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Alhagi honey polysaccharides, extracted from a perennial plant Alhagi pseudalhagi syn, possessed many biological activities such as immune enhancement, anti-tumor effect, and antioxygenation. In this study, we used Alhagi honey polysaccharide encapsulated (poly lactic-co-glycolic acid) (PLGA) nanoparticles to prepare an assembled particles-oil pickering emulsion: PPAS and PEI-PPAS. We investigated the characterization of two pickering emulsions, and the possible mechanism to enhance immune responses. The results showed that PPAS and PEI-PPAS both could load high adsorption of OVA and had ability to sustained controlled release OVA. In vivo experiment, PEI-PPAS/OVA enhanced the levels of IgG and cytokines. Meanwhile, it could effectively target dendritic cells (DCs), promoted the cellular uptake of OVA then activated DCs in lymph nodes. And this effect of PEI-PPAS might be induced through the MHC II and MHC I pathway in DCs. Thus, these findings demonstrated that PEI-PPAS could induce a strong and long-term cellular and humoral immune response, and have potential to applied to vaccine adjuvant delivery system.

Immune checkpoint blockade (ICB) therapy has revolutionized clinical oncology. However, the efficacy of ICB therapy is limited by the ineffective infiltration of T effector (Teff) cells to tumors and the immunosuppressive tumor microenvironment (TME). Here, we report a programmable tumor cells/Teff cells bispecific nano-immunoengager (NIE) that can circumvent these limitations to improve ICB therapy. The peptidic nanoparticles (NIE-NPs) bind tumor cell surface α3β1 integrin and undergo in situ transformation into nanofibrillar network nanofibers (NIE-NFs). The prolonged retained nanofibrillar network at the TME captures Teff cells via the activatable α4β1 integrin ligand and allows sustained release of resiquimod for immunomodulation. This bispecific NIE eliminates syngeneic 4T1 breast cancer and Lewis lung cancer models in mice, when given together with anti-PD-1 antibody. The in vivo structural transformation-based supramolecular bispecific NIE represents an innovative class of programmable receptor-mediated targeted immunotherapeutics to greatly enhance ICB therapy against cancers.

The continuous emergence of multidrug-resistant (MDR) bacteria has posed an increasingly serious public health threat which urges people to develop some alternatives. Gallic acid (GA) is a natural ingredient in many traditional Chinese medicines, which has many biological activities, such as antibacterial, and antiseptic. Here, clinical isolates of MDR Escherichia coli (E. coli) were used to evaluate the antibacterial effect of GA and the underlying mechanism. The results revealed that GA exerted bactericidal activity and inhibited the formation of bacterial biofilm. GA enhanced the activities of ceftiofur sodium or tetracycline against E. coli, and facilitated antibiotic accumulation in bacteria. Further analysis of morphological alterations and efflux pump gene expressions confirmed that GA damaged outer and inner membranes, and suppressed the mRNA expressions of acrA, acrB, tolC, acrD and acrF involved in membrane permeability. In addition, GA showed protective effects against bacterial infection and improved the survival rates of Galleria mellonella and BALB/c mice. These data highlight a better understanding of GA against bacteria and provide an alternative strategy for MDR bacterial infection.

Artesunate (ARS) has been shown to have a protective effect on ulcerative colitis (UC) in mice. However, its lack of targeting and short half-life severely hamper its efficacy. In this study, polylactic acid-glycolic acid copolymer (PLGA) and chitosan (CS) double emulsification solvent volatilisation method was used to prepare a stable nanoemulsion loaded with ARS (CPA). The in vitro drug release profile was detected using dialysis and the potential protective effect was evaluated in an experimental ulcerative colitis (UC) model induced by oral administration of dextran sulphate sodium (DSS). The results suggested that the mean droplet diameter of CPA nanoemulsion is 409.9 ± 9.21 nm, the polydispersity index is 0.17 ± 0.01 and the zeta potential is 40.07 ± 1.65 mV. The cumulative release curve showed the ARS was mainly released at pH 7.4, which is similar to the colonic environment. Oral administration of CPA effectively relieved DSS-induced clinical symptoms by lowering the body weight loss, disease activity index (DAI) score and impressively maintained tight junction protein expression in colon tissue when compared to the blank nanoemulsion control. Meanwhile, CPA remarkably suppressed TLR4/NF-κB pathway activation and mRNA levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) while enhanced levels of IL-10 and CD206. In addition, the effect of CPA was slightly better than that of injecting ARS. Therefore, this study demonstrates a convenient drug delivery system for oral administration of ARS that potentially helps to target colonic tissue and alleviate UC.

Objective Recent studies have revealed the adjuvant activity of cubosomes and their potential utility as an antigen delivery system. In this study, to further enhance the adjuvant activity of cubosomes, two cationic polymers are modified on the surface of cubosomes. Methods Here, we exploit the effects of surface chemistry on the adjuvant activity of Ganoderma lucidum polysaccharide cubosomes (GLPC) by placing two kinds of molecules, that is, cetyltrimethylammonium bromide (CTAB) and poly(diallydimethyl ammonium chloride) (PDDAC), on their surface. Results CTAB- or PDDAC-modified GLPC were found to significantly promote humoral and cellular immune responses, as well as the proliferation of CD3+ CD4+ or CD3+ CD8+T cells through the powerful activation of dendritic cells (DCs). The enhanced immune responses of PDDAC-modified GLPC might be attributed to the maturation of DCs into draining lymph nodes and the activation of spleen and cytokines in serum. Conclusion PDDAC modification is beneficial for enhancing humoral and cellular immune response, suggesting that PDDAC-GLPC-OVA has the ability to be a potential adjuvant for vaccine.

Graphene oxide (GO) and lentinan have received great attention because of their utility in biomedical applications. Graphene oxide is utilized in drug- and vaccine-delivery systems due to its biocompatibility, large surface area, and outstanding adsorption capability, while lentinan has immunity-enhancing effects. In this study, we synthesized and characterized GO grafted with lentinan (LNT) as an adjuvant and investigated how to impact the immune responses. Lentinan-modified GO (GO-LNT) facilitated antigen uptake in macrophages and improved the efficiency of antigen application in vitro. Furthermore, in vivo, compared with GO/OVA, GO-LNT/OVA decreased the release rate of ovalbumin (OVA) to sustain long-term immune responses and boost the levels of IgG and IgG subtypes. Hence, we can infer that the effects of GO-LNT were a result of the increased amounts of antigen uptake by cells. Overall, our studies demonstrated that GO-LNT could suffice for a safe and effective vaccine-delivery system as well as an excellent adjuvant that both elicits a long-term immune memory response and potentiates cellular and humoral immunity.

Artesunate (ARS) has been shown to be highly effective against chloroquine-resistant malaria. In vitro studies reported that ARS has anticancer effects; however, its detrimental action on cancer cells may also play a role in its toxicity toward normal cells and its potential toxicity has not been sufficiently researched. In this study, we investigated the possible cytotoxic effects using normal BRL-3A and AML12 liver cells. The results showed that ARS dose-dependently inhibited cell proliferation and arrested the G0/G1 phase cell cycle in both BRL-3A and AML12 liver cells. Western blotting demonstrated that ARS induced a significant downregulation of cyclin-dependent kinase-2 (CDK2), CDK4, cyclin D1, and cyclin E1 in various levels and then caused apoptosis when the Bcl-2/Bax ratio decreased. Conversely, the levels of intracellular reactive oxygen species (ROS) were increased. The ROS scavenger N-acetylcysteine can significantly inhibit cell cycle arrest and apoptosis induced by ARS. Thus, the data confirmed that ARS exposure impairs normal liver cell proliferation by inducing G0/G1 cell cycle arrest and apoptosis, and this detrimental action may be associated with intracellular ROS accumulation. Collectively, the possible side effects of ARS on healthy normal cells cannot be neglected when developing therapies.

Ganoderma lucidum has been widely used as a fungal, for promoting health and longevity in China and other Asian countries. Polysaccharide (PS) extracted from Ganoderma lucidum exhibits a variety of immunomodulatory activities and has the ability to induce strong immune responses. Liposomes (Lip) have been shown to be useful carriers of vaccine antigens and can be applied as a versatile delivery system for vaccine adjuvants. Here, PS and inactivated porcine circovirus type II (PCV-II) were encapsulated into Lip as a vaccine and inoculated into mice. The magnitude and kinetics of adjuvant activity were investigated. Polysaccharide-loaded liposomes (Lip-PS) could induce more efficient PCV-II-specific immune responses than other single-component formulations. The Lip-PS group displayed robust and higher titers of PCV-II-specific immunoglobulin (Ig)G antibodies and IgG subtypes as well as higher cytokine levels, furthermore, splenocytes were activated by Lip-PS. Thus, Lip-PS formulation produced vigorous humoral and cellular immune responses, with a mixed T-helper (Th)1/Th2/Th17 immune response and slight Th1 polarized cellular immune response. Overall, these results suggested that Lip-PS could provide a universal platform for vaccine design against PCV-II.

Immune checkpoint blockade (ICB) therapy has revolutionized clinical oncology. However, the efficacy of ICB therapy is limited by the ineffective homing of T effector (Teff) cells to tumours and the immunosuppressive tumour microenvironment (TME). Here, we report a programmable tumour cells/Teff cells bispecific nano-immuno-engager (NIE) that can circumvent these limitations to improve ICB therapy. We have developed 28 nm non-toxic peptidic micellar nanoparticles (NIE-NPs) that bind α3β1 integrin on tumour cells membrane and undergo in situ transformation on surface of tumour cells into nanofibrillar network (NIE-NFs). The nanofibrillar network persistently facilitates cytotoxic T cells’ homing to the proximity of tumour cells via activatable α4β1 integrin ligands, and also allows sustained release of resiquimod to reprogram the TME. This bispecific NIE eliminates syngeneic 4T1 breast cancer and Lewis lung cancer models in mice, when given together with anti-PD-1 antibody. The in vivo structural transformation-based supramolecular bispecific NIE represents an innovative class of programmable receptor-mediated targeted immunotherapeutics to greatly enhance ICB therapy against cancers.

Background Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common food-borne pathogen, which has the ability to infect a wide range of hosts. The increasing emergence of drug-resistant strains urgently requires new alternative therapies. Eugenol has been shown to be very effective against drug-resistant strains of Gram-negative and Gram-positive bacteria. The purpose of this study is to explore the effects of eugenol on the virulence factors and pathogenicity of S. Typhimurium. Methods The antibacterial activity of eugenol was investigated via the changes of cell morphology, fimbriae related-genes and virulence factors of S. Typhimurium, then the pathogenicity of S. Typhimurium pretreated by eugenol to chickens was evaluated. Results Susceptibility testing showed that eugenol possessed significant antimicrobial activity. Scanning electron microscope analysis showed eugenol treatment deformed the morphology with damaged fimbriae structure of S. Typhimurium. Real time PCR assay confirmed eugenol significantly down-regulated the expressions of virulence factors (hilA, hilD, sipA, sipC, spiC, misL) of Type III secretion system (T3SS) and adherence genes (fimA, fimH, fimD, fimY, fimZ, stm0551) of Type I fimbriae (TIF). Animal experiment proved that the pathogenicity of S. Typhimurium exposed by eugenol was reduced, which was evidenced by the higher survival rate, weight gains and organs indexes, the lower bacterial loads in organs. Meanwhile, the duodenal histopathological changes were mitigated, with a significantly decline in the expressions of TNF-α, IL-6 and IL-18. Conclusion In summary, eugenol pretreatment may alleviate the pathogenicity of the S. Typhimurium to chickens via wrecking the fimbriae and inhibiting the mRNA expressions of virulence factors and adhesion molecules. These data dedicated the potential mechanisms of eugenol against S. Typhimurium in vitro.

In bovine mammary epithelial cells (BMECs), a cascade of inflammatory reactions induced by lipopolysaccharide (LPS) has been shown to result in cell injury and apoptosis. The present study aims to reveal the protective effect of ferulic acid (FA) on LPS-induced BMEC apoptosis and explore its potential molecular mechanisms. First, we showed that FA had low cytotoxicity to BMECs and significantly decreased cell apoptosis and the proinflammatory response induced by LPS. Next, FA blocked LPS-induced oxidative stress by restoring the balance of the redox state and inhibiting mitochondrial dysfunction, the main contributor to LPS-induced apoptosis and ROS generation. Furthermore, the relief of inflammation and redox disturbance in the FA preconditioning group were accompanied by weaker NF-κB activation, enhanced Nrf2 activation and maintained cell viability compared to the LPS group. When BMECs were treated with FA alone, we observed that Nrf2 activation was induced before the inhibition of NF-κB activation and that the Keap1–Nrf2 relationship was disturbed. We concluded that FA prevented LPS-induced BMEC apoptosis by reversing the dominant relationship between NF-κB and Nrf2.

The purpose of this study was to optimize modification conditions of selenized garlic polysaccharides (sGPS) and investigate its structural characterization, immune and antioxidant activities. Herein, selenized garlic polysaccharides (sGPS) were prepared using by HNO3-Na2SeO3 selenylation method. And then modification conditions of sGPS were optimized through L9 (34) orthogonal test. The structural characterization of sGPS were identified by the Fourier-transform infrared (FT-IR), Solid-State nuclear magnetic resonance (NMR) spectra, X-ray diffraction (XRD) and thermogravimetric (TGA). The morphology of sGPS was detected using scanning electron microscope (SEM) and transmission electron microscope (TEM). In vivo investigation showed that sGPS significantly improved serum hemagglutination-inhibition (HI) antibody titers against Newcastle disease virus, enhanced secretory IgA (sIgA), IFN-γ, IL-2 secretion in jejunum and trachea irrigation compared with vaccine immunized control group. Furthermore, it showed that sGPS had some effects on the antioxidant activities in livers of chickens. In conclusion, the optimal modification conditions of sGPS were as follows: reaction temperature was 70 °C, the dosage of Na2SeO3 was 400 mg and reaction time was 6 h. The selenylation modification of garlic polysaccharides (GPS) could improve its immune and antioxidant activity in chickens.

Diclazuril (DIC) is widely used in the poultry industry to control coccidiosis. However, drug resistance makes it less effective, and the underlying mechanism remains unclear. One DIC-resistant E. tenella (RE) isolate and one sensitive E. tenella (SE) isolate were used to compare the differences in their endogenous development, pathogenicity, invasion-related gene expression and apoptotic characteristics. Chickens were allocated into four groups to receive RE or SE strain and their corresponding DIC treatment or not. Caeca tissues were sampled at 96 h, 120 h and 144 h post-infection (PI) for pathological analysis. Meanwhile, second-generation merozoites (Mz2) were separated at 120 h PI to detect alterations in mitochondrial membrane potential (MMP), apoptotic rate and caspase-3 activity and mRNA expression of protein phosphatase 5 (PP5), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin depolymerizing factor (ADF) and microneme proteins (MICs). Haematoxylin and eosin staining revealed that DIC treatment strictly blocked the development of the SE strain but slightly affected the RE strain. Meanwhile, the number of SE Mz2 and their MMP decreased at the same time the apoptotic rate increased after DIC treatment. Real-time quantitative PCR and caspase-3 activity studies demonstrated that Mz2 from the RE strain had higher mRNA expression of ADF and MICs along with no significant changes in GAPDH and caspase-3 activity under DIC pressure compared to its control; in contrast, the mRNA expression of ADF, MICs and PP5 was markedly suppressed in Mz2 from SE with upregulated caspase-3 activity and GAPDH transcription. In addition, the mRNA expression of GAPDH and PP5 in Mz2 from RE was remarkably higher than that of SE. Taken together, the higher mRNA expression of invasion-related genes and almost unaffected endogenous development provide a better understanding of coccidian resistance to DIC.

Alhagi honey polysaccharide (AHP) exhibit an excellent immune adjuvant effect, but low bioavailability in the body limits its application. Cationic polymer-modified poly (lactic-co-glycolic acid) (PLGA) nanoparticles have been widely investigated as vaccine delivery systems owing to their excellent antigen-loading efficiency. In this study, three kinds of cationic polymer were used to coat AHP-encapsulated PLGA nanoparticles (AHPP) to build positively charged antigen carriers. Among them, H5N1-loaded PEI-AHPP formulation could induce highest hemagglutination inhibition (HI) titer, IgG-subtype, and cytokines, activated dendritic cells (DCs) in lymph nodes, and CD3e+CD4+ and CD3e+CD8a+ T cells in the spleen of immunized mice. PEI-AHPP could stimulate DCs to highly express MHCI and MHCII molecules and had good antigen slow-release effect at the injected site along with lymph node targeting. These findings demonstrate that PEI-AHPP has the potential to be an effective adjuvant to induce strong and long-lasting Th1 and Th2 mixed immune responses.

Nanotheranostics with integrated diagnostic and therapeutic functions show exciting potentials towards precision nanomedicine. However, targeted delivery of nanotheranostics is hindered by several biological barriers. Here, we report the development of a dual size/charge- transformable, Trojan-Horse nanoparticle (pPhD NP) for delivery of ultra-small, full active pharmaceutical ingredients (API) nanotheranostics with integrated dual-modal imaging and trimodal therapeutic functions. pPhD NPs exhibit ideal size and charge for drug transportation. In tumour microenvironment, pPhD NPs responsively transform to full API nanotheranostics with ultra-small size and higher surface charge, which dramatically facilitate the tumour penetration and cell internalisation. pPhD NPs enable visualisation of biodistribution by near-infrared fluorescence imaging, tumour accumulation and therapeutic effect by magnetic resonance imaging. Moreover, the synergistic photothermal-, photodynamic- and chemo-therapies achieve a 100% complete cure rate on both subcutaneous and orthotopic oral cancer models. This nanoplatform with powerful delivery efficiency and versatile theranostic functions shows enormous potentials to improve cancer treatment., Size and charge can significantly affect delivery of therapeutic agents to tumours. Here, the authors report on nanoparticles optimised for delivery to the tumour which release smaller particles and change charge in the tumour microenvironment to optimise tumour penetration and cellular uptake.

Cubosomes, as biocompatible carriers in drug delivery systems, consist of curved bicontinuous lipid bilayers. With a honeycombed structure divided into two internal aqueous channels, cubosomes could be used for many bioactive ingredients. Achyranthes bidentata polysaccharides (ABPs) are isolated from the roots of Achyranthes bidentata, used in Chinese herbal medicine, and present a noticeable effect as an immunomodulator. This study investigates the optimal preparation of combined cubosome-ABP (Cub-ABP) nanoparticles using response surface methodology and explores their characteristics and stability. The encapsulation efficiency of optimized Cub-ABPs was 72.59%. In-vitro stability studies demonstrated the stability of Cub-ABPs and cubosome nanoparticles without ABPs; both were stable for up to 25days. Safe concentrations of Cub-ABPs and cubosome nanoparticles without ABPs are 104.06μg/mL and 208.13μg/mL with comparatively low cytotoxicity against lymphocytes. Moreover, the feasible immunomodulatory effects of Cub-ABPs were determined by evaluating their proliferation and change of CD4+/CD8+ ratio on splenic lymphocytes in vitro. Proliferation and flow cytometry studies revealed that, compared with free ABPs and blank cubosomes, Cub-ABPs proved more effective in promoting lymphocyte proliferation and in triggering the transformation of T-lymphocytes into Th-cells.

The main purpose of this study was the preparation, optimization, characterization, and in vitro immunological activity evaluation of the polysaccharides of Atractylodes macrocephala Koidz-loaded nanostructured lipid carriers (PAMK-NLC). Both the blank NLC and PAMK-NLC were prepared using melt-emulsification and ultra-sonication techniques. The optimization of preparation conditions were a stearic acid to caprylic/capric triglyceride mass ratio of 2:1, a poloxamer 188 to soybean lecithin mass ratio of 2:1, and an ultrasound time of 12 min; these parameters were identified using response surface methodology (RSM). Under these conditions, the encapsulation efficiency was around 76.85% and the polydispersity index was around 0.281, indicating a good dimensional distribution. Furthermore, the potential of PAMK-NLC to enhance immunological activity in vitro was investigated in murine splenic lymphocytes. The results showed that PAMK-NLC could significantly stimulate lymphocyte proliferation and increase the ratio of CD4+ to CD8+ T cells. In conclusion, these findings indicated that PAMK-NLC possessed an efficient immunological activity, and that NLC is suitable for the delivery and protection of PAMK in clinical use.

Tea tree oil (TTO) is a popular topical use to treat skin infections. However, its poor aqueous solubility and stability have substantially limited its widespread application, including oral administration that might be therapeutic for enteric infections. In this study, mechanical ultrasonic methods were used to prepare TTO nanoemulsion (nanoTTO) with a mean droplet diameter of 161.80 nm ± 3.97, polydispersity index of 0.21 ± 0.01, and zeta potential of −12.33 ± 0.72 mV. The potential toxicity of nanoTTO was assessed by studying the oral median lethal dose (LD50) and repeated 28-day oral toxicity to provide a reference for in vivo application. Results showed that nanoTTO had no phase separation under a centrifugation test and displayed good stability during storage at −20, 4 and 25 °C over 60 days. Repeated-dose 28-day oral toxicity evaluation revealed no significant effects on growth and behavior. Assessments of hematology, clinical biochemistry, and histopathology indicated no obvious adverse effects in mice at 50, 100 and 200 mg/mL. These data suggest that nanoTTO can be considered a potential antimicrobial agent by oral administration due to its inhibitory effect on bacteria and relatively lower toxicity.

The success of subunit vaccines has been hampered by the problems of weak or short-term immunity and the lack of availability of nontoxic, potent adjuvants. It would be desirable to develop safe and efficient adjuvants with the aim of improving the cellular immune response against the target antigen. In this study, the targeting and sustained release of simple nanoliposomes containing Lycium barbarum polysaccharides (LBP) as an efficacious immune adjuvant to improve immune responses were explored. LBP liposome (LBPL) with high entrapment efficiency (86%) were obtained using a reverse-phase evaporation method and then used to encapsulate the model antigen, ovalbumin (OVA). We demonstrated that the as-synthesized liposome loaded with OVA and LBP (LBPL-OVA) was stable for 45 days and determined the encapsulation stability of OVA at 4°C and 37°C and the release profile of OVA from LBPL-OVA was investigated in pH 7.4 and pH 5.0. Further in vivo investigation showed that the antigen-specific humoral response was correlated with antigen delivery to the draining lymph nodes. The LBPL-OVA were also associated with high levels of uptake by key dendritic cells in the draining lymph nodes and they efficiently stimulated CD4+ and CD8+ T cell proliferation in vivo, further promoting antibody production. These features together elicited a significant humoral and celluar immune response, which was superior to that produced by free antigen alone.

Nanomedicine, the medical application of nanotechnology, promises a seemingly limitless range of applications from drug delivery to adjuvants and therapeutics. Our current research is focused on natural polymer-based liposome adjuvants. With the aim of inducing protective and long-lasting immunity, the immunological adjuvant activity of Rehmannia glutinosa polysaccharide liposome (RGPL) was investigated. In vivo, the splenic lymphocyte proliferation ratios and ovalbumin-specific immunoglobulin G titers of ovalbumin-RGPL-vaccinated mice were significantly upregulated. In draining lymph nodes, the expression of MHC II+CD11c+ and CD86+CD11c+ was increased by RGPL; in addition, the percentages of central memory cells (TCM) and effector memory cells (TEM) were also elevated. RGPL could effectively provide adequate antigen exposure in lymph nodes. In vitro, RGPL could promote dendritic cell maturation and enhance dendritic cell functions, such as the mixed lymphocyte reaction and antigen presentation. Overall, the results demonstrated that RGPL has the potential to act as an effective controlled release vaccine adjuvant.

In previous researches, the results showed that selenium Hericium erinaceus polysaccharide and Hericium erinaceus polysaccharide-loaded poly (lactic-co-glycolic acid) nanoparticles enhanced immune responses. In order to further enhance the immune adjuvant activity and phagocytosis of the nanoparticles, two way of combination (selenium-HEP loaded PLGA nanoparticles and selenium modified HEP-PLGA nanoparticles) were prepared to investigate the effects on macrophages in vitro. After treatment with the nanoparticles, the effects of phagocytosis, co-stimulatory molecules expression, nitric oxide (NO), and cytokines secretion were evaluated. The results showed that the particle size, PDI and zeta potential of the selenium-HEP loaded PLGA nanoparticles (Se-HEP-PLGA) and selenium modifified HEP-PLGA nanoparticles (HEP-PLGA-Se) were presented. Se-HEP-PLGA and HEP-PLGA-Se nanoparticles significantly stimulated phagocytic activity, CD40 and CD86 expression of macrophages. In addition, the levels of NO, TNF-α, IL-1β and IL-6 were enhanced in the peritoneal macrophages by stimulation with Se-HEP-PLGA and HEP-PLGA-Se nanoparticles. Among them, Se-HEP-PLGA showed the best effects on the expression of co-stimulatory molecules, secretions of NO and cytokines. These results indicated that Se-HEP-PLGA could enhance the activation of macrophages, and it could be potentially used as an HEP delivery system for the induction of strong immune responses.

Nanoparticle-based vaccine delivery systems have been extensively used to promote and induce immune responses to protein antigens. The properties of the nanoparticles, such as size, surface charge, and antigen loading mode, have been proved to significantly influence the adjuvant effect and immunoreactivity of nanoparticle-based vaccine delivery systems. The purpose of the study was to investigate how the surface charge and antigen loading mode of nanoparticles impact the immune responses. In this study, three ovalbumin (OVA)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles with different surface charges and antigen loading modes were developed. The three nanoparticles were designed as antigen encapsulated with negatively charged (Angelica sinensis polysaccharide (ASP)-PLGA/OVA), antigen encapsulated with polyethylenimine (PEI)-coated (ASP-PLGA/OVA-PEI), and antigen adsorbed on PEI-coated (ASP-PLGA-PEI-OVA) nanoparticles. The Angelica sinensis polysaccharide (ASP) was used as the immunopotentiator and encapsulated into three nanoparticles. The results demonstrated that both PEI-coated (positively charged) nanoparticles promoted the antigen escape from the endosome, which led to the cytoplasmic antigen delivery to generate cross presentation, compared to negatively charged nanoparticles. In addition, PEI-coated nanoparticles activated the DCs in lymph nodes 5 days after the primary vaccination. In vivo experiments demonstrated that both antigen-encapsulated nanoparticles induced more potent and long-term antigen-specific antibody responses, compared to that of antigen-adsorbed nanoparticles. Thus, the PEI-coated and antigen-encapsulated nanoparticles (ASP-PLGA/OVA-PEI) as a vaccine adjuvant delivery system have the potential to induce strong and long-term humoral and cellular immune responses.

Liposome-based vaccine delivery systems are known to enhance immune responses. Ganoderma lucidum polysaccharides (GLP) have been widely studied as immunomodulator and it could be as inducers of strong immune responses. In the research, GLP and ovalbumin (OVA) were encapsulated into liposome as vaccine and inoculated to mice. The magnitude and kinetics of the humoral and cellular immune responses were investigated. The results showed that GLP-OVA-loaded liposomes (GLPL/OVA) could induce more powerful antigen-specific immune responses than each single-component formulation. Mice immunized with GLPL/OVA displayed higher antigen-specific IgG antibodies, better splenocytes proliferation, higher cytokine secretion by splenocytes and significant activation of CD3+CD4+ and CD3+CD8+ T cells. Thus the GLPL/OVA formulation produced a heightened humoral and cellular immune response, with an overall Th1 bias. Enhanced immune responses elicited by the GLPL/OVA formulation might be attributed to effective activation and mature of DC in draining lymph nodes. Overall, these findings indicate that GLPL have the potential to enhance immune responses as vaccine delivery systems.

The activation of murine peritoneal macrophages by Ganoderma lucidum polysaccharides liposomes (GLPL) was investigated in vitro. After treatment with GLPL, the changes of the nitric oxide (NO) secretion and iNOS (inducible nitric oxide synthase) activity were evaluated. The results showed that NO production and iNOS activity of macrophages were enhanced compared to GLP and BL group. In addition, both the phagocytic activity and levels of cytokines IL-1β, TNF-α and IFN-γ were enhanced in the peritoneal macrophages of mice by stimulation of GLPL. The expression of the major histocompatibility complex class II molecule (MHC II) on the surface of peritoneal macrophages significantly increased. These indicated that GLPL could enhance the activation of peritoneal macrophages and their potential for use as a delivery system of GLP.

Adjuvants improve vaccine potency by enhancing immunogenicity and sustaining long-term immune responses. Lentinan (LNT), a β-1,3-glucohexaose with β-1,6-branches, is extracted from the mushroom Lentinus edodes and functions as an effective immunostimulatory drug. Previous studies have demonstrated the adjuvant activity of calcium carbonate (CaCO3) microspheres as well as their use as antigen delivery systems. In this study, we successfully loaded CaCO3 microspheres with LNT and evaluated their physicochemical characteristics prior to the adsorption of ovalbumin. Our experimental results demonstrated that LNT-CaCO3 significantly enhanced lymphocyte proliferation, and boosted the frequency of CD69 + B cells and the ratio of CD4+ to CD8 + T cells in spleen lymphocytes. Moreover, LNT-CaCO3 unexpectedly induced the secretion of IgG and Th-associated cytokines (IL-2, IL-4, IFN-γ, and TNF-α) in immunized mice. Therefore, LNT-CaCO3 microspheres induce robust cellular and humoral immune responses and have potential utility as vaccine delivery systems.

Decoquinate (DQ) is used for prophylaxis against coccidian infections within the digestive tract of chickens, but DQ is extremely insoluble in water. Hence, improving the water solubility of DQ is extremely important. First, decoquinate nanoliposomes (DQNLs) were prepared by the thin-film dispersion–ultrasonic method. The preparation conditions of DQNLs were optimized using the orthogonal test. The optimal preparation conditions of DQNLs were: a ratio of egg-yolk lecithin:drug (w/w) of 10:1, ratio of egg-yolk lecithin:cholesterol (w/w) of 5:1, rotary-evaporation temperature of 50 ℃, and ultrasound duration of 15 min. The encapsulation efficiency of DQNLs prepared under these conditions reached 99.24 % and drug loading was 5.67 %. The characterization of optimized DQNLs was also done. Transmission electron microscopy of DQNLs showed that they had the characteristic structure of liposomes. The mean particle size was 115.6 nm. The polydispersity index was 0.175. The zeta potential was −39.1 mV. The stability of DQNLs was high upon storage at 4 ℃. In vivo studies demonstrated that the lower dose (5 mg/L) of DQNLs in drinking water obtained the similar anticoccidial efficacy to that of 40 mg/kg DQ in feed against diclazuril-resistance Eimeria tenella isolate. The in vitro inhibitory effect of DQNLs on the sporulation of Eimeria tenella oocysts was dose-dependent. Therefore, the anticoccidial efficacy of DQ was enhanced significantly after being encapsulated into nanoliposomes.

Superparamagnetic iron oxide (SPIO) nanoparticles were extensively employed for theranostic applications, due to their good biocompatibility and excellent magnetic resonance imaging (MRI) properties. However, these particles typically require surface modification due to their hydrophobic surfaces caused by the oil-phase surfactants used in the fabrication and the drug loading on their surface is usually limited. Here, we provided a novel and facile approach to conveniently perform surface modification of SPIO while simultaneously loading a large amount of drug. By synthesizing an amphiphilic irinotecan-based compound, which hydrophobic tail enabled to insert into the SPIOs assembly, an excellent SPIO-based theranostic nanomedicine (SPIO@IR) was formed. The SPIO@IR can not only extensively improve the drug efficacy, but also enable to visualize themselves by MRI in the biological system.

Dendritic cells (DCs) are professional antigen-presenting cells (APC) that play a central role in the initiation and regulation of immune responses. We have previously demonstrated that Lycium barbarum polysaccharides liposomes (LBPL) as immune adjuvant elicits strong antigen-specific Th1 immune responses. The purpose of this study was to investigate underlying mechanism of liposomes promoting effect of Lycium barbarum polysaccharides (LBP) on activating DCs. LBP were loaded with high entrapment efficiency (86%) into liposomes using reverse phase evaporation. LBPL activation of phenotypic and functional maturation of DCs was explored through mechanistic studies of the TLR4-MyD88-NF-κB signaling pathway and amount of proinflammatory cytokines released. We found that LBPL indeed activated immature DCs and induced DCs maturation characterized by up-regulation of co-stimulatory molecules (MHCII, CD80, CD86), production of cytokines (IL-12p40, TNF-α), and enhancement of antigen uptake. Additionally, we demonstrated that liposomes could promote LBP up-regulation of TLR4, MyD88, TRAF6, NF-κB gene and protein expression.

Sisi Zheng,1,* Tao Qin,2,* Yu Lu,3 Yifan Huang,2 Li Luo,1 Zhenguang Liu,1 Ruonan Bo,1 Yuanliang Hu,1 Jiaguo Liu,1 Deyun Wang1,2 1Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 2Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal, Fujian Agriculture and Forestry University, Fuzhou, 3National Veterinary Product Engineering Research Center, Jiangsu Academy of Agricultural Sciences, Nanjing, People’s Republic of China *These authors contributed equally to this work Background: Poly lactide (PLA) was proved in the last years to be good for use in sustained drug delivery and as carriers for vaccine antigens. In our previous research, pachyman (PHY)-encapsulated PLA (PHYP) nanospheres were synthesized and their function of controlling drug release was demonstrated.Purpose: In order to modify the fast drug-release rate of PHY when inoculated alone, the maturation of bone marrow dendritic cells (BMDCs) in vitro and their immunological enhancement in vivo were explored using PHYP nanospheres.Methods: The maturation and antigen uptake of BMDCs were evaluated, both alone and with formulated antigen PHYP nanospheres, ie, ovalbumin (OVA)-loaded PHYP nanospheres, as an antigen delivery system, to investigate antigen-specific humoral and cellular immune responses.Results: The results indicated that, when stimulated by PHYP, the BMDCs matured as a result of upregulated expression of co-stimulatory molecules; the mechanism was elucidated by tracing fluorescently labeled antigens in confocal laser scanning microscopy images and observing the uptake of nanospheres by transmission electron microscopy. It was further revealed that mice inoculated with OVA-PHYP had augmented antigen-specific IgG antibodies, increased cytokine secretion by splenocytes, increased splenocyte proliferation, and activation of cluster of differentiation (CD)4+ and CD8+ T cells in vivo. Elevated immune responses were produced by OVA-PHYP, possibly owing to the activation and maturation of dendritic cells (in draining lymph nodes).Conclusion: It was corroborated that PHY- and/or OVA-encapsulated PLA nanospheres elicited prominent antigen-presenting effects on BMDCs and heightened humoral and cellular immune responses compared with other formulations. Keywords: PHYP, bone marrow dendritic cell, antigen delivery system, immune response

The purpose of this study was to optimize the preparation conditions of Lycium barbarum polysaccharides liposome (LBPL) by response surface methodology (RSM) and to investigate the effect of LBPL activating function of peritoneal macrophages. LBPL was prepared using the reverse-phase evaporation method. The optimal preparation conditions of LBPL by RSM were as follows: the ratio of lipid to drug (w/w) of 25:1, the ultrasound time of 14 min and the ratio of soybean phospholipids to cholesterol (w/w) of 2.4:1. Under these conditions, the experimental encapsulation efficiency of LBPL was 86.37±0.63%, which was close to the predicted value. These indicated that LBPL with high entrapping efficiency and small particle size could be prepared by the reverse-phase evaporation method, which is applied easily. Furthermore, macrophages are the key players in the innate immune system. LBPL could effectively enhance peritoneal macrophages phagocytosis and resulted in inducing NO (nitric oxide) production in mouse peritoneal macrophages.

The success of subunit vaccines has been hampered by the problems of weak or short-term immunity and the lack of availability of nontoxic, potent adjuvants. It would be desirable to develop safe and efficient adjuvants with the aim of improving the cellular immune response against the target antigen. In this study, the targeting and sustained release of simple nanoliposomes containing Lycium barbarum polysaccharides (LBP) as an efficacious immune adjuvant to improve immune responses were explored. LBP liposome (LBPL) with high entrapment efficiency (86%) were obtained using a reverse-phase evaporation method and then used to encapsulate the model antigen, ovalbumin (OVA). We demonstrated that the as-synthesized liposome loaded with OVA and LBP (LBPL-OVA) was stable for 45 days and determined the encapsulation stability of OVA at 4°C and 37°C and the release profile of OVA from LBPL-OVA was investigated in pH 7.4 and pH 5.0. Further in vivo investigation showed that the antigen-specific humoral response was correlated with antigen delivery to the draining lymph nodes. The LBPL-OVA were also associated with high levels of uptake by key dendritic cells in the draining lymph nodes and they efficiently stimulated CD4+ and CD8+ T cell proliferation in vivo, further promoting antibody production. These features together elicited a significant humoral and celluar immune response, which was superior to that produced by free antigen alone.