Construction of fatty acid-ovalbumin binary complexes to improve the water dispersibility, thermal/digestive stability and bioaccessibility of lutein: A comparative study of different fatty acids.

Lipids are increasingly being incorporated into delivery systems due to their ability to facilitate intestinal absorption of lipid-soluble nutrients through molecular solubilization and micellization. In this work, self-assembled complexes of ovalbumin (OVA) and nine dietary fatty acids (FAs) were constructed to improve the processability and absorbability of lutein (LUT). Results showed that all FAs could form stable hydrophilic particles with OVA under the optimized ultrasound-coupled pH conditions. Fourier infrared spectroscopy and transmission electron microscopy analysis showed that these binary complexes effectively encapsulated LUT with an encapsulation rate > 90.0 %. Stability experiments showed that these complexes protected LUT well, which could improve thermal stability and in vitro digestive stability by 1.66-3.58-fold and 1.27-2.74-fold, respectively. Besides, the bioaccessibility of LUT was also enhanced by 7.16-24.99-fold. The chain length and saturation of FAs affected the stability and absorption of LUT. Therefore, these results provided some reference for the selection of FAs for efficient delivery of lipid-soluble nutrients.

Near-infrared-II responsive ovalbumin functionalized gold-genipin nanosystem cascading photo-immunotherapy of cancer.

Synergistic cancer therapies have attracted wide attention owing to their multi-mode tumor inhibition properties. Especially, photo-responsive photoimmunotherapy demonstrates an emerging cancer treatment paradigm that significantly improved treatment efficiency. Herein, near-infrared-II responsive ovalbumin functionalized Gold-Genipin nanosystem (Au-G-OVA NRs) was designed for immunotherapy and deep photothermal therapy of breast cancer. A facile synthesis method was employed to prepare the homogeneous Au nanorods (Au NRs) with good dispersion. The nanovaccine was developed further by the chemical cross-linking of Au-NRs, genipin and ovalbumin. The Au-G-OVA NRs outstanding aqueous solubility, and biocompatibility against normal and cancer cells. The designed NRs possessed enhanced localized surface plasmon resonance (LSPR) effect, which extended the NIR absorption in the second window, enabling promising photothermal properties. Moreover, genipin coating provided complimentary red fluorescent and prepared Au-G-OVA NRs showed significant intracellular encapsulation for efficient photoimmunotherapy outcomes. The designed nanosystem possessed deep photothermal therapy of breast cancer and 90% 4T1 cells were ablated by Au-G-OVA NRs (80µg ml-1concentration) after 1064 nm laser irradiation. In addition, Au-G-OVA NRs demonstrated outstanding vaccination phenomena by facilitating OVA delivery, antigen uptake, maturation of bone marrow dendritic cells, and cytokine IFN-γsecretion for tumor immunosurveillance. The aforementioned advantages permit the utilization of fluorescence imaging-guided photo-immunotherapy for cancers, demonstrating a straightforward approach for developing nanovaccines tailored to precise tumor treatment.

Self-assembled protein vesicles as vaccine delivery platform to enhance antigen-specific immune responses.

Self-assembling protein nanoparticles are beneficial platforms for enhancing the often weak and short-lived immune responses elicited by subunit vaccines. Their benefits include multivalency, similar sizes as pathogens and control of antigen orientation. Previously, the design, preparation, and characterization of self-assembling protein vesicles presenting fluorescent proteins and enzymes on the outer vesicle surface have been reported. Here, a full-size model antigen protein, ovalbumin (OVA), was genetically fused to the recombinant vesicle building blocks and incorporated into protein vesicles via self-assembly. Characterization of OVA protein vesicles showed room temperature stability and tunable size. Immunization of mice with OVA protein vesicles induced strong antigen-specific humoral and cellular immune responses. This work demonstrates the potential of protein vesicles as a modular platform for delivering full-size antigen proteins that can be extended to pathogen antigens to induce antigen specific immune responses.

Changes of structure properties and potential allergenicity of ovalbumin under high hydrostatic pressures.

Egg proteins, notably ovalbumin (OVA), contribute to a prevalent form of food allergy, particularly in children. This study aims to investigate the impact of high hydrostatic pressure (HHP) treatment at varying levels (300, 400, 500, and 600 MPa) on the molecular structure and allergenicity of OVA. The structure of HHP-treated OVA was assessed through fluorescence spectroscopy, circular dichroism spectroscopy, and molecular dynamics (MD) simulation. HHP treatment (600 MPa) altered OVA structures, such as α-helix content decreased from 28.07 % to 19.47 %, and exogenous fluorescence intensity increased by 8.8 times compared to that of the native OVA. The free sulfhydryl groups and zeta potential value were also increased with HHP treatment (600 MPa). ELISA analysis and MD simulation unveiled a noteworthy reduction in the allergenicity of OVA when subjected to 600 MPa for 10 min. Overall, this study suggests that the conformational changes in HHP-treated OVA contribute to its altered allergenicity.

Induction of antigen-specific immunity by mesoporous silica nanoparticles incorporating antigen peptides.

Mesoporous silica nanoparticles (MSNs) are physically and chemically stable inorganic nanomaterials that have been attracting much attention as carriers for drug delivery systems in the field of nanomedicine. In the present study, we investigated the potential of MSN vaccines that incorporate antigen peptides for use in cancer immunotherapy. In vitro experiments demonstrated that fluorescently labeled MSNs accumulated in a line of mouse dendritic cells (DC2.4 cells), where the particles localized to the cytosol. These observations could suggest that MSNs have potential for use in delivering the loaded molecules into antigen-presenting cells, thereby stimulating the host acquired immune system. In vivo experiments demonstrated prolonged survival in mice implanted with ovalbumin (OVA)-expressing lymphoma cells (E.G7-OVA cells) following subcutaneous inoculation with MSNs incorporating OVA antigen peptides. Furthermore, OVA-specific immunoglobulin G antibodies and cytotoxic T lymphocytes were detected in the serum and the spleen cells, respectively, of mice inoculated with an MSN-OVA vaccine, indicating the induction of antigen-specific responses in both the humoral and cellular immune systems. These results suggested that the MSN therapies incorporating antigen peptides may serve as novel vaccines for cancer immunotherapy.

Oral biomimetic virus vaccine hydrogel for robust abscopal antitumour efficacy.

Remarkable progress has been made in tumour immunotherapy in recent decades. However, the clinical outcomes of therapeutic interventions remain unpredictable, largely because of inefficient immune responses. To address this challenge and optimise immune stimulation, we present a novel administration route for enhancing the bioavailability of immunotherapeutic drugs. Our approach involves the development of an oral tumour vaccine utilising virus-like particles derived from the Hepatitis B virus core (HBc) antigen. The external surfaces of these particles are engineered to display the model tumour antigen OVA, whereas the interiors are loaded with cytosine phosphoguanosine oligodeoxynucleotide (CpG ODN), resulting in a construct called CpG@OVAHBc with enhanced antigenicity and immune response. For oral delivery, CpG@OVAHBc is encapsulated in a crosslinked dextran hydrogel called CpG@OVAHBc@Dex. The external hydrogel shield safeguards the biomimetic virus particles from degradation by gastric acid and proteases. Upon exposure to intestinal flora, the hydrogel disintegrates, releasing CpG@OVAHBc at the intestinal mucosal site. Owing to its virus-like structure, CpG@OVAHBc exhibits enhanced adhesion to the mucosal surface, facilitating uptake by microfold cells (M cells) and subsequent transmission to antigen-presenting cells. The enzyme-triggered release of this oral hydrogel ensures the integrity of the tumour vaccine within the digestive tract, allowing targeted release and significantly improving bioavailability. Beyond its efficacy, this oral hydrogel vaccine streamlines drug administration, alleviates patient discomfort, and enhances treatment compliance without the need for specialised injection methods. Consequently, our approach expands the horizons of vaccine development in the field of oral drug administration.

Biomimetic Multilayered Lipid Nanovesicles for Potent Protein Vaccination.

Lipid vesicles are widely used for drug and gene delivery, but their structural instability reduces in vivo efficacy and requires specialized handling. To address these limitations, strategies like lipid cross-linking and polymer-lipid conjugation are suggested to enhance stability and biological efficacy. However, the in vivo metabolism of these altered lipids remains unclear, necessitating further studies. A new stabilization technique without chemical modification is urgently needed. Here, a bio-mimetic approach for fabricating robust multilamellar lipid vesicles to enhance in vivo delivery and stabilization of protein antigens is presented. This method leverages 1-O-acylceramide, a natural skin lipid, to facilitate the self-assembly of lipid nanovesicles. Incorporating 1-O-acylceramide, anchoring lipid bilayers akin to its role in the stratum corneum, provides excellent stability under environmental stresses, including freeze-thaw cycles. Encapsulating ovalbumin as a model antigen and the adjuvant monophosphoryl lipid A demonstrates the vesicle's potential as a nanovaccine platform. In vitro studies show enhanced immune responses with both unilamellar and multilamellar vesicles, but in vivo analyses highlight the superior efficiency of multilamellar vesicles in inducing higher antibody and cytokine levels. This work suggests ceramide-induced multilamellar lipid vesicles as an effective nanovaccine platform for enhanced antigen delivery and stability.

PLGA micro/nanoparticle vaccination elicits non-tumor antigen specific resident memory CD8+ T cell protection from hepatocellular carcinoma.

Together, tumor and virus-specific tissue-resident CD8+ memory T cells (TRMs) of hepatocellular carcinoma (HCC) patients with Hepatitis B virus (HBV) infection can provide rapid frontline immune surveillance. The quantity and activity of CD8+ TRMs were correlated with the relapse-free survival of patients with improved health. However, HBV-specific CD8+ TRMs have a more exhausted phenotype and respond more actively under anti-PDL1 or PD1 treatment of HBV+HCC patients. Vaccination strategies that induce a strong and sustained CD8+ TRMs response are quite promising. Herein, a biodegradable poly(D,L-lactide-co-glycolide) microsphere and nanosphere particle (PLGA N.M.P) delivery system co-assembled by anti-PD1 antibodies (aPD1) and loaded with ovalbumin (OVA-aPD1 N.M.P) was fabricated and characterized for size (200 nm and 1 µm diameter), charge (-15 mV), and loading efficiencies of OVA (238 µg mg-1 particles) and aPD1 (40 µg mg-1 particles). OVA-aPD1 N.M.P could stimulate the maturation of BMDCs and enhance the antigen uptake and presentation by 2-fold compared to free OVA. The nanoparticles also induced the activation of macrophages (RAW 264.7) to produce a high level of cytokines, including TNF-α, IL-6 and IL-10. In vivo stimulation of mice using OVA-aPD1 N.M.P robustly enhanced IFN-γ-producing-CD8+ T cell infiltration in tumor tissues and the secretion of IgG and IgG2a/IgG1 antibodies. OVA-aPD1 N.M.P delivered OVA to increase the activation and proliferation of OVA-specific CD8+ TRMs, and its combination with anti-PD1 antibodies promoted complete tumor rejection by the reversal of tumor-infiltrating CD8+ T cell exhaustion. Thus, PLGA N.M.P could induce a strong CD8+ TRMs response, further highlighting its therapeutic potential in enhancing an antitumor immune response.

Fabrication of chitosan-based emulsion as an adjuvant to enhance nasal mucosal immune responses.

Nasal vaccine is a non-invasive vaccine that activates systemic and mucosal immunity in the presence of an adjuvant, thereby enhancing immune function. In this work, chitosan/oligochitosan/tween 80 (CS-COS-T80) co-stabilized emulsion was designed and further used as the nasal adjuvant. CS-COS-T80 emulsion exhibited outstanding stability under pH 6-8 with uniformly dispersed droplets and nano-scale particle size (<0.25 µm), and maintained stable at 4 °C for 150-day storage. Addition of model antigen ovalbumin (OVA) had no effect on the stability of CS-COS-T80 emulsion. In vivo nasal immunity indicated that CS-COS-T80 emulsion prolonged the retention time of OVA in the nasal cavity (from 4 to 8 h to >12 h), as compared to T80-emulsion. CS-COS-T80 emulsion produced a stronger mucosal immune response to OVA, with secretory IgA levels 5-fold and 2-fold higher than those of bare OVA and commercial adjuvant MF59, respectively. Compared to MF59, CS-COS-T80 induced a stronger humoral immune response and a mixed Th1/Th2 immune response of OVA after immunization. Furthermore, in the presence of CS-COS-T80 emulsion, the secretion of IL-4 and IFN-γ and the activation of splenocyte memory T-cell differentiation increased from 173.98 to 210.21 pg/mL and from 75.46 to 104.01 pg/mL, respectively. Therefore, CS-COS-T80 emulsion may serve as a promising adjuvant platform.

Spray drying the Pickering emulsions stabilized by chitosan/ovalbumin polyelectrolyte complexes for the production of oxidation stable tuna oil microcapsules.

The microencapsulation of polysaturated fatty acids by spray drying remains a challenge due to their susceptibility to oxidation. In this work, antioxidant Pickering emulsions were attempted as feeds to produce oxidation stable tuna oil microcapsules. The results indicated that the association between chitosan (CS) and ovalbumin (OVA) was a feasible way to fabricate antioxidant and wettable complexes and a high CS percentage favored these properties. The particles could yield tuna oil Pickering emulsions with enhanced oxidation stability through high-pressure homogenization, which were successfully spray dried to produce microcapsules with surface oil content of 8.84 % and microencapsulation efficiency of 76.65 %. The microcapsules exhibited significantly improved oxidation stability and their optimum peroxide values after storage at 50 °C, 85 % relative humidity, or natural light for 15 d were 48.67 %, 60.07 %, and 39.69 % respectively lower than the powder derived from the OVA-stabilized emulsion. Hence, Pickering emulsions stabilized by the CS/OVA polyelectrolyte complexes are potential in the production of oxidation stable polyunsaturated fatty acid microcapsules by spray drying.

Construction of lymph nodes-targeting tumor vaccines by using the principle of DNA base complementary pairing to enhance anti-tumor cellular immune response.

Tumor vaccines, a crucial immunotherapy, have gained growing interest because of their unique capability to initiate precise anti-tumor immune responses and establish enduring immune memory. Injected tumor vaccines passively diffuse to the adjacent draining lymph nodes, where the residing antigen-presenting cells capture and present tumor antigens to T cells. This process represents the initial phase of the immune response to the tumor vaccines and constitutes a pivotal determinant of their effectiveness. Nevertheless, the granularity paradox, arising from the different requirements between the passive targeting delivery of tumor vaccines to lymph nodes and the uptake by antigen-presenting cells, diminishes the efficacy of lymph node-targeting tumor vaccines. This study addressed this challenge by employing a vaccine formulation with a tunable, controlled particle size. Manganese dioxide (MnO2) nanoparticles were synthesized, loaded with ovalbumin (OVA), and modified with A50 or T20 DNA single strands to obtain MnO2/OVA/A50 and MnO2/OVA/T20, respectively. Administering the vaccines sequentially, upon reaching the lymph nodes, the two vaccines converge and simultaneously aggregate into MnO2/OVA/A50-T20 particles through base pairing. This process enhances both vaccine uptake and antigen delivery. In vitro and in vivo studies demonstrated that, the combined vaccine, comprising MnO2/OVA/A50 and MnO2/OVA/T20, exhibited robust immunization effects and remarkable anti-tumor efficacy in the melanoma animal models. The strategy of controlling tumor vaccine size and consequently improving tumor antigen presentation efficiency and vaccine efficacy via the DNA base-pairing principle, provides novel concepts for the development of efficient tumor vaccines.

Improved biodistribution and enhanced immune response of subunit vaccine using a nanostructure formed by self-assembly of ascorbyl palmitate.

New adjuvant strategies are needed to improve protein-based subunit vaccine immunogenicity. We examined the potential to use nanostructure of 6-O-ascorbyl palmitate to formulate ovalbumin (OVA) protein and an oligodeoxynucleotide (CpG-ODN) (OCC). In mice immunized with a single dose, OCC elicited an OVA-specific immune response superior to OVA/CpG-ODN solution (OC). Rheological studies demonstrated OCC's self-assembling viscoelastic properties. Biodistribution studies indicated that OCC prolonged OVA and CpG-ODN retention at injection site and lymph nodes, reducing systemic spread. Flow-cytometry assays demonstrated that OCC promoted OVA and CpG-ODN co-uptake by Ly6ChiCD11bhiCD11c+ monocytes. OCC and OC induced early IFN-γ in lymph nodes, but OCC led to higher concentration. Conversely, mice immunized with OC showed higher serum IFN-γ concentration compared to those immunized with OCC. In mice immunized with OCC, NK1.1+ cells were the IFN-γ major producers, and IFN-γ was essential for OVA-specific IgG2c switching. These findings illustrate how this nanostructure improves vaccine's response.

Polymeric monolithic columns based on natural wood for rapid purification of targeted protein.

With multiscale hierarchical structure, wood is suitable for a range of high-value applications, especially as a chromatographic matrix. Here, we have aimed to provide a weak anion-exchange polymeric monolithic column based on natural wood with high permeability and stability for effectively separating the targeted protein. The wood-polymeric monolithic column was synthesized by in situ polymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in wood, and coupled with diethylaminoethyl hydrochloride. The wood-polymeric monolithic column can be integrated with fast-protein liquid chromatography for large-scale protein purification. According to the results, the wood-polymeric monolithic column showed high hydrophilicity, permeability and stability. Separation experiments verified that the wood-polymeric monolithic column could purify the targeted protein (spike protein of SARS-COV-2 and ovalbumin) from the mixed proteins by ion exchange, and the static adsorption capacity was 33.04 mg mL-1 and the dynamic adsorption capacity was 24.51 mg mL-1. In addition, the wood-polymerized monolithic column had good stability, and a negligible decrease in the dynamic adsorption capacity after 20 cycles. This wood-polymerized monolithic column can provide a novel, efficient, and green matrix for monolithic chromatographic columns.

Probiotic-derived amphiphilic exopolysaccharide self-assembling adjuvant delivery platform for enhancing immune responses.

Enhancing immune response activation through the synergy of effective antigen delivery and immune enhancement using natural, biodegradable materials with immune-adjuvant capabilities is challenging. Here, we present NAPSL.p that can activate the Toll-like receptor 4 (TLR4) pathway, an amphiphilic exopolysaccharide, as a potential self-assembly adjuvant delivery platform. Its molecular structure and unique properties exhibited remarkable self-assembly, forming a homogeneous nanovaccine with ovalbumin (OVA) as the model antigen. When used as an adjuvant, NAPSL.p significantly increased OVA uptake by dendritic cells. In vivo imaging revealed prolonged pharmacokinetics of NAPSL. p-delivered OVA compared to OVA alone. Notably, NAPSL.p induced elevated levels of specific serum IgG and isotype titers, enhancing rejection of B16-OVA melanoma xenografts in vaccinated mice. Additionally, NAPSL.p formulation improved therapeutic effects, inhibiting tumor growth, and increasing animal survival rates. The nanovaccine elicited CD4+ and CD8+ T cell-based immune responses, demonstrating the potential for melanoma prevention. Furthermore, NAPSL.p-based vaccination showed stronger protective effects against influenza compared to Al (OH)3 adjuvant. Our findings suggest NAPSL.p as a promising, natural self-adjuvanting delivery platform to enhance vaccine design across applications.

Cold plasma for enhancing covalent conjugation of ovalbumin-gallic acid and its functional properties.

The utilization of cold plasma (CP) treatment to promote covalent conjugation of ovalbumin (OVA) and gallic acid (GA), as well as its functionality, were investigated. Results demonstrated that CP significantly enhanced the covalent grafting of OVA and GA. The maximum conjugation of GA, 24.33 ± 2.24 mg/g, was achieved following 45 s of CP treatment. Covalent conjugation between GA and OVA were confirmed through analyses of total sulfhydryl (-SH) group, Fourier transform infrared (FTIR) spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Unfolding of the OVA molecule occurred upon conjugation with GA, as evidenced by multiple spectroscopy analyses. Additionally, conjugation with GA resulted in significant improvements in the antioxidant activity and emulsifying properties of OVA. This study demonstrated that CP is a robust and sustainable technique for promoting the covalent conjugate of polyphenols and proteins, offering a novel approach to enhance the functional properties of proteins.

Remarkable Enhancement of Antioxidant Activity of the Ovalbumin-EGCG Conjugate through a Novel Preceding Selective Protection Grafting Strategy.

Conventional radical grafting of proteins with catechins consumed the most antioxidant-active hydroxyls during grafting, thus failing to effectively retain antioxidant activity in conjugates. In this study, a novel strategy of selective protection of the most reactive hydroxyls before grafting was developed to preserve the most reactive hydroxyls and effectively retain antioxidant activity in conjugates. Selective protection of the most reactive hydroxyls of (-)-epigallocatechin-3-gallate (EGCG) was successfully realized in a yield of 87% applying trimethyl orthopropionate and catalytic calcium triflate at 40 °C. The novel ovalbumin (OVA)-EGCG conjugate with 93% grafting ratio was prepared by radical grafting with the selectively protected EGCG and subsequent deprotection. Substantially enhanced antioxidant performance of the novel OVA-EGCG conjugate in liposomes was unveiled with notably reduced curcumin degradation and leakage. The strategy and approaches developed in this study will be valuable to effectively improve the antioxidant activities of protein-catechin grafting conjugates.

Effects of polysaccharides on colonic targeting and colonic fermentation of ovalbumin-ferulic acid based emulsion.

Rutin is a polyphenol with beneficial pharmacological properties. However, its bioavailability is often compromised due to low solubility and poor stability. Encapsulation technologies, such as emulsion systems, have been proven to be promising delivery vehicles for enhancing the bioavailability of bioactive compounds. Thus, this study was proposed and designed to investigate the colonic targeting and colonic fermentation characteristics of rutin-loaded ovalbumin-ferulic acid-polysaccharide (OVA-FA-PS) complex emulsions. The results indicate that OVA-FA-PS emulsion effectively inhibits the degradation of rutin active substances and facilitates its transport of rutin to the colon. The analysis revealed that the OVA-FA-κ-carrageenan emulsion loaded with rutin exhibited superior elasticity and colon targeting properties compared to the OVA-FA-hyaluronic acid or OVA-FA-sodium alginate emulsions loaded with rutin in the composite emulsion. Additionally, it was observed that the rutin loaded within the OVA-FA-κ-carrageenan emulsion underwent degradation and was converted to 4-hydroxybenzoic acid during colonic fermentation.

Acidity-responsive polyphenol-coordinated nanovaccines for improving tumor immunotherapy via bidirectional reshaping of the immunosuppressive microenvironment and controllable release of antigens.

The tumor immunosuppressive microenvironment (TIME) and uncontrollable release of antigens can lower the efficacy of nanovaccine-based immunotherapy (NBI). Therefore, it is necessary to develop a new strategy for TIME reshaping and controllable release of antigens to improve the NBI efficacy. Herein, an acidity-responsive Schiff base-conjugated polyphenol-coordinated nanovaccine was constructed for the first time to realize bidirectional TIME reshaping and controllable release of antigens for activating T cells. In particular, an acidity-responsive tannic acid-ovalbumin (TA-OVA) nanoconjugate was prepared via a Schiff base reaction. FeIII was coordinated with TA-OVA to produce a FeIII-TA-OVA nanosystem, and 1-methyltryptophan (1-MT) as an indoleamine 2,3-dioxygenase inhibitor was loaded to form a polyphenol-coordinated nanovaccine. The coordination between FeIII and TA could cause photothermal ablation of primary tumors, and the acidity-triggered Schiff base dissociation of TA-OVA could controllably release OVA to realize lysosome escape, initiating the body's immune response. More importantly, oxidative stress generated by a tumor-specific Fenton reaction of Fe ions could promote the polarization of tumor-associated macrophages from the M2 to M1 phenotype, resulting in the upregulation of cytotoxic T cells and helper T cells. Meanwhile, 1-MT could downregulate immunosuppressive regulatory T cells. Overall, such skillful combination of bidirectional TIME reshaping and controllable antigen release into one coordination nanosystem could effectively enhance the NBI efficacy of tumors.

Microfluidic-oriented green synthesis of pepsin-doped gold nanoparticles for colorimetric and photothermal dual-readout detection of food hazards.

Gold nanoparticles (AuNPs)-based immunochromatographic assay has gained popularity as a rapid detection method for food hazards. Synthesizing highly stable AuNPs in a rapid, simple and environmentally friendly manner is a key focus in this field. Here, we present a green microfluidic strategy for the rapid, automated, and size-controllable synthesis of pepsin-doped AuNPs (AuNPs@Pep) by employing glucose-pepsin as a versatile reducing agent and stabilizer. Through combining the colorimetric and photothermal (PoT) properties of AuNPs@Pep, both "signal-off" and "signal-on" formats of microfluidic paper analytical devices (PADs) were developed for detection of a small molecule antibiotic, florfenicol, and an egg allergen, ovalbumin. Compared to the colorimetric mode, a 4-fold and 3-fold improvement in limit of detection was observed in the "signal-off" detection of florfenicol and the "signal-on" detection of ovalbumin, respectively. The results demonstrated the practicality of AuNPs@Pep as a colorimetric/PoT dual-readout probe for immunochromatographic detection of food hazards at different molecular scales.

Identification and Molecular Mechanism of Novel Two-Way Immunomodulatory Peptides from Ovalbumin: In Vitro Cell Experiments, De Novo Sequencing, and Molecular Docking.

The purpose of this study was to identify ovalbumin-derived immunomodulatory peptides by in vitro cell experiments, de novo sequencing, and molecular docking. Ovalbumin hydrolysates were prepared by two enzymes (alkaline protease and papain) individually, sequentially, or simultaneously, respectively. The simultaneous enzymatic hydrolysate (OVAH) had a high degree of hydrolysis (38.12 ± 0.48%) and exhibited immune-enhancing and anti-inflammatory activities. A total of 160 peptides were identified by LC-MS/MS in OVAH. Three novel peptides NVMEERKIK, ADQARELINS, and WEKAFKDE bound to TLR4-MD2 through hydrogen bonds and hydrophobic interactions with high binding affinity and binding energies of -181.40, -178.03, and -168.12 kcal/mol, respectively. These three peptides were synthesized and validated for two-way immunomodulatory activity. NVMEERKIK exhibiting the strongest immunomodulatory activity, increased NO and TNF-α levels by 128.69 and 38.01%, respectively, in normal RAW264.7 cells and reduced NO and TNF-α levels by 27.31 and 39.13%, respectively, in lipopolysaccharide-induced inflammatory RAW264.7 cells. Overall, this study first revealed that ovalbumin could be used as an immunomodulatory source for controlling inflammatory factor secretion.