting L. lactis (LLRANKL) as a candidate oral vaccine in pregnant sows. Pregnant sows were vaccinated twice (with a 2 week interval between doses) at 4 weeks before farrowing. Titers of virus-specific IgA antibodies in colostrum, and neutralizing antibodies in serum, of sows vaccinated with HPMCP (aP2) plus LL RANKL increased significantly at 4 weeks post-first vaccination. Furthermore, the survival rate of newborn suckling piglets delivered by sows vaccinated with HPMCP (aP2) plus LL RANKL was similar to that of piglets delivered by sows vaccinated with a commercial killed porcine epidemic diarrhea virus (PED) vaccine. The South Korean government promotes a PED vaccine program (live-killed-killed) to increase the titers of IgA and IgG antibodies in pregnant sows and prevent PEDV. The oral vaccine strategy described herein, which is based on a safe and efficient recombinant subunit antigen, is an alternative PED vaccination strategy that could replace the traditional strategy, which relies on attenuated live oral vaccines or artificial infection with virulent PEDV.

Novel production of natural bacteriocin via internalization of dextran nanoparticles into probiotics.

Recently, probiotics has drawn much attention as an alternative of antibiotics because overuse use of antibiotics has caused widespread bacterial resistance. Given that prebiotics enhance the activity of probiotics, we prepared phthalyl dextran nanoparticles (PDNs) by conjugation of phthalic anhydride with dextran to form a prebiotic and checked its effects on the cellular and antimicrobial properties of the probiotics. First, we found that the internalization of PDNs by probiotics was dependent on temperature, time, and glucose transporters. Internalization of PDNs enhanced the production of antimicrobial peptides by probiotics through self-defense mechanism and resulted in higher antimicrobial activities against Gram-positive and -negative pathogens compared to probiotics themselves. Moreover, pediocin produced by PDN-internalized probiotics was able to suppress pathogenic gut infections and alter the population of gut microbiome in vivo. The enhanced antimicrobial property of Pediococcus acidilactidi internalized with PDNs could decrease the number of pathogens and increase beneficial bacteria species in mice. Furthermore, the composition of gut microbiome was changed, and resulted in preventing reduction of the diversity of the microflora. Our results indicate that PDNs as a new type of prebiotic can regulate probiotic bacterial metabolism, suggesting a new avenue for probiotic modulation and their use in addressing the challenge of bacterial resistance.

HDAC6 regulates thermogenesis of brown adipocytes through activating PKA to induce UCP1 expression.

Mitochondrial uncoupling protein 1 (UCP1) is responsible for nonshivering thermogenesis in brown adipose tissue (BAT). UCP1 increases the conductance of the inner mitochondrial membrane (IMM) for protons to make BAT mitochondria generate heat rather than ATP. HDAC6 is a cytosolic deacetylase for non-histone substrates to regulate various cellular processes, including mitochondrial quality control and dynamics. Here, we showed that the body temperature of HDAC6 knockout mice is slightly decreased in normal hosing condition. Interestingly, UCP1 was downregulated in BAT of HDAC6 knockout mice, which extensively linked mitochondrial thermogenesis. Mechanistically, we showed that cAMP-PKA signaling plays a key role in HDAC6-dependent UCP1 expression. Notably, the size of brown adipocytes and lipid droplets in HDAC6 knockout BAT is increased. Taken together, our findings suggested that HDAC6 contributes to mitochondrial thermogenesis in BAT by increasing UCP1 expression through cAMP-PKA signaling pathway.

A New Method of Producing a Natural Antibacterial Peptide by Encapsulated Probiotics Internalized with Inulin Nanoparticles as Prebiotics.

Synbiotics are a combination of probiotics and prebiotics, which lead to synergistic benefits in host welfare. Probiotics have been used as an alternative to antibiotics. Among the probiotics, Pediococcus acidilactici (PA) has shown excellent antimicrobial activity against Salmonella Gallinarum (SG) as a major poultry pathogen and has improved the production performances of animals. Inulin is widely used as a prebiotic for the improvement of animal health and growth. The main aim of this study is to investigate the effect of the antimicrobial activity of inulin nanoparticles (INs)-internalized PA encapsulated into alginate/chitosan/alginate (ACA) microcapsules (MCs) in future in vivo application. The prepared phthalyl INs (PINs) were characterized by DLS and FE-SEM. The contents of phthal groups in phthalyl inulin were estimated by ¹H-NMR measurement as 25.1 mol.-%. The sizes of the PINs measured by DLS were approximately 203 nm. Internalization into PA was confirmed by confocal microscopy and flow cytometry. Antimicrobial activity of PIN-internalized probiotics encapsulated into ACA MCs was measured by co-culture antimicrobial assays on SG. PIN-internalized probiotics had a higher antimicrobial ability than that of ACA MCs loaded with PA/inulin or PA. Interestingly, when PINs were treated with PA and encapsulated into ACA MCs, as a natural antimicrobial peptide, pediocin was produced much more in the culture medium compared with other groups inulin-loaded ACA MCs and PA-encapsulated into ACA MCs.

HDAC6 deficiency induces apoptosis in mesenchymal stem cells through p53 K120 acetylation.

The acetylation of p53 is critical in modulating its pro-apoptotic roles. However, its regulatory mechanism and physiological significance are unclear. Here, we show HDAC6 negatively regulates pro-apoptotic acetylation of p53 at lysine residue 120 (K120) in mesenchymal stem cells (MSCs). The loss of HDAC6 expression in MSCs increases K120 acetylation of p53, which is successfully reversed by the wild-type but not by catalytically dead HDAC6. Deletion of HDAC6 induces caspase-dependent apoptosis by promoting transactivation of Bax and suppression of Bcl-2. Moreover, HDAC6 deficiency leads to mitochondrial dysfunction characterized by aberrant reactive oxygen species production and defective oxidative phosphorylation, which is reversed by ectopic expression of wild-type or acetylation mimetic p53. This study demonstrates that HDAC6 is a critical regulator of a pro-apoptotic p53 K120 acetylation and mitochondrial function in MSCs, suggesting that the modulation of HDAC6 activity could be a novel approach to improve MSC- based therapies.

Combination therapy with doxorubicin-loaded galactosylated poly(ethyleneglycol)-lithocholic acid to suppress the tumor growth in an orthotopic mouse model of liver cancer.

Despite advances in technology, neither conventional anti-cancer drugs nor current nanoparticle (NP) drugs have gained substantial success in cancer treatment. While conventional chemotherapy drugs have several limitations such as low potency, poor in vivo stability and limited bioavailability, non-specific targeting of NP drugs diminishes their potency at actual target sites. In addition, the development of drug resistance to anti-cancer drugs is another challenging problem. To overcome these limitations, we aimed to develop a polymer-drug conjugate, which functions as an active NP drug and drug carrier both, to deliver a chemotherapeutic drug for combination therapy. Accordingly, we made targeting NP carrier of lithocholic acid-poly(ethylene glycol)-lactobionic acid (LPL) loading doxorubicin (Dox) to produce Dox/LPL NPs. The cellular uptake of Dox/LPL NPs was relatively higher in human liver cancer cell line (SK-HEP-1) due to galactose ligand-asialoglycoprotein receptor interaction. Consequently, the cellular uptake of Dox/LPL NPs led to massive cell death of SK-HEP-1 cells by two different mechanisms, particularly apoptotic activity by LPL and mitotic catastrophe by Dox. Most importantly, Dox/LPL NPs, when administered to orthotopic xenograft model of liver cancer, greatly reduced proliferation, invasion, migration, and angiogenesis of liver tumor in vivo. Thus, this study exemplifies the superiority of combination therapy over individual NP drug or conventional small molecule drug for cancer therapy. Overall, we present a promising approach of combinatorial therapy to inhibit the hepatic tumor growth and metastasis in the orthotopic xenograft model mice, thus representing an effective weapon for cancer treatment.

Systemic administration of RANKL overcomes the bottleneck of oral vaccine delivery through microfold cells in ileum.

A successful delivery of antigen through oral route requires to overcome several barriers, such as enzymatic barrier of gastrointestinal tract and epithelial barrier that constitutes of microfold cells (M cells) for antigen uptake. Although each barrier represents a critical step in determining the final efficiency of antigen delivery, the transcytosis of antigen by M cells in the follicle-associated epithelium (FAE) to Peyer's patches appears to be a major bottleneck. Considering the systemic administration of receptor activator of nuclear factor (NF)-ĸB ligand (RANKL) induces differentiation of receptor activator of nuclear factor (NF)-ĸB (RANK)-expressing enterocytes into M cells, here, we illustrated a promising approach of antigen delivery using full length transmembrane RANKL (mRANKL). The results showed that the intraperitoneal injection of mRANKL increased the population of dendritic cells and macrophages in mesenteric lymph nodes and spleen. Subsequently, systemic administration of mRANKL resulted in significantly higher number of functional GP2(+) M cells leading higher transcytosis of fluorescent beads through them. To corroborate the effect of mRANKL in antigen delivery through M cells, we orally delivered microparticulate antigen to mice treated with mRANKL. Oral immunization induced strong protective IgA and systemic IgG antibody responses against orally delivered antigen in mRANKL-treated mice. The higher antibody responses are attributed to the higher transcytosis of antigens through M cells. Ultimately, the higher memory B cells and effector memory CD4 T cells after oral immunization in RANKL-treated mice confirmed potency of RANKL-mediated antigen delivery. To the best of our knowledge, this is the first study to demonstrate significant induction of mucosal and humoral immune responses to M cell targeted oral vaccines after the systemic administration of RANKL.

Cloning and Characterization of an Endoglucanase Gene from Actinomyces sp. Korean Native Goat 40.

A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-ß-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) DH5α. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli DH5α harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine-Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was 55°C, but it retained over 90% of maximum activity in a broad temperature range (40°C to 60°C). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, Km and Vmax of rEG1 were 0.39% CMC and 143 U/mg, respectively.

Influence of Flaxseed Oil on Fecal Microbiota, Egg Quality and Fatty Acid Composition of Egg Yolks in Laying Hens.

Although there have been many attempts to produce ω-3 fatty acid-rich eggs using alpha-linolenic acid (ALA) that is a popular fatty acid in the poultry feed industry, only limited knowledge about the effects of ALA-enriched diets on chicken fecal microbiota is currently available. Herein we examined the changes in the fecal microbiota composition, egg quality traits and fatty acid composition of the egg yolks of laying hens fed ALA-rich flaxseed oil for 8 weeks. The animals fed the experimental diets that contained 0 % (group C), 0.5 % (group T1), and 1.0 % (group T2) of flaxseed oil, respectively, and eggs and feces were obtained for the analyses. ω-3 fatty acids, including ALA, were increased in T1 and T2 compared with C. Furthermore, the freshness of eggs was improved with no side effects on the eggs. The diet also changed the fecal microbiota; Firmicutes was increased in T1 and T2 (48.6 to 83 and 79.6 %) and Bacteroidetes was decreased (40.2 to 8.8 and 4.2 %). Principal coordinate analysis revealed that Lactobacillus, among the 56 examined genera, was the most influenced bacterial group in terms of the fecal microbial community shifts. These results indicate that ALA-rich diets influenced both the egg and fecal microbiota in beneficial manners in laying hens although the association between the fatty acid composition of the egg yolk and the fecal microbiota was not clear. This study is a first step to understand the effect of flaxseed oil as well as intestinal microbiota of laying hens.

Soluble RANKL expression in Lactococcus lactis and investigation of its potential as an oral vaccine adjuvant.

BACKGROUND: To initiate mucosal immune responses, antigens in the intestinal lumen must be transported into gut-associated lymphoid tissue through M cells. Recently, it has been increasingly recognized that receptor activator of NF-kB ligand (RANKL) controls M cell differentiation by interacting with RANK expressed on the sub-epithelium of Peyer's patches. In this study, we increased the number of M cells using soluble RANKL (sRANKL) as a potent mucosal adjuvant. RESULTS: For efficient oral delivery of sRANKL, we constructed recombinant Lactococcus lactis (L. lactis) IL1403 secreting sRANKL (sRANKL-LAB). The biological activity of recombinant sRANKL was confirmed by observing RANK-RANKL signaling in vitro. M cell development in response to oral administration of recombinant L. lactis was determined by 1.51-fold higher immunohistochemical expression of M cell marker GP-2, compared to that of non-treatment group. In addition, an adjuvant effect of sRANKL was examined by immunization of mice with M-BmpB as a model antigen after treatment with sRANKL-LAB. Compared with the wild-type L. lactis group, the sRANKL-LAB group showed significantly increased systemic and mucosal immune responses specific to M-BmpB. CONCLUSIONS: Our results show that the M cell development by sRANKL-LAB can increase the antigen transcytotic capability of follicle-associated epithelium, and thereby enhance the mucosal immune response, which implies that oral administration of sRANKL is a promising adjuvant strategy for efficient oral vaccination.

Image-Guided Nanoparticle-Based siRNA Delivery for Cancer Therapy.

With the discovery of RNA interference technology, small-interfering RNA (siRNA) has emerged as new powerful tool for gene therapy because of its high targeting specificity and selectivity. However, one of the limitations to successful gene therapy is the inability to monitor delivery of genes and therapeutic responses at the targeted site. Hence, a combinatorial approach of gene therapy with molecular imaging has been crucial in optimizing gene therapy. Recent advances in nanotechnology have made tremendous efforts to develop multifunctional nanoparticles that contain imaging and therapeutic agents together for image-guided therapy. The nanoparticles serve as contrast agents in imaging for disease detection with simultaneous delivery of therapeutics to cure the diseases. The therapy also helps to monitor the drug accumulation and assimilation in the body, thereby facilitating the evaluation of treatment effects. Here, we present an overview of polymer and lipid-based carriers for siRNA delivery, along with imaging agents as image guided therapy, in the treatment of breast, lung, liver, ovarian, cervical, and prostate cancers.

Nanoparticle-mediated delivery of siRNA for effective lung cancer therapy.

Lung cancer is one of the most lethal diseases worldwide, and the survival rate is less than 15% even after the treatment. Unfortunately, chemotherapeutic treatments for lung cancer are accompanied by severe side effects, lack of selectivity and multidrug resistance. In order to overcome the limitations of conventional chemotherapy, nanoparticle-mediated RNA interference drugs represent a potential new approach due to selective silencing effect of oncogenes and multidrug resistance related genes. In this review, we provide recent advancements on nanoparticle-mediated siRNA delivery strategies including lipid system, polymeric system and rigid nanoparticles for lung cancer therapies. Importantly, codelivery of siRNA with conventional anticancer drugs and recent theranostic agents that offer great potential for lung cancer therapy is covered.

Recombinant interleukin 6 with M cell-targeting moiety produced in Lactococcus lactis IL1403 as a potent mucosal adjuvant for peroral immunization.

Development and application of safe and effective mucosal adjuvants are important to improve immunization efficiency in oral vaccine. Here, we report a novel mucosal adjuvant, IL-6-CKS9, a recombinant cytokine generated by conjugating an M cell-targeting peptide (CKS9) with c-terminus of the murine interleukin 6 (IL-6), which facilitated enhancement of mucosal immune response. Lactococcus lactis IL1403, a food-grade strain of lactic acid bacteria (LAB) which is widely used in dairy industry, was used as a host cell to express and secrete the IL-6-CKS9 for a mucosal vaccine adjuvant. The recombinant L. lactis IL1403 secreting IL-6-CKS9 was orally administered with a model antigen protein, M-BmpB (Brachyspira membrane protein B conjugated with CKS9), to BALB/c mice for mucosal immunization. ELISA analyses showed consistent enhancement tendencies in induction of anti-M-BmpB antibody levels both with mucosal (IgA) and systemic (IgG) immune responses in IL-6-CKS9-LAB treated group compared with other groups tested by conducting two separated mice immunization assays. In addition, we characterized that the oral administration of model protein antigen with live LAB producing IL-6-CKS9 could induce both Th1 and Th2 type immune responses by analysis of the specific anti-BmpB IgG1 and IgG2a isotypes in the sera and also investigated possible oral tolerance in our vaccine strategy. Collectively, our results showed successful production and secretion of recombinant murine IL-6 with M cell-targeting moiety (IL-6-CKS9) from L. lactis IL1403 and demonstrated the live recombinant LAB producing IL-6-CKS9 could have a potential to be used as an efficient adjuvant for peroral vaccination.

Galactosylated poly(ethyleneglycol)-lithocholic Acid selectively kills hepatoma cells, while sparing normal liver cells.

Delivering drugs selectively to cancer cells but not to nearby normal cells is a major obstacle in drug therapy. In this study, lithocholic acid (LCA), a potent anti-cancer drug, is converted to two forms of poly(ethyleneglycol) (PEG) conjugates, viz., PEG-LCA (PL) and lactobionic acid (LBA) conjugated PEG-LCA (LPL). The latter form contains a galactose ligand in LBA to target the hepatocytes. Both forms are self-assembled to form nanoparticle formulation, and they have high potency than LCA to kill HepG2 cancer cells, sparing normal LO2 cells. Besides, LPL has high specificity to mouse liver cells in vivo. Western blot results confirm that the cell death is occurred through apoptosis induced by LPL nanoparticles. In conclusion, the induction of apoptosis and cell death is much more efficient with LPL nanoparticles than LCA molecules.

Tuning the buffering capacity of polyethylenimine with glycerol molecules for efficient gene delivery: staying in or out of the endosomes.

Endosomal escape is a major bottleneck for efficient non-viral gene delivery. This paper presents the development of two novel non-viral vectors by cross-linking glycerol molecules with low molecular weight polyethylenimine (PEI). The vectors, namely, HG-PEI (45 mol% glycerol content) and LG-PEI (9 mol% glycerol content) have apparently similar DNA binding, DNA unpacking and cellular uptake abilities but differ in buffering capacity. The cellular uptake and subsequent transfection efficiency of LG-PEI is superior to commercially available PEI 25 k. Interestingly, although the cellular uptake of HG-PEI is higher than that of PEI 25 k, the transgene expression by HG-PEI-mediated transfection is very low. Inhibitor and co-localization studies demonstrate the mechanism of endocytosis and formation of endosomes prone to lysosomal lysis of HG-PEI polyplexes as a consequence of its weak buffering capacity. Importantly, when the lysosomal lysis is inhibited, the transgene expression of HG-PEI-mediated transfection increases by 9-fold of its initial capacity which is comparable to the transfection efficiency of PEI 25 k. These results indicated that the buffering capacity of the polymers primarily impacts endosomal escape and subsequent transfection efficiency. Furthermore, this study highlights the significance of cross-linkers in optimizing the buffering capacity when designing polymers for gene delivery.

Production of recombinant human growth hormone conjugated with a transcytotic peptide in Pichia pastoris for effective oral protein delivery.

Among the possible delivery routes, the oral administration of a protein is simple and achieves high patient compliance without pain. However, the low bioavailability of a protein drug in the intestine due to the physical barriers of the intestinal epithelia is the most critical problem that needs to be solved. To overcome the low bioavailability of a protein drug in the intestine, we aimed to construct a recombinant Pichia pastoris expressing a human growth hormone (hGH) fusion protein conjugated with a transcytotic peptide (TP) that was screened through peroral phage display to target goblet cells in the intestinal epithelia. The TP-conjugated hGH was successfully produced in P. pastoris in a secreted form at concentrations of up to 0.79 g/l. The function of the TP-conjugated hGH was validated by in vitro and in vivo assays. The transcytotic function of the TP through the intestinal epithelia was verified only in the C terminus conjugated hGH, which demonstrated the induction of IGF-1 in a HepG2 cell culture assay, a higher translocation of recombinant hGH into the ileal villi after oral administration in rats and both IGF-1 induction and higher body weight gain in rats after oral administration. The present study introduces the possibility for the development of an effective oral protein delivery system in the pharmaceutical and animal industries through the introduction of an effective TP into hGH.

Targeted delivery of vaccine to dendritic cells by chitosan nanoparticles conjugated with a targeting peptide ligand selected by phage display technique.

The paper presents a novel dendritic cells (DC)-targeting peptide, TPAFRYS (TP) identified by phage display technique and conjugated to chitosan in order to develop an efficient DC-targeting vaccine delivery carrier. TP-conjugated chitosan nanoparticles (TPC-NPs) were prepared with ovalbumin (OVA) as a model vaccine by ionic gelation. Flow cytometry and immunocytochemistry studies demonstrated the higher targeting ability of TPC-NPs to DCs in compared to chitosan NPs. Moreover, TPC-NPs exhibited higher targeting specificity in DCs than macrophage and myoblasts. Furthermore, immunization of mice with OVA-loaded TPC-NPs enhanced OVA-specific serum IgG and IgG isotype antibodies production. Thus, DC-targeting strategy demonstrates a potential approach to enhance the effectiveness of vaccines.

Induction of long-term immunity against respiratory syncytial virus glycoprotein by an osmotic polymeric nanocarrier.

Respiratory syncytial virus (RSV) is one of the most common causes of viral deaths in infants worldwide, yet no effective vaccines are available. Here, we report an osmotically active polysaccharide-based polysorbitol transporter (PST) prepared from sorbitol diacrylate and low-molecular-weight polyethylenimine (PEI) showing a potent, yet safe, adjuvant activity and acting as an effective delivery tool for RSV glycoprotein (RGp) antigen. PST showed no toxicity in vitro or in vivo, unlike PEI and the well-known experimental mucosal adjuvant cholera toxin (CT). PST formed nano-sized complexes with RGp by simple mixing, without affecting antigenic stability. The complexes exhibited negative surface charges that made them highly efficient in the selective activation of phagocytic cells and enhancement of phagocytic uptake. This resulted in an improved cytokine production and in the significant augmentation of RGp-specific antibody production, which persisted for over 200 days. Interestingly, PST/RGp enhanced phagocytic uptake owing to the osmotic property of PST and its negative zeta potential, suggesting that PST could selectively stimulate phagocytic cells, thereby facilitating a long-lived antigen-specific immune response, which was presumably further enhanced by the polysaccharide properties of PST.

Exploring codon optimization and response surface methodology to express biologically active transmembrane RANKL in E. coli.

Receptor activator of nuclear factor (NF)-κB ligand (RANKL), a master cytokine that drives osteoclast differentiation, activation and survival, exists in both transmembrane and extracellular forms. To date, studies on physiological role of RANKL have been mainly carried out with extracellular RANKL probably due to difficulties in achieving high level expression of functional transmembrane RANKL (mRANKL). In the present study, we took advantage of codon optimization and response surface methodology to optimize the soluble expression of mRANKL in E. coli. We optimized the codon usage of mRANKL sequence to a preferred set of codons for E. coli changing its codon adaptation index from 0.64 to 0.76, tending to increase its expression level in E. coli. Further, we utilized central composite design to predict the optimum combination of variables (cell density before induction, lactose concentration, post-induction temperature and post-induction time) for the expression of mRANKL. Finally, we investigated the effects of various experimental parameters using response surface methodology. The best combination of response variables was 0.6 OD600, 7.5 mM lactose, 26°C post-induction temperature and 5 h post-induction time that produced 52.4 mg/L of fusion mRANKL. Prior to functional analysis of the protein, we purified mRANKL to homogeneity and confirmed the existence of trimeric form of mRANKL by native gel electrophoresis and gel filtration chromatography. Further, the biological activity of mRANKL to induce osteoclast formation on RAW264.7 cells was confirmed by tartrate resistant acid phosphatase assay and quantitative real-time polymerase chain reaction assays. Importantly, a new finding from this study was that the biological activity of mRANKL is higher than its extracellular counterpart. To the best of our knowledge, this is the first time to report heterologous expression of mRANKL in soluble form and to perform a comparative study of functional properties of both forms of RANKL.

Highly efficient gene transfection by a hyperosmotic polymannitol based gene tranporter through regulation of caveolae and COX-2 induced endocytosis.

The regulation of cellular uptake to cross the cell membrane is one of the key strategies of importance for efficient gene transfection of non-viral vectors. Hyperosmotic activity of polyplexes may facilitate crossing of the membrane barrier by elevating the osmolarity of the extracellular matrix. In this study, we demonstrated that a polymannitol based gene transporter (PMGT) utilizes the hyperosmoticity contributed by the polymannitol backbone leading to accelerated cellular uptake and enhanced gene transfection. Mannitol dimethacrylate (MDM) monomer was synthesized by esterification of mannitol and methacryloyl chloride. The prepared MDM was then cross-linked with low molecular weight (LMW) branched polyethyleneimine (bPEI) by Michael addition reaction to produce PMGT. PMGT provided polyplex stability in serum, low cytotoxicity, and degradability due to the ester linkages present in the polymannitol backbone. Elevated transfection activity and efficiency, both in vitro and in vivo, were achieved by modulating the mode of cellular uptake due to the effect of the hyperosmotic properties of PMGT. Cyclooxygenase-2 (COX-2) inhibition by SC58236 revealed the up-regulation of this osmoprotectant molecule against the hyperosmotic activity of polymannitol, inducing rapid endocytosis of PMGT in order to re-balance the hyperosmotic environment. Various inhibition studies of endocytosis showed caveolae-mediated endocytosis to be the main route of cellular internalization to account for the enhanced transgene expression.