Hyaluronic acid-poly(glyceryl)10-stearate nanoemulsion for co-delivery of fish oil and resveratrol: Enhancing bioaccessibility and antioxidant potency.

Hyaluronic acid (HA), an endogenous polysaccharide comprising alternating D-glucuronic acid and N-acetylglucosamine units, is renowned for its high hydrophilicity, biocompatibility, and biodegradability. These attributes have rendered HA invaluable across medical and drug delivery fields. HA can be altered through physical, chemical, or enzymatic methods to improve the properties of the modified substances. In this work, we synthesized a derivative via the esterification of HA with poly(glyceryl)10-stearate (PG10-C18), designated as HA-PG10-C18. This novel derivative was employed to fabricate a nano co-delivery system (HA-PG10-C18@Res-NE) for fish oil and resveratrol (Res), aiming to enhance their stability and bioaccessibility. An exhaustive investigation of HA-PG10-C18@Res-NE revealed that the HA-modified system displayed superior physicochemical stability, notably in withstanding oxidation and neutralizing free radicals. Moreover, in vitro simulated digestion underscored the system's enhanced bioaccessibility of Res and more efficient release of free fatty acids. These outcomes underscore the strategic advantage of HA in modifying PG10-C18 for nanoemulsion formulation. Consequently, HA-PG10-C18 stands as a promising emulsifier for encapsulating lipophilic bioactives in functional foods, nutraceuticals, and pharmaceuticals.

A conjugate vaccine strategy that induces protective immunity against arecoline.

Betel-quid chewing addiction is the leading cause of oral submucous fibrosis and oral cancer, resulting in significant socio-economic burdens. Vaccination may serve as a promising potential remedy to mitigate the abuse and combat accidental overdose of betel nut. Hapten design is the crucial factor to the development of arecoline vaccine that determines the efficacy of a candidate vaccine. Herein, we reported that two kinds of novel arecoline-based haptens were synthesized and conjugated to Bovine Serum Albumin (BSA) to generate immunogens, which generated antibodies with high affinity for arecoline but reduced binding for guvacoline and no affinity for arecaidine or guvacine. Notably, vaccination with Arec-N-BSA, which via the N-position on the tetrahydropyridine ring (tertiary amine group), led to a higher antibody affinity compared to Arec-CONH-BSA, blunted analgesia and attenuated hypothermia for arecoline.

Improved physicochemical stability of fish oil nanoemulsion via a dense interfacial layer formed by hyaluronic acid-poly(glyceryl)10-stearate.

This study aimed to synthesize a novel emulsifier, hyaluronic acid-poly(glyceryl)10-stearate (HA-PG10-C18), and employ it for the fabrication of nanoemulsions incorporating deep-sea fish oil to improve their apparent solubility and physicochemical stability. 1H NMR and FT-IR analyses indicated successful synthesis of HA-PG10-C18. Nanoemulsions of deep-sea fish oil loaded with HA-PG10-C18 (HA-PG10-C18@NE) were successfully fabricated by ultrasonic emulsification. The fixed aqueous layer thickness (FALT) of PG10-C18@NE and HA-PG10-C18@NE was determined and the FALT of both nanoemulsions was similar, while the surface density of HA-PG10-C18@NE (4.92 × 10-12 ng/nm2) is 60 % higher than that of PG10-C18@NE (3.07 × 10-12 ng/nm2). Notably, HA-PG10-C18@NE demonstrated an exceptional physicochemical stability when exposed to various stressed environmental conditions, especially its freeze-thaw stability. Moreover, after simulated in vitro digestion, the HA-PG10-C18@NE exhibited a comparatively greater liberation of free fatty acids (94.0 ± 1.7 %) when compared to the release observed in PG10-C18@NE (85.5 ± 2.2 %).

Hyaluronic Acid Poly(glyceryl)10-Stearate Derivatives: Novel Emulsifiers for Improving the Gastrointestinal Stability and Bioaccessibility of Coenzyme Q10 Nanoemulsions.

Fish oils are a rich source of polyunsaturated fatty acids, including eicosapentaenoic acid and docosahexaenoic acid, which are reported to exhibit therapeutic effects in a variety of human diseases. However, these oils are highly susceptible to degradation due to oxidation, leading to rancidity and the formation of potentially toxic reaction products. The aim of this study was to synthesize a novel emulsifier (HA-PG10-C18) by esterifying hyaluronic acid with poly(glyceryl)10-stearate (PG10-C18). This emulsifier was then used to formulate nanoemulsion-based delivery systems to co-deliver fish oil and coenzyme Q10 (Q10). Q10-loaded fish oil-in-water nanoemulsions were fabricated, and then their physicochemical properties, digestibility, and bioaccessibility were measured. The results indicated that the environmental stability and antioxidant activity of oil droplets coated with HA-PG10-C18 surpassed those coated with PG10-C18 due to the formation of a denser interfacial layer that blocked metal ions, oxygen, and lipase. Meanwhile, the lipid digestibility and Q10 bioaccessibility of nanoemulsions formulated with HA-PG10-C18 (94.9 and 69.2%) were higher than those formulated with PG10-C18 (86.2 and 57.8%), respectively. These results demonstrated that the novel emulsifier synthesized in this study could be used to protect chemically labile fat-soluble substances from oxidative damage, while still retaining their nutritional value.

Self-Adjuvanting Protein Vaccine Conjugated with a Novel Synthetic TLR4 Agonist on Virus-Like Liposome Induces Potent Immunity against SARS-CoV-2.

Exploring potent adjuvants and new vaccine strategies is crucial for the development of protein vaccines. In this work, we synthesized a new TLR4 agonist, structurally simplified lipid A analogue GAP112, as a potent built-in adjuvant to improve the immunogenicity of SARS-CoV-2 spike RBD protein. The new TLR4 agonist GAP112 was site-selectively conjugated on the N-terminus of RBD to construct an adjuvant-protein conjugate vaccine in a liposomal formulation. It is the first time that a TLR4 agonist is site-specifically and quantitatively conjugated to a protein antigen. Compared with an unconjugated mixture of GAP112/RBD, a two-dose immunization of the GAP112-RBD conjugate vaccine strongly activated innate immune cells, elicited a 223-fold increase in RBD-specific antibodies, and markedly enhanced T-cell responses. Antibodies induced by GAP112-RBD also effectively cross-neutralized SARS-CoV-2 variants (Delta/B.1.617.2 and Omicron/B.1.1.529). This conjugate strategy provides an effective method to greatly enhance the immunogenicity of antigen in protein vaccines against SARS-CoV-2 and other diseases.

Adjuvant-Protein Conjugate Vaccine with Built-In TLR7 Agonist on S1 Induces Potent Immunity against SARS-CoV-2 and Variants of Concern.

With the global pandemic of the new coronavirus disease (COVID-19), a safe, effective, and affordable mass-produced vaccine remains the current focus of research. Herein, we designed an adjuvant-protein conjugate vaccine candidate, in which the TLR7 agonist (TLR7a) was conjugated to S1 subunit of SARS-CoV-2 spike protein, and systematically compared the effect of different numbers of built-in TLR7a on the immune activity for the first time. As the number of built-in TLR7a increased, a bell-shaped reaction was observed in three TLR7a-S1 conjugates, with TLR7a(10)-S1 (with around 10 built-in adjuvant molecules on one S1 protein) eliciting a more potent immune response than TLR7a(2)-S1 and TLR7a(18)-S1. This adjuvant-protein conjugate strategy allows the built-in adjuvant to provide cluster effects and prevents systemic toxicity and facilitates the co-delivery of adjuvant and antigen. Vaccination of mice with TLR7a(10)-S1 triggered a potent humoral and cellular immunity and a balanced Th1/Th2 immune response. Meanwhile, the vaccine induces effective neutralizing antibodies against SARS-CoV-2 and all variants of concern (B.1.1.7/alpha, B.1.351/beta, P.1/gamma, B.1.617.2/delta, and B.1.1.529/omicron). It is expected that the adjuvant-protein conjugate strategy has great potential to construct a potent recombinant protein vaccine candidate against various types of diseases.

A protein vaccine with Alum/c-GAMP/poly(I:C) rapidly boosts robust immunity against SARS-CoV-2 and variants of concern.

Adjuvants are important components in vaccines to increase the immunogenicity of proteins and induce optimal immunity. In this study, we designed a novel ternary adjuvant system Alum + c-GAMP + poly(I:C) with STING agonist 3,3'-c-GAMP (c-GAMP) and TLR3 agonist poly(I:C) co-adsorbed on the conventional adjuvant aluminum gel (Alum), and further constructed an S1 protein vaccine. Two doses of vaccination with the ternary adjuvant vaccine were sufficient to induce a balanced Th1/Th2 immune response and robust humoral and cellular immunity. Additionally, the ternary adjuvant group had effective neutralizing activity against live virus SARS-CoV-2 and pseudovirus of all variants of concern (alpha, beta, gamma, delta and omicron). These results indicate that the ternary adjuvants have a significant synergistic effect and can rapidly trigger potent immune responses; the combination of the ternary adjuvant system with S1 protein is a promising COVID-19 vaccine candidate.

MPLA-Adjuvanted Liposomes Encapsulating S-Trimer or RBD or S1, but Not S-ECD, Elicit Robust Neutralization Against SARS-CoV-2 and Variants of Concern.

Safe and effective vaccines are the best method to defeat worldwide SARS-CoV-2 and its circulating variants. The SARS-CoV-2 S protein and its subunits are the most attractive targets for the development of protein-based vaccines. In this study, we evaluated three lipophilic adjuvants, monophosphoryl lipid A (MPLA), Toll-like receptor (TLR) 1/2 ligand Pam3CSK4, and α-galactosylceramide (α-GalCer), in liposomal and nonliposomal vaccines. The immunological results showed that the MPLA-adjuvanted liposomal vaccine induced the strongest humoral and cellular immunity. Therefore, we further performed a systematic comparison of S-trimer, S-ECD, S1, and RBD as antigens in MPLA-adjuvanted liposomes and found that, although these four vaccines all induced robust specific antibody responses, only S-trimer, S1, and RBD liposomes, but not S-ECD, elicited potent neutralizing antibody responses. Moreover, RBD, S-trimer, and S1 liposomes effectively neutralized variants (B.1.1.7/alpha, B.1.351/beta, P.1/gamma, B.1.617.2/delta, and B.1.1.529/omicron). These results provide important information for the subunit vaccine design against SARS-CoV-2 and its variants.

RBD conjugate vaccine with a built-in TLR1/2 agonist is highly immunogenic against SARS-CoV-2 and variants of concern.

The coronavirus 2019 (COVID-19) pandemic is causing serious impacts in the world, and safe and effective vaccines and medicines are the best methods to combat the disease. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein plays a key role in interacting with the angiotensin-converting enzyme 2 (ACE2) receptor, and is regarded as an important target of vaccines. Herein, we constructed the adjuvant-protein conjugate Pam3CSK4-RBD as a vaccine candidate, in which the N-terminal of the RBD was site-selectively oxidized by transamination and conjugated with the TLR1/2 agonist Pam3CSK4. This demonstrated that the conjugation of Pam3CSK4 significantly enhanced the anti-RBD antibody response and cellular response. In addition, sera from the Pam3CSK4-RBD immunized group efficiently inhibited the binding of the RBD to ACE2 and protected cells from SARS-CoV-2 and four variants of concern (alpha, beta, gamma and delta), indicating that this adjuvant strategy could be one of the effective means for protein vaccine development.

Self-Adjuvanting Lipoprotein Conjugate αGalCer-RBD Induces Potent Immunity against SARS-CoV-2 and its Variants of Concern.

Safe and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are the best approach to successfully combat the COVID-19 pandemic. The receptor-binding domain (RBD) of the viral spike protein is a major target to develop candidate vaccines. α-Galactosylceramide (αGalCer), a potent invariant natural killer T cell (iNKT) agonist, was site-specifically conjugated to the N-terminus of the RBD to form an adjuvant-protein conjugate, which was anchored on the liposome surface. This is the first time that an iNKT cell agonist was conjugated to the protein antigen. Compared to the unconjugated RBD/αGalCer mixture, the αGalCer-RBD conjugate induced significantly stronger humoral and cellular responses. The conjugate vaccine also showed effective cross-neutralization to all variants of concern (B.1.1.7/alpha, B.1.351/beta, P.1/gamma, B.1.617.2/delta, and B.1.1.529/omicron). These results suggest that the self-adjuvanting αGalCer-RBD has great potential to be an effective COVID-19 vaccine candidate, and this strategy might be useful for designing various subunit vaccines.

Synthesis and Evaluation of Liposomal Anti-GM3 Cancer Vaccine Candidates Covalently and Noncovalently Adjuvanted by αGalCer.

GM3, a typical tumor-associated carbohydrate antigen, is considered as an important target for cancer vaccine development, but its low immunogenicity limits its application. αGalCer, an iNKT cell agonist, has been employed as an adjuvant via a unique immune mode. Herein, we prepared and investigated two types of antitumor vaccine candidates: (a) self-adjuvanting vaccine GM3-αGalCer by conjugating GM3 with αGalCer and (b) noncovalent vaccine GM3-lipid/αGalCer, in which GM3 is linked with lipid anchor and coassembled with αGalCer. This demonstrated that ßGalCer is an exceptionally optimized lipid anchor, which enables the noncovalent vaccine candidate GM3-ßGalCer/αGalCer to evoke a comparable antibody level to GM3-αGalCer. However, the antibodies induced by GM3-αGalCer are better at recognition B16F10 cancer cells and more effectively activate the complement system. Our study highlights the importance of vaccine constructs utilizing covalent or noncovalent assembly between αGalCer with carbohydrate antigens and choosing an appropriate lipid anchor for use in noncovalent vaccine formulation.

Liposomal Antitumor Vaccines Targeting Mucin 1 Elicit a Lipid-Dependent Immunodominant Response.

The tumor-associated antigen mucin 1 (MUC1) has been pursued as an attractive target for cancer immunotherapy, but the poor immunogenicity of the endogenous antigen hinders the development of vaccines capable of inducing effective anti-MUC1 immunodominant responses. Herein, we prepared synthetic anti-MUC1 vaccines in which the hydrophilic MUC1 antigen was N-terminally conjugated to one or two palmitoyl lipid chains (to form amphiphilic Pam-MUC1 or Pam2 -MUC1). These amphiphilic lipid-tailed MUC1 antigens were self-assembled into liposomes containing the NKT cell agonist αGalCer as an adjuvant. The lipid-conjugated antigens reshaped the physical and morphological properties of liposomal vaccines. Promising results showed that the anti-MUC1 IgG antibody titers induced by the Pam2 -MUC1 vaccine were more than 30- and 190-fold higher than those induced by the Pam-MUC1 vaccine and the MUC1 vaccine without lipid tails, respectively. Similarly, vaccines with the TLR1/2 agonist Pam3 CSK4 as an adjuvant also induced conjugated lipid-dependent immunological responses. Moreover, vaccines with the αGalCer adjuvant induced significantly higher titers of IgG antibodies than vaccines with the Pam3 CSK4 adjuvant. Therefore, the non-covalent assembly of the amphiphilic lipo-MUC1 antigen and the NKT cell agonist αGalCer as a glycolipid adjuvant represent a synthetically simple but immunologically effective approach for the development of anti-MUC1 cancer vaccines.

Peptide-free Synthetic Nicotine Vaccine Candidates with α-Galactosylceramide as Adjuvant.

Peptides are generally needed as T-helper epitopes in nicotine vaccines to induce effective antibody responses, but the highly polymorphic property of major histocompatibility complex (MHC) molecules may limit opportunities of B cell to receive CD4+ T-cell help. Invariant natural killer T (iNKT) cells recognize lipid antigens presented by the nonpolymorphic CD1d molecule that is conserved in mammals to a great extent. iNKT cells also display some similar functions to conventional CD4+ T-helper cells, especially they license dendritic cells stimulate antibody isotype switching by B cells. Herein, α-galactosylceramide (αGalCer), a classical iNKT cell agonist, serves as an adjuvant in synthetic nicotine vaccine candidates absent of peptide or protein. Our study reveals that αGalCer displays better adjuvant activity than Pam3CSK4 (a commonly used lipopeptide TLR agonist). Remarkably, the covalent linker between the nicotine hapten and αGalCer is not critical. Self-assembly of the lipid-tailed nicotine and αGalCer into the liposome represents a structurally simple but immunologically effective way to develop nicotine vaccines. This is the first time to introduce the iNKT cell agonist as an adjuvant to an antidrug vaccine. This discovery may contribute to improving the efficacy of clinical candidate nicotine vaccines in the future.

Efficient access to chiral dihydrobenzoxazinones via Rh-catalyzed hydrogenation.

Rh/(S)-DTBM-SegPhos-catalyzed asymmetric hydrogenation of prochiral (Z)-2-(2-oxo-2H-benzo[b][1,4]oxazin-3(4H)-ylidene)acetate esters was successfully developed. A series of chiral dihydrobenzoxazinones were prepared through this efficient methodology with good to excellent results (up to >99% conversion, 93% yield and >99% ee), which are important motifs in the biologically active molecules.

Iridium-Catalyzed Asymmetric Hydrogenation of Tetrasubstituted α-Fluoro-ß-enamino Esters: Efficient Access to Chiral α-Fluoro-ß-amino Esters with Two Adjacent Tertiary Stereocenters.

An iridium-catalyzed highly efficient asymmetric hydrogenation of challenging tetrasubstituted α-fluoro-ß-enamino esters was successfully developed using bisphosphine-thiourea (ZhaoPhos) as the ligand, which prepared a series of chiral α-fluoro-ß-amino esters containing two adjacent tertiary stereocenters with high yields and excellent diastereoselectivities/enantioselectivities (73%-99% yields, >25:1 dr, 91%->99% ee, and turnover number (TON) values up to 8600), and no defluorinate byproduct was detected.

Enantioselective Access to Chiral 2-Substituted 2,3-Dihydrobenzo[1,4]dioxane Derivatives through Rh-Catalyzed Asymmetric Hydrogenation.

Rh-catalyzed asymmetric hydrogenation of various benzo[ b][1,4]dioxine derivatives was successfully developed to prepare chiral 2-substituted 2,3-dihydrobenzo[1,4]dioxane derivatives using ZhaoPhos and N-methylation of ZhaoPhos ligands with high yields and excellent enantioselectivities (up to 99% yield, >99% enantiomeric excess (ee), turnover number (TON) = 24 000). Moreover, this asymmetric hydrogenation methodology, as the key step with up to 10 000 TON, was successfully applied to develop highly efficient synthetic routes for the construction of some important biologically active molecules, such as MKC-242, WB4101, BSF-190555, and ( R)-doxazosin·HCl.

Rh/SPO-WudaPhos-Catalyzed Asymmetric Hydrogenation of α-Substituted Ethenylphosphonic Acids via Noncovalent Ion-Pair Interaction.

Asymmetric hydrogenation of α-substituted ethenylphosphonic acids has been successfully achieved by Rh/ferrocenyl chiral bisphosphorus ligand (SPO-Wudaphos) through noncovalent ion-pair interaction between the substrate and catalyst under mild reaction conditions without base. A series of chiral phosphonic acids were obtained with excellent results (up to 98% ee, >99% conversion, 2000 TON). Moreover, the control experiments showed that the noncovalent ion-pair interaction was critical in this asymmetric hydrogenation.

Rh/Wudaphos-Catalyzed Asymmetric Hydrogenation of Sodium α-Arylethenylsulfonates: A Method To Access Chiral α-Arylethylsulfonic Acids.

A highly enantioselective hydrogenation of various sodium α-arylethenylsulfonates catalyzed by Rh/chiral ferrocenyl bisphosphorus ligand (Wudaphos) was successfully developed to construct a series of chiral α-arylethylsulfonic acids in the presence of CF3SO3H with full conversion and good to excellent enantioselectivity (>99% conversion, up to 96% ee) under mild reaction conditions for the first time. Moreover, the control experiment results showed that the non-covalent ion pair interaction between the α-arylethenylsulfonic acid and the Wudaphos ligand plays an important role in this asymmetric hydrogenation system.

Rh-Catalyzed Asymmetric Hydrogenation of α-Substituted Vinyl Sulfones: An Efficient Approach to Chiral Sulfones.

Rh/(S)-(+)-DTBM-Segphos complex catalyzed asymmetric hydrogenation of α-substituted vinyl sulfones has been achieved, furnishing the desired products in high yields and excellent enantioselectivities (>90% yield, up to 99% ee). This method provided an efficient approach to α-substituted chiral sulfones under mild conditions and has potential applications in organic synthesis.

IgG Antibody Response Elicited by a Fully Synthetic Two-Component Carbohydrate-Based Cancer Vaccine Candidate with α-Galactosylceramide as Built-in Adjuvant.

A fully synthetic self-adjuvanting cancer vaccine candidate was constructed through covalent conjugation of invariant natural killer T (iNKT) cell ligand α-galactosylceramide (αGalCer) with sialyl Tn (STn), a representative tumor-associated carbohydrate antigen (TACA). This two-component vaccine STn-αGalCer is devoid of antigenic peptide, featuring the well-defined structure with high simplicity. STn-αGalCer showed remarkable efficacy in inducing antibody class switching from IgM to STn-specific IgG. Subtypes of IgG antibody were primarily IgG1 and IgG3.