Rational Design of PLGA Nanoparticle Vaccine Delivery Systems To Improve 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.

Chitosan-coated PLGA microemulsion loaded with tannic acid against Escherichia coli in vitro and in vivo.

The overuse of antibiotics has resulted in a surge of drug-resistant bacteria, making the pursuit of natural antimicrobials an urgent and significant trend. Encapsulation and nanoparticulation are effective ways to enhance the antibacterial properties of natural drugs. In this study, we encapsulated tannic acid (TA) with chitosan (CS) and poly (lactide-co-glycolide) (PLGA) using the emulsion-solvent evaporation method to enhance the antimicrobial effect of TA. We prepared a bilayer membrane spherical nanoemulsion of TA-PLGA-CS (TPC) with uniform size of 559.87 ± 1.16 nm, and zeta potential of 59.53 ± 1.07 mV. TPC could be stably stored for 90 days at 4°C without affecting the properties of the emulsion, and the minimum bactericidal concentration against four strains of Escherichia coli (E. coli) remained unchanged for 60 d. The results indicated that TPC enhanced the inhibitory effect of TA against E. coli. Scanning electron microscope images revealed that TPC treatment caused damage to the bacterial cell membrane. In addition, in vivo experiments indicated that TPC exhibited a superior therapeutic effect on artificial colibacillosis in chickens infested with Avian pathogenic Escherichia coli, as evidenced by the changes in body weight and a reduction bacterial load in heart. Furthermore, TPC reversed the down-regulation of catalase, glutathione peroxidase1 (GPX1), and GPX7 gene expression levels in intestinal tissues. Compared to the model group, TPC treatment elevated serum glutathione peroxidase activities and lowered myeloperoxidase and lactate dehydrogenase levels, offering antioxidant protection that was slightly better than that of doxycycline hydrochlorid group. In summary, we prepared a novel TA antimicrobial preparation with significant antioxidant potential and inhibitory effect against E. coli both in vitro and in vivo.

Alhagi honey polysaccharides encapsulated into PLGA nanoparticle-based pickering emulsion as a novel adjuvant to induce strong and long-lasting immune responses.

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.

Evaluation of optimum conditions for decoquinate nanoliposomes and their anticoccidial efficacy against diclazuril-resistant Eimeria tenella infections in broilers.

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.

Surface-Engineered Cubosomes Serve as a Novel Vaccine Adjuvant to Modulate Innate Immunity and Improve Adaptive Immunity in vivo.

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.

Lentinan-Functionalized Graphene Oxide Is an Effective Antigen Delivery System That Modulates Innate Immunity and Improves Adaptive Immunity.

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.

Designing selenium polysaccharides-based nanoparticles to improve immune activity of Hericium erinaceus.

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-1ß 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.

Adjuvanticity of Ganoderma lucidum polysaccharide liposomes on porcine circovirus type-II in mice.

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.

Mechanism of Lycium barbarum polysaccharides liposomes on activating murine dendritic cells.

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.

Maturation of dendritic cells in vitro and immunological enhancement of mice in vivo by pachyman- and/or OVA-encapsulated poly(d,l-lactic acid) nanospheres.

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.

Simple nanoliposomes encapsulating Lycium barbarum polysaccharides as adjuvants improve humoral and cellular immunity in mice.

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.

Exploring the immunopotentiation of Chinese yam polysaccharide poly(lactic-co-glycolic acid) nanoparticles in an ovalbumin vaccine formulation in vivo.

Biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) has been approved by the US Food and Drug Administration and has frequently been used to develop potential vaccine delivery systems. The immunoregulation and immunopotentiation of Chinese yam polysaccharide (CYP) have been widely demonstrated. In the current study, cell uptake mechanisms in dendritic cells (DCs) were monitored in vitro using confocal laser scanning microscopy, transmission electron microscopy, and flow cytometry. To study a CYP-PLGA nanoparticle-adjuvanted delivery system, CYP and ovalbumin (OVA) were encapsulated in PLGA nanoparticles (CYPPs) to act as a vaccine, and the formulation was tested in immunized mice. The CYPPs more easily underwent uptake by DCs in vitro, and CYPP/OVA could stimulate more effective antigen-specific immune responses than any of the single-component formulations in vivo. Mice immunized using CYPP/OVA exhibited more secretion of OVA-specific IgG antibodies, better proliferation, and higher cytokine secretion by splenocytes and significant activation of CD3+CD4+ and CD3+CD8+ T cells. Overall, the CYPP/OVA formulation produced a stronger humoral and cellular immune response and a mixed Th1/Th2 immune response with a greater Th1 bias in comparison with the other formulations. In conclusion, the data demonstrate that the CYPP-adjuvanted delivery system has the potential to strengthen immune responses and lay the foundation for novel adjuvant design.

Preparation and characterization of Chinese yam polysaccharide PLGA nanoparticles and their immunological activity.

This paper first provides that Chinese yam polysaccharide (CYP) is encapsulated by PLGA using a double emulsion solvent evaporation method and aims to screen the optimal preparation of CYP-PLGA nanoparticles (CYPP) using response surface methodology (RSM). The volume ratio of the internal water phase to the organic phase (W1:O), the volume ratio of the primary emulsion to the external water phase (PE:W2) and the concentration of Poloxamer 188 (F68) are deemed key variables for the encapsulation efficiency of CYPP. The results demonstrated that the data were accurately fitted into the RSM model. According to the RSM, the optimal scheme was a volume ratio of W1:O of 1:9, a volume ratio of PE: W2 of 1:10 and a concentration of F68 (W/V) of 0.7%. TEM and SEM images demonstrated that the nanoparticles had a spherical shape and smooth surface. The CYP and CYPP in vitro release studies demonstrated that the CYPP showed a release rate 53.41% lower than the release rate of CYP after 48h. The result of pro-proliferation and flow cytometry emerged that the CYPP were more effective compared with the free CYP and blank PLGA nanoparticles in promoting lymphocyte proliferation and triggering the transformation of T lymphocytes into Th cells.

Evaluation of optimum conditions for pachyman encapsulated in poly(d,l-lactic acid) nanospheres by response surface methodology and results of a related in vitro study.

This study aimed to optimize the preparation conditions of pachyman (PHY)-loaded poly(d,l-lactic acid) (PLA) (PHYP) nanospheres by response surface methodology, explore their characteristics, and assess their effects on splenic lymphocytes. Double emulsion solvent evaporation was used to synthesize PHYP nanospheres, and the optimal preparation conditions were identified as a concentration of poloxamer 188 (F68) (w/v) of 0.33%, a concentration of PLA of 30 mg/mL, and a ratio of PLA to drug (w/w) of 10.25:1 required to reach the highest encapsulation efficiency, which was calculated to be 59.10%. PHYP had a spherical shape with a smooth surface and uniform size and an evident effect of sustained release and relative stability. Splenic lymphocytes are crucial and multifunctional cells in the immune system, and their immunological properties could be enhanced significantly by PHYP treatment. This study confirmed that PHY encapsulated in PLA nanospheres had comparatively steady properties and exerted obvious immune enhancement.

Ganoderma lucidum polysaccharides encapsulated in liposome as an adjuvant to promote Th1-bias immune response.

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.

Rehmannia glutinosa polysaccharide liposome as a novel strategy for stimulating an efficient immune response and their effects on dendritic cells.

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.

Activation effect of Ganoderma lucidum polysaccharides liposomes on murine peritoneal macrophages.

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-1ß, 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.

The enhanced immune response of PCV-2 vaccine using Rehmannia glutinosa polysaccharide liposome as an adjuvant.

Liposomes, one kind of vaccine adjuvants, have been demonstrated as effective adjuvants and vaccine delivery system. Immunization against PCV-2 has been studied intensely and found to be the most effective strategy for protecting pigs against PCV-2 infection. Inactivated vaccines represent a complex mixture of viral antigens closely resembling the native virion. In the present study, PCV-2 attenuated antigen was encapsulated within Rehmannia glutinosa polysaccharide liposome, instead of oil adjuvant which is the mainstream adjuvant. Our results showed that RGPL could elicit a strong IgG response and significantly increased the production of Th1 and Th2 associated IgG subtypes and cytokines. R. glutinosa polysaccharide liposome showed excellent particle stability. In vitro, R. glutinosa polysaccharide liposome could also significantly promote phagocytic activity of macrophage and the levels of cytokines it produced. Overall, the results demonstrated that R. glutinosa polysaccharide liposome has the potential to be developed into a more effective and safer vaccine adjuvant.

The immunological activity of Lycium barbarum polysaccharides liposome in vitro and adjuvanticity against PCV2 in vivo.

In previous researches, the results showed that Lycium barbarum polysaccharides (LBP) encapsulated with liposome could enhance the immune activity of LBP. Therefore, the present study was designed to investigate the effects of LBPL on spleen lymphocytes and macrophages of mice in vitro and evaluate the immunological adjuvant activity of PCV2 vaccine in vivo. The results showed that LBPL could significantly promote splenocyte proliferation synergistically with PHA or LPS, increase the ratio of CD4(+) to CD8(+) T cells and promote the cytokine secretion of macrophages; enhance PCV2-specific IgG antibody responses, promote Th1 cytokines (IFN-γ and TNF-a) and Th2 cytokine (IL-4) secretion. The histomorphological observation of spleen demonstrated that LBPL as a vaccine adjuvant also has good improvement and stimulating effect on the immune organ.

Optimization on conditions of Lycium barbarum polysaccharides liposome by RSM and its effects on the peritoneal macrophages function.

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.