Synthetic Particles for Cancer Vaccines: Connecting the Inherent Supply Chain.

Cancer vaccines have opened a new paradigm for safe and effective antitumor therapy, but they still suffer from shortcomings such as insufficient immunogenicity and immune tolerance, which seldom makes them the first choice in clinic. In fact, similar to providing a high-end product, a robust antitumor effect depends on the inherent supply chain, which attains, processes, and presents tumor-associated antigens via antigen presenting cells to T cells, which then leads to lysis of the cancer cells to release more antigens to complete the supply chain. Under these circumstances, the failure of cancer vaccines can be treated as a blockade or chain rupture. Thus, for effective tumor treatment, the key is to rationally design logistic systems to restore the supply chain.Under these circumstances, this Account summarizes our recent attempts to exploit the immunogenic trait of synthetic particles to enhance the distribution, presentation, and immune activations of the whole priming process in cancer vaccines: (1) Raw material (tumor antigen/signals) procurement: We illustrated the efforts to deliver antigens to antigen presenting cells (APCs) and draining lymph nodes for potent internalizations, and put more emphasis on the structural effect of sizes, charges, shapes, and assembly strategies for the antigen depot, lymph node transfer, and APC endocytosis. (2) Manufacture of cytotoxic T lymphocytes (CTLs) via APC recognition and presentation: We centered on exploiting the softness of two-dimensional graphene and Pickering emulsions to dynamically potentiate the immune recognition, and demonstrating the recent advances in lysosome escape strategies for enhanced antigen cross-presentations. (3) Marketing the accumulations of CTLs and the reversal of an immunosuppressive microenvironment within the tumor: We demonstrated the previous attempts to inherently cultivate the tumor tropism of the T cells via the multiantigenic repertoire and discussed the advances and challenges of combinatory cancer vaccines with an immune checkpoint blockade to reinforce the antitumor efficacy. Collectively, this Account aims to illustrate the potential of the particulate cancer vaccines to recapitalize the inherent host immune responses for the maximum antitumor effect. And by integrating the antitumor supply chain, optimized synthetic particles may shed light on the development of safe and effective particulate cancer vaccines.

A Novel Particulate Delivery System Based on Antigen-Zn2+ Coordination Interactions Enhances Stability and Cellular Immune Response of Inactivated Foot and Mouth Disease Virus.

The interactions between antigen and adjuvant were among the most significant factors influencing the immunogenicity of vaccines, especially for unstable antigens like inactivated foot and mouth disease virus (iFMDV). Here we propose a novel antigen delivery pattern based on the coordination interaction between transition metal ions Zn2+ chelated to chitosan nanoparticles and iFMDV, which is known to be rich in histidine. The zinc chelated chitosan particles (CP-PEI-Zn) were prepared by cross-linking chitosan particles (CP) with sodium tripolyphosphate (TPP), modifying with metal chelator polyethylenimine (PEI), and subsequent chelating of Zn2+. The coordination interaction was confirmed by analyzing the adsorption and desorption behavior of iFMDV on CP-PEI-Zn by high-performance size exclusion chromatography (HPSEC), while the CP-PEI without chelating Zn2+ loads iFMDV mainly through electrostatic interactions. The iFMDV loaded on CP-PEI-Zn showed better thermal stability than that on CP-PEI, as revealed by a slightly higher transition temperature (Tm) related to iFMDV dissociation. After subcutaneous immunization in female Balb/C mice, antigens loaded on CP-PEI and CP-PEI-Zn all induced higher specific antibody titers, better activation of B lymphocytes, and more effector-memory T cells proliferation than the free antigen and iFMDV adjuvanted with ISA 206 emulsion did. Moreover, CP-PEI-Zn showed superior efficacy to CP-PEI in promoting the proliferation of effector-memory T cells and secretion of cytokines, indicating a more potent cellular immune response. In summary, the CP-PEI-Zn stabilized the iFMDV after loading and promoted both humoral and cellular immune responses, thus reflecting its potential to be a promising adjuvant for the iFMDV vaccine and other unstable viral antigens.

Positively Charged Polyprodrug Amphiphiles with Enhanced Drug Loading and Reactive Oxygen Species-Responsive Release Ability for Traceable Synergistic Therapy.

Due to the vast differences in chemical properties among small molecule drugs, nucleotide drugs, and superparamagnetic iron oxide nanocubes (SPIONs), such as charge and hydrophobicity, entrapment of these within a single carrier for traceable synergistic therapy has been proven difficult. Herein, we synthesize positively charged polyprodrug amphiphiles. The hydrophobic polyprodrug unit of the amphiphiles is positively charged, which can simultaneously load hydrophobic SPIONs and absorb negative let-7b antisense oligonucleotide to construct traceable co-delivery nanoparticles (NPs). This characteristic avoids the use of inert materials and enhances drug loading of the traceable NPs. The traceable NPs can achieve controlled release of drugs to reduce the differentiation of exogenous neural stem cells (NSCs) and enhance their secretion of brain-derived neurotrophic factor (BDNF) synergistically. Exogenous NSCs treated with the NPs significantly rescue the memory deficits in 2xTg-AD mice. In addition, the transplantation site and migration of exogenous NSCs can be traced using the SPIONs with high r2 value for magnetic resonance imaging. Therefore, traceable NPs self-assembled from the positively charged polyprodrug amphiphiles may have the potential to open up a new avenue for treatment of Alzheimer's disease (AD), as well as other neurodegenerative disorders.

Adjuvanticity Regulation by Biodegradable Polymeric Nano/microparticle Size.

Polymeric nano/microparticles as vaccine adjuvants have been researched in experimental and clinical studies. A more profound understanding of how the physicochemical properties regulate specific immune responses has become a vital requirement. Here we prepared poly(d,l-lactic-co-glycolic acid) (PLGA) nano/microparticles with uniform sizes (500 nm, 900 nm, 2.1 µm, and 4.9 µm), and the size effects on particle uptake, activation of macrophages, and antigen internalization were evaluated. Particle uptake kinetic studies demonstrated that 900 nm particles were the easiest to accumulate in cells. Moreover, they could induce macrophages to secrete NO and IL-1ß and facilitate antigen internalization. Furthermore, 900 nm particles, mixed with antigen, could exhibit superior adjuvanticity in both humoral and cellular immune responses in vivo, including offering the highest antibody protection, promoting the maximum secretion levels of IFN-γ and IL-4 than particles with other sizes. Overall, 900 nm might be the optimum choice for PLGA particle-based vaccine adjuvants especially for recombinant antigens. Understanding the effect of particle size on the adjuvanticity based immune responses might have important enlightenments for rational vaccine design and applications.

Pathogen-Mimicking Polymeric Nanoparticles based on Dopamine Polymerization as Vaccines Adjuvants Induce Robust Humoral and Cellular Immune Responses.

Aiming to enhance the immunogenicity of subunit vaccines, a novel antigen delivery and adjuvant system based on dopamine polymerization on the surface of poly(D,L-lactic-glycolic-acid) nanoparticles (NPs) with multiple mechanisms of immunity enhancement is developed. The mussel-inspired biomimetic polydopamine (pD) not only serves as a coating to NPs but also functionalizes NP surfaces. The method is facile and mild including simple incubation of the preformed NPs in the weak alkaline dopamine solution, and incorporation of hepatitis B surface antigen and TLR9 agonist unmethylated cytosine-guanine (CpG) motif with the pD surface. The as-constructed NPs possess pathogen-mimicking manners owing to their size, shape, and surface molecular immune-activating properties given by CpG. The biocompatibility and biosafety of these pathogen-mimicking NPs are confirmed using bone marrow-derived dendritic cells. Pathogen-mimicking NPs hold great potential as vaccine delivery and adjuvant system due to their ability to: 1) enhance cytokine secretion and immune cell recruitment at the injection site; 2) significantly activate and maturate dendritic cells; 3) induce stronger humoral and cellular immune responses in vivo. Furthermore, this simple and versatile dopamine polymerization method can be applicable to endow NPs with characteristics to mimic pathogen structure and function, and manipulate NPs for the generation of efficacious vaccine adjuvants.

Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with ß-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

Superior intratumoral penetration of paclitaxel nanodots strengthens tumor restriction and metastasis prevention.

Recently discovered intratumoral diffusion resistance, together with poor solubility and nontargeted distribution of chemotherapeutic drugs, has significantly impaired the performance of cancer treatments. By developing a well-designed droplet-confined/cryodesiccation-driven crystallization approach, we herein report the successful preparation of nanocrystallites of insoluble chemotherapeutic drug paclitaxel (PTX) in forms of nanodots (NDs, ≈10 nm) and nanoparticles (NPs, ≈70 nm) with considerably high drug loading capacity. Superficially coated Pluronic F127 is demonstrated to endow the both PTX nanocrystallites with excellent water solubility and prevent undesired phagocyte uptake. Further decoration with tumor-penetrating peptide iRGD, as expected, indiscriminatively facilitates tumor cell uptake in traditional monolayer cell culture model. On the contrary, distinctly enhanced performances in inward penetration and ensuing elimination of 3D multicellular tumor spheroids are achieved by iRGD-NDs rather than iRGD-NPs, revealing the significant influence of particle size variation in nanoscale. In vivo experiments verify that, although efficient tumor enrichment is achieved by all nanocrystallites, only the iRGD-grafted nanocrystallites of ultranano size realize thorough intratumoral delivery and reach cancer stem cells, which are concealed inside the tumor core. Consequently, much strengthened restriction on progress and metastasis of orthotopic 4T1 mammary adenocarcinoma is achieved in murine model, in sharp contrast to commercial PTX formulation Taxol.

A hydrophobic interaction chromatography strategy for purification of inactivated foot-and-mouth disease virus.

A purification scheme based on hydrophobic interaction chromatography was developed to separate inactivated foot-and-mouth disease virus (FMDV) from crude supernatant. About 92% recovery and 8.8-fold purification were achieved on Butyl Sepharose 4 FF. Further purification on Superdex 200 resulted in another 29-fold purification, with 92% recovery. The columns were coupled through an intermediate ultrafiltration unit to concentrate the virus. The entire process was completed in about 3.5h, with 75% final FMDV recovery, and 247-fold purification. The final product had purity above 98%, with over 99.5% of host cell DNA removed. High-performance size exclusion chromatography (HPSEC), Western blot, dynamic light scattering (DLS), and transmission electron microscopy (TEM) indicated that the purified virus contained the required antigen, and was structurally intact with a spherical shape and a particle size of 28 nm.

Microcosmic mechanism of dication for inhibiting acylation of acidic Peptide.

PURPOSE: For long-effective peptide formulation based on poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, acylation often leads to peptide instability during its release and reduced drug efficacy. Among the reported solving strategies, adding dication such as Ca(2+) and Mn(2+) in the formulation was the most convenient method for inhibiting basic peptide acylation. However, the strategies for the acidic peptide still remain unexplored, possibly due to the peptide's changeable charge state in acid environment within degraded PLGA microspheres. Moreover, the previous studies mainly focusing on the macroscopical adsorption of peptide to PLGA cannot demonstrate the inhibition mechanism. METHODS: Acylation inhibition for acidic peptide (exenatide) by dications (Ca(2+), Mn(2+) and Zn(2+)) was studied for the first time, and Quartz Crystal Microbalance with Dissipation (QCM-D) was innovatively employed to analyze microcosmic mechanism of the inhibition. RESULTS: These dications played different roles in acylation inhibition of acidic peptide. The effects of dications on acylation outside or inside PLGA microspheres indicated that Ca(2+) did not work, Mn(2+) played a weak role, and Zn(2+) possessed the greatest inhibition. CONCLUSIONS: Zn(2+) was the most effective dication for the acylation inhibition because of the complex formation and its steric-hindrance effect, which was a new function for this dication.

Immunopotentiator-Loaded Polymeric Microparticles as Robust Adjuvant to Improve Vaccine Efficacy.

PURPOSE: Adjuvants are required to ensure the efficacy of subunit vaccines. Incorporating molecular immunopotentiators within particles could overcome drawbacks of molecular adjuvants (such as solubility and toxicity), and improve adjuvanticity of particles, achieving stronger adjuvant activity. Aim of this study is to evaluate the adjuvanticity of immunopotentiator-loaded polymeric particles for subunit vaccine. METHODS: PLGA microparticles (PMPs) and imiquimod (TLR-7 ligand)-loaded PLGA microparticles (IPMPs) were prepared by SPG premix membrane emulsification. In vitro and in vivo studies were performed to their adjuvant activity, using ovalbumin and H5N1 influenza split vaccine as antigens. RESULTS: Incorporating imiquimod into microparticles significantly improved the efficacy of PLGA microparticles in activating BMDCs and pMΦs, and antigen uptake by pMΦs was also promoted. IPMPs showed stronger adjuvanticity to augment OVA-specific immune responses than PMPs. IgG subclass profiles and cytokine secretion levels by splenocytes indicated that IPMPs elicited more Th1-polarized immune response, compared to PMPs. In vivo study using H5N1 influenza split vaccine as antigen also confirmed the effects of IPMPs on antigen-specific cellular immunity. CONCLUSIONS: Considering adjuvanticity and safety profiles (PLGA and IMQ, both approved by FDA), we conclude that IMQ-loaded PLGA microparticles are promising robust adjuvant for subunit vaccines.

Comparison of covalent and physical immobilization of lipase in gigaporous polymeric microspheres.

Lipase (EC 3.1.1.3) is a versatile enzyme which has been widely used in ester-reaction industries. We have previously discovered that gigaporous polystyrene (PST) microspheres can be used as a novel immobilization carrier for lipase. In this work, a series of gigaporous microspheres with different densities of epoxy group including poly(glycidyl methacrylate) (PGMA) and poly(styrene-co-glycidyl methacrylate) [P(ST-GMA)] were evaluated as lipase immobilization carriers, which were also compared with gigaporous PST microspheres and the commercial immobilized lipase Novozym 435. Lipase immobilized in gigaporous PGMA microspheres showed the highest activity yield, reusability, and stability as well as the best affinity for the substrate. The characterizations of adsorption curves, the change of epoxy group amounts, and hydrophobic-hydrophilic properties of the microspheres were carried out to investigate the interaction between lipase molecules and carriers. It was found that covalent binding played a key role in improving the properties of lipase immobilized in gigaporous PGMA microspheres.

Comparison of site-specific PEGylations of the N-terminus of interferon beta-1b: selectivity, efficiency, and in vivo/vitro activity.

PEGylation, including nonspecific and site-directed approaches, is a well-established and validated strategy to increase the stability, in vivo plasma retention time, and efficacy of protein pharmaceutics together with a reduction in immunogenicity and hydrophobicity. Site-directed conjugation by PEG-aldehyde is the most widely used method for N-terminal modification; however, the generation of multimodified products is inevitable because of lysine chemistry, which always leads to difficulties in purification and quantification. In this study, we developed a specific PEGylation strategy through the periodation of the N-terminus of interferon beta-1b (IFN-ß-1b) followed by the coupling of PEG-hydrazide. The prolonged elimination half-life and significantly diminished immunogenicity of the PEG-hydrazide-modified protein indicated the development of an effective process for improving the pharmacology and immunogenicity of IFN-ß-1b. We further conducted comparisons on the selectivity, velocity, yield, and pharmacokinetics of the two methods. The results demonstrated that the hydrazide-based conjugation was a highly specific coupling reaction that only produced homogeneous N-terminal mono-PEGylated conjugate but also generated heterogeneous multimodified products in the aldehyde-based process. In addition, a better PEGylation yield was found for the hydrazide conjugation compared with that of the aldehyde strategy. These investigations supply a practical approach for the site-specific modification of proteins with an N-terminal serine or threonine to achieve improved homogeneity of the conjugates as well as enhanced pharmacological properties.

Nanoengineering of stimuli-responsive protein-based biomimetic protocells as versatile drug delivery tools.

We present a general strategy to nanoengineer protein-based colloidal spheres (biomimetic protocells) as versatile delivery carriers with stimuli responsiveness by the electrostatic assembly of binary components (proteins and polypeptides) in association with intermolecular disulfide cross-linking. The size of the colloidal spheres, ranging from nanoscale to microscale, is readily tuned through parameters like protein and polypeptide concentration, the ratio between both, pH, and so on. Moreover, such colloidal spheres show versatile encapsulation of various guest molecules including small organic molecules and biomacromolecules. The pH and redox dual-responsiveness facilitates the rapid release of the payload in an acidic and reductant-enriched ambient such as in lysosomes. Thus, nanoengineering of protein-based biomimetic protocells opens a new alternative avenue for developing delivery vehicles with multifunctional properties towards a range of therapeutic and diagnostic applications.

Enhanced humoral and cell-mediated immune responses generated by cationic polymer-coated PLA microspheres with adsorbed HBsAg.

Surface-engineered particulate delivery systems for vaccine administration have been widely investigated in experimental and clinical studies. However, little is known about charge-coated microspheres as potential recombinant subunit protein antigen delivery systems in terms of adsorption and related immune responses. In the present study, cationic polymers, including chitosan (CS), chitosan chloride (CSC), and polyethylenimine (PEI), were used to coat PLA microspheres to build positively charged surfaces. Antigen adsorption capacity was enhanced with increased surface charge of coated microspheres. In macrophages, HBsAg adsorbed on the surface of cationic microspheres specifically enhanced antigen uptake and augmented CD86, MHC I, and MHC II expression and IL-1ß, IL-6, TNF-α, and IL-12 release. Antigens were more likely to localize independent of lysosomes after phagocytosis in antigen-attached cationic microsphere formulations. After intraperitoneal immunization, cationic microsphere-based vaccine formulations generated a rapid and efficient humoral immune response and cytokine release as compared with aluminum-adsorbed vaccine and free antigens in vivo. Moreover, microspheres coated with cationic polymers with relatively high positive charges and higher antigen adsorption exhibited strong stimulation of the Th1 response. In conclusion, PLA microspheres coated with cationic polymers may be a potential recombinant antigen delivery system to induce strong cell and humoral immune responses.

Liquid marbles stabilized by charged polymer latexes: how does the drying of the latex particles affect the properties of liquid marbles?

The coating of solid particles on the surface of liquid in air makes liquid marbles a promising approach in the transportation of a small amount of liquid. The stabilization of liquid marbles by polymeric latex particles imparts extra triggers such as pH and temperature, leading to the remote manipulation of droplets for many potential applications. Because the functionalized polymeric latexes can exist either as colloidally stable latex or as flocculated latex in a dispersion, the drying of latex dispersions under different conditions may play a significant role in the stabilization of subsequent liquid marbles. This article presents the investigation of liquid marbles stabilized by poly(styrene-co-methacrylic acid) (PS-co-MAA) particles drying under varied conditions. Protonation of the particles before freeze drying makes the particles excellent liquid marble stabilizers, but it is hard to stabilize liquid marbles for particles dried in their deprotonated states. The static properties of liquid marbles with increasing concentrations of protonating reagent revealed that the liquid marbles are gradually undermined by protonating the stabilizers. Furthermore, the liquid marbles stabilized by different particles showed distinct behaviors in separation and merging manipulated by tweezers. This study shows that the initial state of the particles should be carefully taken into account in formulating liquid marbles.

Preparation of uniform particle-stabilized emulsions using SPG membrane emulsification.

Various aspects of particle-stabilized emulsions (or so-called Pickering emulsions) have been extensively investigated during the last two decades, but the preparation of uniform Pickering emulsion droplets via a simple and scalable method has been sparingly realized. We report the preparation of uniform Pickering emulsions by Shirasu porous glass (SPG) membrane emulsification. The size of the emulsion droplets ranging from 10-50 µm can be precisely controlled by the size of the membrane pore. The emulsion droplets have a high monodispersity with coefficients of variation (CV) lower than 15% in all of the investigated systems. We further demonstrate the feasibility of locking the assembled particles at the interface, and emulsion droplets have been shown to be excellent templates for the preparation of monodisperse colloidosomes that are necessary in drug-delivery systems.

Comparative studies on the influences of primary emulsion preparation on properties of uniform-sized exenatide-loaded PLGA microspheres.

PURPOSE: It is well known that primary emulsion (W1/O) preparation process (by ultrasonication or homogenization) plays an important role in the properties of drug-loaded microspheres, such as encapsulation efficiency, release behavior and pharmacodynamics. However, its involved mechanism has not been intensively and systematically studied, partly because that broad size distribution of the resultant particles prepared by conventional preparation can greatly disturb the analysis and reliability of the results. Here, we focused on the relevant studies. METHODS: In order to eliminate the disturbance caused by broad size distribution, uniform-sized exenatide-loaded poly(DL-lactic-co-glycolic acid) (PLGA) microspheres were prepared by Shirasu Porous Glass (SPG) premix membrane emulsification. The properties of microspheres whose W1/O was formed by ultrasonication (UMS) and homogenization (HMS) were compared including in vitro release, pharmacology and so forth. RESULTS: HMS exhibited fast release rate and hyperglycemic efficacy within first 14 days, but declined afterwards. Comparatively, UMS showed slower polymer degradation, more acidic microclimate pH (µpH) in vitro, and stable drug release with sustained efficacy during 1 month in vivo. CONCLUSIONS: HMS was desirable for the 2-week-sustained release in vivo, while UMS was more appropriate for the longer time release (about 1 month). These comparative researches can provide guidance for emulsion-microsphere preparation routs in pharmaceutics.

Comparison of PLA microparticles and alum as adjuvants for H5N1 influenza split vaccine: adjuvanticity evaluation and preliminary action mode analysis.

PURPOSE: To compare the adjuvanticity of polymeric particles (new-generation adjuvant) and alum (the traditional and FDA-approved adjuvant) for H5N1 influenza split vaccine, and to investigate respective action mode. METHODS: Vaccine formulations were prepared by incubating lyophilized poly(lactic acid) (PLA) microparticles or alum within antigen solution. Antigen-specific immune responses in mice were evaluated using ELISA, ELISpot, and flow cytometry assay. Adjuvants' action modes were investigated by determining antigen persistence at injection sites, local inflammation response, antigen transport into draining lymph node, and activation of DCs in secondary lymphoid organs (SLOs). RESULTS: Alum promoted antigen-specific humoral immune response. PLA microparticles augmented both humoral immune response and cell-mediated-immunity which might enhance cross-protection of influenza vaccine. With regard to action mode, alum adjuvant functions by improving antigen persistence at injection sites, inducing severe local inflammation, slightly improving antigen transport into draining lymph nodes, and improving the expression of MHC II on DCs in SLOs. PLA microparticles function by slightly improving antigen transport into draining lymph nodes, and promoting the expression of both MHC molecules and co-stimulatory molecules on DCs in SLOs. CONCLUSIONS: Considering the adjuvanticity and side effects (local inflammation) of both adjuvants, we conclude that PLA microparticles are promising alternative adjuvant for H5N1 influenza split vaccine.

"Ready-to-use" hollow nanofiber membrane-based glucose testing strips.

A novel "ready-to-use" glucose test strip based on a polyurethane hollow nanofiber membrane was fabricated through facile co-axial electrospinning. By utilizing glucose oxidase and horseradish peroxidase in the core-phase solution, and a chromogenic agent either in the core solution (in which case 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) was used) or in the shell-phase solution (in which case o-dianisidine was used) for co-axial electrospinning, in situ co-encapsulation of the two enzymes within the hollow nano-chamber and incorporation of chromogenic agents either inside the nano-chamber or in the shell of the hollow nanofibers was realized. Such unique "all-in-one" feature enabled the prepared hollow nanofiber membrane-based test strips to be applied either as colorimetric sensors in solution or as an optical biosensor operated in the "dip-and-read" mode. When used as a colorimetric biosensor in solution, the test strip with o-dianisidine as chromogenic agent shows an excellent linear response range between 0.01 mM to 20 mM and a high apparent lumped activity recovery of 62.1% as compared to the reaction rate of the free bi-enzyme system. While the activity recovery of the test strip with ABTS as chromogenic agent is only 18.0%, and the test strip is found to be unstable due to spontaneous-oxidation of the ABTS. The o-dianisidine test strip was also applied as an optical biosensor, visible rufous color was quickly developed on the surface of the membrane upon dropping 10 µL of glucose sample, and an excellent correlation between differential diffusive reflectance of the test strip at 440 nm and glucose concentration was obtained in the range of 0.5-50 mM. The test strips also exhibited excellent long-term storage stability with a half-life at 25 °C as long as four months.

Dictating the spatial-temporal delivery of molecular adjuvant and antigen for the enhanced vaccination.

The incorporation of molecular adjuvants has revolutionized vaccine by boosting overall immune efficacy. While traditional efforts have been concentrated on the quality and quantity of vaccine components, the impact of adjuvant and antigen delivery kinetics on immunity remains to be fully understood. Here, we employed poly (lactic-co-glycolic acid) nanoparticle (PLGA NP) -stabilized Pickering emulsion (PPE) to refine the delivery kinetics of molecular adjuvant CpG and antigen, aiming to optimize immune responses. The hierarchical structure of PPE enabled spatially differential loading of CpG and antigen. The component inserted on the oil-water interphase exhibited a rapid release profile, while the one encapsulated in the PLGA NPs demonstrated a sustained release. This led to distinct intracellular spatial-temporal release kinetics. Compared to the PPE with sustained CpG release and burst release of antigen, we found that the PPE with rapid CpG release and sustained antigen release triggered an early and robust activation of Toll-like receptor 9 (TLR9) in direct way. This fostered a more immunogenic microenvironment, significantly outperforming the inverted delivery profile in dendritic cells (DCs) activation, resulting in higher CD40 expression, elevated proinflammatory cytokine levels, sustained antigen cross-presentation, an enhanced Th1 response, and increased CD8+ T cells. Moreover, prior exposure of CpG led to suppressed tumor growth and enhanced efficacy in Varicella-zoster virus (VZV) vaccine. Our findings underscore the importance of tuning adjuvant and antigen delivery kinetics in vaccine design, proposing a novel path for enhancing vaccination outcomes.