Hongwu Sun

Development and characterization of a novel nanoemulsion drug-delivery system for potential application in oral delivery of protein drugs

Background: The stability of protein drugs remains one of the key hurdles to their success in the market. The aim of the present study was to design a novel nanoemulsion drug-delivery system (NEDDS) that would encapsulate a standard-model protein drug – bovine serum albumin (BSA) – to improve drug stability. Methods: The BSA NEDDS was prepared using a phase-inversion method and pseudoternary phase diagrams. The following characteristics were studied: morphology, size, zeta potential, drug loading, and encapsulation efficiency. We also investigated the stability of the BSA NEDDS, bioactivity of BSA encapsulated within the NEDDS, the integrity of the primary, secondary, and tertiary structures, and specificity. Results: The BSA NEDDS consisted of Cremophor EL-35, propylene glycol, isopropyl myristate, and normal saline. The average particle diameter of the BSA NEDDS was about 21.8 nm, and the system showed a high encapsulation efficiency (>90%) and an adequate drug-loading capacity (45 mg/mL). The thermodynamic stability of the system was investigated at different temperatures and pH levels and in room-temperature conditions for 180 days. BSA NEDDS showed good structural integrity and specificity for the primary, secondary, and tertiary structures, and good bioactivity of the loaded BSA. Conclusions: BSA NEDDS showed the properties of a good nanoemulsion-delivery system. NEDDS can greatly enhance the stability of the protein drug BSA while maintaining high levels of drug bioactivity, good specificity, and integrity of the primary, secondary, and tertiary protein structures. These findings indicate that the nanoemulsion is a potential formulation for oral administration of protein drugs.

Induction of systemic and mucosal immunity against methicillin-resistant Staphylococcus aureus infection by a novel nanoemulsion adjuvant vaccine.

The Gram-positive bacterial pathogen methicillin-resistant Staphylococcus aureus (MRSA) can cause infections in the bloodstream, endocardial tissue, respiratory tract, culture-confirmed skin, or soft tissue. There are currently no effective vaccines, and none are expected to become available in the near future. An effective vaccine capable of eliciting both systemic and mucosal immune responses is also urgently needed. Here, we reported a novel oil-in-water nanoemulsion adjuvant vaccine containing an MRSA recombination protein antigen, Cremophor EL-35(®) as a surfactant, and propylene glycol as a co-surfactant. This nanoemulsion vaccine, whose average diameter was 31.34±0.49 nm, demonstrated good protein structure integrity, protein specificity, and good stability at room temperature for 1 year. The intramuscular systemic and nasal mucosal immune responses demonstrated that this nanoemulsion vaccine could improve the specific immune responses of immunoglobulin (Ig)G and related subclasses, such as IgG1, IgG2a, and IgG2b, as well as IgA, in the serum after Balb/c mice intramuscular immunization and C57 mice nasal immunization. Furthermore, this nanoemulsion vaccine also markedly enhanced the interferon-γ and interleukin-17A cytokine cell immune response, improved the survival ratio, and reduced bacterial colonization. Taken together, our results show that this novel nanoemulsion vaccine has great potential and is a robust generator of an effective intramuscular systemic and nasal mucosal immune response without the need for an additional adjuvant. Thus, the present study serves as a sound scientific foundation for future strategies in the development of this novel nanoemulsion adjuvant vaccine to enhance both the intramuscular systemic and nasal mucosal immune responses.

Genotyping analysis of Helicobacter pylori using multiple-locus variable-number tandem-repeats analysis in five regions of China and Japan

BackgroundH. pylori (Helicobacter pylori) is the major causative agent of chronic active gastritis. The population of H. pylori shows a high genomic variability among isolates. And the polymorphism of repeat-units of genomics had participated the important process of evolution. Its long term colonization of the stomach caused different clinical outcomes, which may relate to the high degree of genetic variation of H. pylori. A variety of molecular typing tools have been developed to access genetic relatedness in H. pylori isolates. However, there is still no standard genotyping system of this bacterium. The MLVA (Multi-locus of variable number of tandem repeat analysis) method is useful for performing phylogenetic analysis and is widely used in bacteria genotyping; however, theres little application in H. pylori analysis. This article is the first application of the MLVA method to investigate H. pylori from different districts and ethnic groups of China.ResultsMLVA of 12 VNTR loci with high discrimination power based on 30 candidates were performed on a collection of 202 strains of H. pylori which originated from five regions of China and Japan. Phylogenetic tree was constructed using MLVA profiles. 12 VNTR loci presented with high various polymorphisms, and the results demonstrated very close relationships between genotypes and ethnic groups.ConclusionsThis study used MLVA methodology providing a new perspective on the ethnic groups and distribution characteristics of H. pylori.

A novel nitro-dexamethasone inhibits agr system activity and improves therapeutic effects in MRSA sepsis models without antibiotics

Methicillin-resistant Staphylococcus aureus (MRSA) sepsis is a life-threatening medical condition that involves systemic inflammation throughout the body. Glucocorticoids are widely used in combination with antibiotics in the treatment of MRSA sepsis to fight the overwhelming inflammation. Here, we describe the improved anti-inflammatory properties of a nitric oxide (NO)-releasing derivative of dexamethasone, ND8008. ND8008 affected MRSA biofilm formation, caused biofilm cell death, and reduced the effects of virulence factors, such as α-toxin, by inhibiting the activity of the Staphylococcus aureus accessory gene regulator (agr) system. Dosing of mice with ND8008 (127.4 nmol/kg, i.p.) alone greatly reduced the inflammatory response caused by MRSA blood stream infection and considerably increased the survival rate of septic mice. These findings suggest that this novel NO-releasing derivative of dexamethasone ND8008 could be helpful in the treatment of MRSA sepsis.

Immune response effects of diverse vaccine antigen attachment ways based on the self-made nanoemulsion adjuvant in systemic MRSA infection

Nanoemulsion adjuvants-based vaccines have potent induced immune responses against methicillin-resistant Staphylococcus aureus (MRSA) infection. However, the efficacies and immune responses of different antigen-attaching ways on self-made nanoemulsion adjuvants remain unknown. In this study, we designed three formulations of nanoemulsion adjuvants (encapsulation, mixture, and combination) to explore their immune response-enhancing effects and their underlying mechanism in a systemic infection model of MRSA. Our results showed that the three nanoemulsion-attachment ways formulated with a fusion antigen of MRSA (HlaH35LIsdB348–465) all improved humoral and cellular immune responses. When compared with the mixture and combination formulations, the nanoemulsion-encapsulation group effectively promoted the antigen uptake of dendritic cells (DCs) in vitro, the activation of DC in draining lymph nodes and the delayed release of antigen at injection sites in vivo. Moreover, the encapsulation group induced a more ideal protective efficacy in a MRSA sepsis model by inducing more potent antibody responses and a Th1/Th17 biased CD4+ T cell response when compared with the other two attachment ways. Our findings suggested that the encapsulated formulation of vaccine with nanoemulsion adjuvant is an effective attachment way to provide protective immunity against MRSA infection.

Intranasal immunization with immunodominant epitope peptides derived from HpaA conjugated with CpG adjuvant protected mice against Helicobacter pylori infection

HpaA is considered to be an effective protective antigen for vaccination against Helicobacter pylori (H. pylori) infection. Oral immunization with HpaA significantly decreases bacterial colonization in H. pylori infected mice. In this study, we investigated whether subcutaneous or intranasal immunization with HpaA could protect against H. pylori infection. Mice immunized subcutaneously with HpaA in Complete Freunds adjuvant, but not mice intranasally immunized with HpaA in CpG adjuvant acquired protection against H. pylori infection. However, intranasal immunization with immunodominant epitope peptides in CpG adjuvant protected mice against H. pylori infection, and immunodominant epitope peptides stimulated stronger Th1 responses and mediated more robust protection against H. pylori infection than subdominant ones. Our results suggest that the length of a candidate antigen is critical for particular vaccination routes, and that immunodominant epitope peptides are promising candidates for protection against H. pylori infection through nasal vaccination.

Protective effects of a nanoemulsion adjuvant vaccine (2C-Staph/NE) administered intranasally against invasive Staphylococcus aureus pneumonia

No licensed Staphylococcus aureus (S. aureus) vaccine is currently available. To develop an effective S. aureus vaccine, we selected the recombinant proteins staphylococcal enterotoxin B (rSEB) and manganese transport protein C (rMntC) as vaccine candidates and formulated a 2C-Staph vaccine. Based on the optimised formation of nanoemulsion (NE) technology, we constructed a novel NE adjuvant vaccine, 2C-Staph/NE. The 2C-Staph/NE particles showed a suitable diameter (24.9 ± 0.14 nm), a good protein structure of integrity and specificity, and high thermodynamic stability. 2C-Staph formulated with an NE adjuvant induced higher survival rates than a 2C-Staph/MF59 vaccine in sepsis and pneumonia models. Moreover, intramuscular vaccination with 2C-Staph/NE yielded protection efficacy in a sepsis model, and the intranasal vaccination route induced a potent protective effect in a pneumonia model. Intranasal vaccination with 2C-Staph/NE induced a strong mucosal response with high levels of IgA and IL-17A in bronchoalveolar lavage fluid (BALF), and the IgG levels in the BALF were comparable to those induced by the intramuscular vaccination route. Furthermore, the serum and BALF induced by intranasal administration showed potent opsonophagocytic activity against S. aureus. And, the IL-17A played a protective role in the pneumonia model demonstrated by a cytokine neutralization test. Taken together, our results showed that intranasal administration of 2C-Staph formulated with an NE adjuvant yielded ideal protection in a murine S. aureus pneumonia model.

Chitosan-polyvinyl alcohol nanoscale liquid film-forming system facilitates MRSA-infected wound healing by enhancing antibacterial and antibiofilm properties

Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most predominant and fatal pathogens at wound infection sites. MRSA is difficult to treat because of its antibiotic resistance and ability to form biofilms at the wound site. Methods In this study, a novel nanoscale liquid film-forming system (LFFS) loaded with benzalkonium bromide was produced based on polyvinyl alcohol and chitosan. Results This LFFS showed a faster and more potent effect against MRSA252 than benzalkonium bromide aqueous solution both in vitro and in vivo. Additionally, the LFFS had a stronger ability to destroy biofilms (5 mg/mL) and inhibit their formation (1.33 μg/mL). The LFFS inflicted obvious damage to the structure and integrity of MRSA cell membranes and caused increases in the release of alkaline phosphate and lactate dehydrogenase in the relative electrical conductivity and in K+ and Mg2+ concentrations due to changes in the MRSA cell membrane permeability. Conclusion The novel LFFS is promising as an effective system for disinfectant delivery and for application in the treatment of MRSA wound infections.

Development of a safety and efficacy nanoemulsion delivery system encapsulated gambogic acid for acute myeloid leukemia in vitro and in vivo

&NA; This study aimed to improve the solubility, reduce the side effects and enhance the efficacy of gambogic acid against acute myeloid leukemia in vitro and in vivo. This oil‐in‐water nanoemulsion (average size 17.20 ± 0.11 nm, zeta potential 4.17 ± 0.82 mV) containing Tween‐80, glycol, squalene and gambogic acid with improving 4000 times solubility was prepared by pseudoternary phase diagrams. We found that this nanoemulsion successfully encapsulated gambogic acid; it was stable and showed an obvious delayed release effect for the drug in three different phosphate‐buffered saline (pH = 2.0, 5.8 and 7.4). The half inhibiting concentration (IC50) of this nanoemulsion (480.7 &mgr;g/mL and 408 &mgr;g/mL) were 1.67 times and 1.98 times higher than those of its water solution (287 &mgr;g/mL and 206 &mgr;g/mL) after acting on the toxicity standard cell line (L929 line) for 24 h and 48 h, respectively. Importantly, acute injection toxicity indicated that the half lethal dose (LD50) of this nanoemulsion (23.25 mg/kg, 95% LD50, 21.7–25.16 mg/kg) was 1.26 times higher than that of its water solution (18.59 mg/kg, 95% LD50, 16.84–20.53 mg/kg). Compared with its suspension, the bioavailability of this nanoemulsion was 318.2%. Furthermore, this nanoemulsion had a better efficacy against the acute myeloid leukemia in vitro and in vivo by improving the time and percent of survival (MV4‐11 engrafts mice) and reducing half inhibiting concentration values in acute myeloid leukemia such as Jurket, HL‐60 and MV4‐11 cells. Our studies suggested that this nanoemulsion may be a promising therapeutic medicine for acute myeloid leukemia.

An immunopotentiator, ophiopogonin D, encapsulated in a nanoemulsion as a robust adjuvant to improve vaccine efficacy

As an ingredient of vaccines, adjuvants are indispensable for enhancing and directly inducing robust and extensive adaptive immune responses associated with vaccine antigens. In this study, we initially determined that a new molecular immunopotentiator, ophiopogonin D (OP-D), enhanced the antibody response to antigen. Because OP-D has certain disadvantages, including poor solubility, we next encapsulated OP-D in a nanoemulsion adjuvant (nanoemulsion-encapsulated OP-D, NOD) using low-energy emulsification methods. The NOD thus produced was small, with an average size of 76.45 nm, and exhibited good distribution (PdI value 0.16), significantly increasing the solubility of OP-D. Furthermore, NOD exhibited reduced cellular toxicity and acute toxicity. Our results showed that a fusion antigen of MRSA (HlaH35LIsdB348-465) formulated with NOD significantly improved humoral and cellular immune responses compared to those observed in the antigen/OP-D and antigen/AlPO4 groups. Compared with antigen/OP-D, the antigen formulated with NOD more effectively promoted antigen uptake by dendritic cells (DCs) and the activation of antigen-presenting cells (APCs). Moreover, the NOD-formulated antigen had ideal protective efficacy in a MRSA sepsis model by inducing more potent antibody responses and a Th1/Th17-biased CD4+ T cell immune response. Therefore, these results suggest that NOD is a promising and robust adjuvant platform for a MRSA vaccine. STATEMENT OF SIGNIFICANCE We first identified a new powerful immunopotentiator, Ophiopogonin D, among dozens of natural products and then used nanotechnology to construct a highly efficient and low toxic adjuvant system (NOD). Our approach intersects natural medicinal chemistry, nanomaterials and immunology, revealing that a strong adjuvant activity of this adjuvant system was verified in vitro and in vivo, and the application of MRSA subunit vaccine model for survival experiments achieved a 100% protection rate. This research illustrate that NOD is a promising and robust adjuvant platform for subunit vaccines.