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Parenteral vaccinations are not able to elicit effective systemic and gastrointestinal immune protection simultaneously because the lymphocytes are typically restricted to primed tissues. Although all-trans retinoic acid (atRA) was reported to trigger the gut-homing of immunocytes, the bioavailability and systemic immune responses remain limited for use in robust enteric vaccinations. Here, we show that co-delivery of atRA, CpG oligodeoxynucleotides (CpG), and antigens via engineered polymer/lipid nanoparticles (PLNPs) could exploit the amplifying function of draining lymph nodes (DLNs) for potent gut tropism and immune activations. After intramuscular injection, forming an immune-potentiated environment at the injection site, the PLNPs induced the designated transfer of primed dendritic cells (DCs) to the DLNs instead of the gastrointestinal tissues. Within the DLNs, the immune-potentiated environment markedly amplified the antigen presentation and homing receptor switch among immunocytes, which simultaneously stimulated the preferential dissipation of activated lymphocytes in the peripheral and gastrointestinal tissues, that is, exerted a DLN-amplifying effect. Compared with current atRA-containing formulations, the PLNPs not only boosted potent IgG secretions and T cell activations in the peripheral tissue but also provoked robust T cell homing and antigen-specific IgA levels in the gastrointestinal tracts in both ovalbumin and EV71 vaccinations. These data indicate that exploiting DLN amplification can stimulate potent systemic and gastrointestinal responses for more efficient enteric vaccinations.

A new type of single-chromophore-based photoactivatable prodrug (B-Cbl-3) enabling green-light-triggered chemotherapy and simultaneous photodynamic therapy with superb therapeutic efficacy was developed by conjugating a photoactive BODIPY derivative with an antitumor chlorambucil moiety. The optimized BODIPY moiety markedly enabled high efficient photogeneration of 1O2 and fluorescence emission with distinct colors before and after photorelease of chlorambucil. The preliminary biological experiment results have verified the efficient photorelease of chlorambucil from B-Cbl-3 and the huge contrast in cytotoxicity between them, superior combined therapeutic performance based on extraordinary low doses of drug and light irradiation, and ratiometric fluorescence imaging for in situ monitoring drug release. The salient superiority of B-Cbl-3 regarding alleviating the attenuation of triggering light caused by optically turbid tissue that short-wavelength lights typically encounters has also been verified.

A scalable and versatile strategy was developed for the fabrication of uniform polymeric microspheres with controllable interconnected porous structures. Uniform water-in-oil-in-water emulsions with linear poly(methyl methacrylate–glycidyl methacrylate) in the oil phase were generated by two-step premix membrane emulsification and used for constructing the microspheres. During the emulsion solidification process, internal water droplets were packed densely together, forming a thin oil film between the internal and external water phases. After solvent diffusion, the thin film can be ruptured and pores can be templated from the internal water droplets to form interconnected porous structures. Membranes with various pore sizes were obtained. The osmotic pressure and Laplace pressure balance were used to control the porosity and pore size precisely. The proposed method enables the fabrication of functional polymeric microspheres with uniform and controllable porous structures and particle sizes. This improves their performance and broadens the scope of their applications, especially in chromatographic separation.

Vaccine adjuvants play an increasingly important role in the development of modern vaccines. Among of them, particle adjuvants have received wide attention due to their unique properties. In order to develop effective particle adjuvants, mimicking natural pathogens is one of the important strategies. By adopting different materials, preparation methods and design ideas, the researchers obtained various particle adjuvants that mimic the shape, surface properties and action mechanisms of natural pathogens. This mini-review outlines some selected relevant recent literatures, discusses several bionic design strategies of particle adjuvants and some important barriers to further study are described.

Chinese biochemical engineering is committed to supporting the chemical and food industries, to advance science and technology frontiers, and to meet major demands of Chinese society and national economic development. This paper reviews the development of biochemical engineering, strategic deployment of these technologies by the government, industrial demand, research progress, and breakthroughs in key technologies in China. Furthermore, the outlook for future developments in biochemical engineering in China is also discussed.

Ropivacaine loaded microspheres offer an attractive alternative for prolonging the duration of local anesthetics for the pain management. However, traditional microcapsulation methods could not provide microspheres with desired drug loading efficiency and a narrow size distribution, leading to poor patient compliance as well as preparation repeatability. In this study, we proposed to combine O/W emulsion method with novel premix membrane emulsification technique. After the optimization of fabrication procedure, ropivacaine loaded microspheres with narrow size distribution (span 0.469), high drug loading efficiency (49%) and an ideal constant release behavior have been obtained. Especially, it was found that solidification process affected the internal structure of microspheres and the physical state of encapsulated ropivacaine. Furthermore, it was interested that lower polymer molecular weight resulted in higher encapsulation efficiency, which was not consistent with reported results. It was found that this was ascribed to the interaction of PLGA-ropivacaine. All these aspects had considerable influences on the characteristics of microspheres. In addition, the cytotoxicity on different cell lines (SHSY5Y and HaCaT cells) was detected qualitatively and quantitatively. It showed that ropivacaine loaded microspheres did reduce the Reactive oxygen species (ROS) and cytotoxicity prominently compared to free ropivacaine, which could be attributed to the sustained and steady drug release behavior. This study provided some pioneering ideas for encapsulating small molecule drug with poor solubility. The optimized microspheres could be chosen as an ideal candidate for the ropivacaine sustained release with its better drug adherence, lower toxicity of drug burst release and best therapeutic effect.

The Central Solenoid Model Coil (CSMC) project has been launched at the Institute of Plasma and Physics Chinese Academy of Sciences (ASIPP) for China Fusion Engineering Test Reactor (CFETR). Electrical joints are important aspect for CSMC, because the local heating from resistive losses can drive the superconductor into a less stable regime and result in premature quench. So part of the project is to develop joints with low DC resistance and AC loss. AC loss power and local heating of the full-size prototype conductor joint in the preliminary test scenario of CSMC are evaluated experimentally and numerically in this paper. The AC loss of the joint sample is measured in SULTAN facility with an applied harmonic field and wider frequency range. Based on the experimental results, the AC loss of the joint is characterized by Multizones Partial Shielding (MPAS) model. Then, the AC loss power of the joint is calculated in the test scenario. The temperature rise of joint is investigated numerically due to the simulated AC loss in the test scenario. The simulation results show the temperature rise in the joint is relatively higher which affects the stability greatly not only in joints, but also the coils. So the joint structure and test scenario of CSMC need to be optimized to reduce the AC loss and ensure safe operation. The way to reduce the AC loss deposit in the joint is also discussed.

Inactivated virus and virus-like particles (VLPs) are important classes of biopharmaceuticals for vaccines, immunotherapy and oncotherapy. Their complex particle structures are easily denatured during processing and storage, leading to loss in their biofunctionality. Ionic liquids (ILs) as stabilizing excipients have garnered interest in protein-based pharmaceutical research, but their stabilizing capacity for inactivated virus antigens remains unknown. Here, three biocompatible choline-based ILs, including [Cho][H2PO4], [Cho][Cl], and [Cho][SO4], were tested as potential stabilizers for the inactivated foot-and-mouth disease virus (iFMDV), which are extremely unstable virus particles easily dissociating into smaller pentamers named 12S. Based on differential scanning fluorimetry technology for thermal stability analysis, together with high-performance size-exclusion chromatography for quantitative determination of 146S, it was found that [Cho][Cl] and [cho][SO4] can improve the thermo- and long-term storage stability of iFMDV particles, while [Cho][H2PO4] showed a destabilizing effect. Animal experiments indicated that the immunogenicity of iFMDV antigens was not attenuated in all three ILs. By monitoring the microenvironmental pH of the virus particles in different ILs, a relatively lower proton intensity was observed in [Cho][Cl] and [Cho][SO4] than in buffers and [Cho][H2PO4]. Therefore, the stabilizing mechanism was supposed to be mainly due to suppression of protonation of histidine residues in the inter-pentamer interface of virus particles in [Cho][Cl] and [Cho][SO4], which is distinct from the mechanism reported for other proteins with relatively simple structures. The results suggest that the choline-based ILs with appropriate anions are promising stabilizing excipients for iFMDV or other vaccine antigens.

Size exclusion chromatography (SEC) of biomacromolecules using large pore size media and long column are usually necessary to obtain a satisfactory separation. However, the SEC separation of inactivated foot and mouth disease virus (FMDV) was found to induce some subtle but important conformational changes of FMDV in a pore-size and column length dependent manner. Here three Sephacryl media including S-300 HR, S-400 HR, and S-500 HR were tested, whose pore sizes were smaller than, similar to, and larger than the FMDV particles, respectively. High performance size exclusion chromatography (HPSEC) analyses showed that the FMDV after all these three SEC processes had earlier retention time, compared with that before SEC, but had no detectable difference in particle integrity. Longer SEC column led to more significant peak shifting in subsequent HPSEC analysis of FMDV. Further analyses indicated the SEC using larger pore size media induced more remarkable conformational changes and decrease in thermostability of FMDV, as well as decrease in immunogenicity in animal test. Fluorescence probe diffusion study suggested compared to SEC by S300, the compactness of the viral capsid after SEC by S400 and S500 was decreased, possibly due to more shear-induced FMDV particle rotation and inter-particle collision inside the media pores, as well as their interactions with the pore walls of the media during flowing through the column. Finally, a stabilization strategy by appending 5 mM CaCl

A novel nanodisc-based immobilization method was developed for high-efficient purification and reconstitution of cytochrome P450 in one step. Using membrane scaffold protein containing a histidine tag, charged-nanodiscs were prepared in the form of self-assembly of lipid-protein nanoparticles. Their properties including the particle diameter and its distribution and Zeta potential were controlled well by adjusting molar ratios of phospholipids to membrane scaffold protein. At an optimum lipid-to-membrane scaffold protein molar ratio of 60:1, uniformly regular-shaped and discoidal nanodiscs with an average particle diameter of 10 nm and Zeta potential of -19 mV were obtained. They can be well fractionated by size exclusion chromatography. Charged-nanodiscs were successfully immobilized onto Ni-chelating microspheres via histidine tags with a density of 6.6 mg membrane scaffold protein/mL gel. After being packed in a column, chromatography studies demonstrated that this nanodisc-immobilized chromatographic medium had a specific binding to cytochrome P450 in rat liver microsome. Nanodiscs containing cytochrome P450 can be furthermore eluted from the column with a diameter of about 87.0 nm and height of about 8.0 nm, respectively. The purity of cytochrome P450 after purification increased 25 folds strikingly. This nanodisc-immobilized chromatography method is promising for the one-step purification and reconstitution of membrane protein.

In article number 2002787, Yugang Wang, Wei Wei, Guanghui Ma, and co‐workers develop an exosome‐based platform for smart drug delivery and personalized antitumor therapy. They discover that the exosomes released from low‐pH treated tumor cells have the strong uptake efficiency and targeting ability for their parent cells. Such “magical” characteristics are based on a glycerolipid selfaggregation‐enhanced uptake mechanism. These reprogrammed exosomes are further utilized as smart carriers for dual‐loading of photosensitizer and chemodrug, which are capable of specifically accumulating in tumor tissue, selectively releasing chemodrug in response to the near‐infrared irradiance and achieving safe and potent antitumor effects. [Image: see text]

Immunotherapy that utilizes the human immune system to fight cancer represents a revolutionary method for cancer treatment. Immunotherapeutic agents that trigger the immune response should be carefully delivered to the desired site to maximize immunotherapy effectiveness and minimize side effects. Vesicles offer the possibility of encapsulating both hydrophilic and hydrophobic drugs and thus serve as a promising delivery tool. As multiple irreconcilable requirements exist at different transport stages, developing vesicles transformable in response to given stimuli is of great significance. In this review, we first introduced various vesicle types used for immunotherapy. Furthermore, the typical stimuli that trigger vesicle transformation and the usually generated transformation styles were described. Focusing on three aspects of antigen-presenting cell (APC)/T cell activation, tumor microenvironment (TME) amelioration, and immunogenic cell death (ICD)-induced immunotherapy, we reviewed recently reported transformable vesicles for tumor treatment. Finally, we put forward possible directions for future research and clinical translation.

In the combat against pathogens, the immune systems were evolved with the immune recognitions against the various danger signals, which responded vigorously upon the pathogen invasions and elicited potent antibodies or T cell engagement against the re-infections. Envisage with the prevailing pandemics and increasing demands for cancer vaccines, bio-mimic particles were developed to imitate the natural traits of the pathogens, which conferred the optimal strategies to stimulate the immune engagement and let to the increased vaccine efficacy. Here, the recent development in bio-mimic particles, as well as the natural cues from the pathogens were discussed. As such, the designing principles that adapted from the physiochemical properties of the pathogens were unfolded as the surface characteristics (hydrophobic, nano-pattern, antigen display, charge), properties (size, shape, softness) and the delivered components (peptide, protein, nuclear acids, toll-like receptor (TLR) agonist, antibody). Additionally, the strategies for the efficient delivery, regarding the biodistribution, internalization and presentation of the antigens were also illustrated. Through reviewing the state-of-art in biomimetic particles, the lesson learnt from the natural traits and pathogenic invasion may shed light on the rational design for the enhanced vaccinations.

In recent years, personalized cancer vaccines have gained increasing attention as emerging immunotherapies with the capability to overcome interindividual differences and show great benefits for individual patients in the clinic due to the highly tailored vaccine formulations. A large number of materials have been studied as delivery vectors to enhance the therapeutic performance of personalized cancer vaccines, including artificial materials, engineered microorganisms, cells and cell derivatives. These delivery vectors with distinct features are employed to change antigen biodistributions and to facilitate antigen uptake, processing and presentation, improving the strength, velocity, and duration of the immune response when delivered by different strategies. Here, we provide an overview of personalized cancer vaccine delivery vectors, describing their materials, physicochemical properties, delivery strategies and challenges for clinical transformation.

We herein developed a core-shell type antitumor nanoagent based on the synergistically reinforced oxidative stress and calcium overload to mitochondria, both of which were triggered by near-infrared (NIR) light. The folic acid (FA) moiety decorated on MOF shells enabled efficient cellular uptake of nanoagents. The upconversion nanoparticle (UCNP) core converted NIR light to ultraviolet (UV) light with the latter catalyzed Fe3+-to-Fe2+ reduction and simultaneously activated the photoacid generator encapsulated in the cavities of MOFs, which enabled the release of free Fe2+ and photoacidification of intracellular microenvironment, respectively. The overexpressed H2O2 in the mitochondria, highly reactive Fe2+ and acidic milieu synergistically reinforced Fenton reactions for producing lethal hydroxyl radicals in mitochondria. Moreover, the photoacidification of plasma induced calcium influx, leading to calcium overload in the mitochondria. The therapeutic potency of the nanoagent based on the dual mitochondrial damage has been unequivocally confirmed in cell- and patient-derived tumor xenograft models in vivo.

Targeting subcellular organelle with multilevel damage has shown great promise for antitumor therapy. Here, we report a core-shell type of nanoagent with iron (III) carboxylate metal-organic frameworks (MOFs) as shell while upconversion nanoparticles (UCNPs) as core, which enables near-infrared (NIR) light-triggered synergistically reinforced oxidative stress and calcium overload to mitochondria. The folate decoration on MOFs shells enables efficient cellular uptake of nanoagents. Based on the upconversion ability of UCNPs, NIR light mediates Fe3+-to-Fe2+ reduction and simultaneously activates the photoacid generator (pHP) encapsulated in MOFs cavities, which enables release of free Fe2+ and acidification of intracellular microenvironment, respectively. The overexpressed H2O2 in mitochondria, highly reactive Fe2+ and acidic milieu synergistically reinforce Fenton reactions for producing lethal hydroxyl radicals (•OH) while plasma photoacidification inducing calcium influx, leading to mitochondria calcium overload. The dual-mitochondria-damage-based therapeutic potency of the nanoagent has been unequivocally confirmed in cell- and patient-derived tumor xenograft models in vivo., Targeting damage to mitochondria has become an effective strategy antitumor therapies. Here, the authors report on nanoagents with upconversion nanoparticles as cores and photoacid-loaded MOFs as shells for NIR triggered Fenton reaction, acidification and calcium overload to provide synergistic mitochondrial damage.

Graphical abstract, Prophylactic vaccines have evolved from traditional whole-cell vaccines to safer subunit vaccines. However, subunit vaccines still face problems, such as poor immunogenicity and low efficiency, while traditional adjuvants are usually unable to meet specific response needs. Advanced delivery vectors are important to overcome these barriers; they have favorable safety and effectiveness, tunable properties, precise location, and immunomodulatory capabilities. Nevertheless, there has been no systematic summary of the delivery systems to cover a wide range of infectious pathogens. We herein summarized and compared the delivery systems for major or epidemic infectious diseases caused by bacteria, viruses, fungi, and parasites. We also included the newly licensed vaccines (e.g., COVID-19 vaccines) and those close to licensure. Furthermore, we highlighted advanced delivery systems with high efficiency, cross-protection, or long-term protection against epidemic pathogens, and we put forward prospects and thoughts on the development of future prophylactic vaccines.

A major challenge in vaccine delivery is to achieve robust lymph-node (LN) accumulation, which can capitalize on concentrated immunocytes and cytokines in LNs to stimulate the onset and persistence of adaptive immune responses. Previous attempts at developing vaccine delivery systems have focused on the sizes, charges, or surface ligands but not on their deformability. In fact, the LN homing of antigen-presenting cells depends on deformability to pass through the cellular gaps. Herein, the deformability of albumin-stabilized emulsions is engineered. Owing to self-adaptive deformability, the droplets (≈330 nm) can attach to and deform between cells and adjust their sizes to pass through the endothelial gaps (20-100 nm), favoring direct LN transfer (intercellular pathway). Additionally, owing to relatively large sizes, some emulsions can be retained at the administration sites for potent antigen uptake and activation of APCs as well as LN-targeted delivery of vaccines (intracellular pathway). Compared with solid particles, the dual LN transfer strategy evidently enhances antigen accumulation and activation of LN drainage, potently stimulates cellular immune responses, and increases the survival rate of tumor-bearing mice. Thus, the deformability of albumin-stabilized droplets may offer an efficient strategy for potent LN targeting and enhanced vaccinations.

Customers can raise various aspects of concerns and anxieties during their participation in gamified social mobile marketing campaigns. To gain insights into how to address these anxieties, this research explores the impact of group-formation gamification mechanisms on users’ emotional anxiety and their intentions to participate in such campaigns. A research model depicting group-formation gamification strategies, user anxieties and participation intentions in gamified social mobile marketing campaigns was proposed and examined through experiments involving 232 participants, which were triangulated by electroencephalogram (EEG) tests and follow-up interviews. The results of this study showed that compared with weak-tie group-formation mechanisms, strong-tie mechanisms can result in a lower level of user emotional anxiety, including reductions in user manipulation anxiety, user privacy anxiety and social image anxiety, which in turn led to higher user intentions to participate in the gamified social mobile marketing campaign. Moreover, it was found that user gender and disposable incomes had significant moderating effects on user anxiety during their interactions with the campaign.

The targeted nanocomplex efficiently treats Parkinson’s disease by clearing α-synuclein and immune activation., The most critical problem in the treatment of neurodegenerative diseases is brain neuronal protection, which can be overcome by clearing pathological substances and regulating the immune environment. In the above treatment strategies, the traditional poor drug delivery problem is inevitable. Here, we show an engineering core-shell hybrid system named rabies virus glycoprotein (RVG) peptide–modified exosome (EXO) curcumin/phenylboronic acid-poly(2-(dimethylamino)ethyl acrylate) nanoparticle/small interfering RNA targeting SNCA (REXO-C/ANP/S). It is a nanoscavenger for clearing α-synuclein aggregates and reducing their cytotoxicity in Parkinson’s disease neurons. The motor behavior of Parkinson’s disease mice is substantially improved after REXO-C/ANP/S treatment. In particular, we demonstrate that REXO-C/ANP/S is also a nanoscavenger for clearing immune activation due to its natural immature dendritic cell EXO coating. Our findings show that REXO-C/ANP/S may serve as a platform for neurodegenerative diseases treatment.

Fast-throughput and cost reduction of current purification platforms are becoming increasing requests during antibody manufacture. The macroporous-matrix absorbents have presented extensive potentiality in improving operational throughput during purification of macromolecule. And meanwhile the peptide ligand has become a promising alternative to recombinant protein ligands for cost reduction of chromatographic purification. Therefore, here we designed a functionalized microspheres resin with both macroporous matrix of polymerized glycidyl methacrylate and ethylene glycol dimethacrylate (PGMA-EDMA) and peptide ligand of hexapeptide (FYEILH). In order to circumvent the steric effect of peptides and amplify the binding sites on macroporous matrix, the peptide ligand was coupled on a liner PGMA polymer brushes grafted on microspheres. Comparing to the conventional agarose-matrix resin and the general peptide-grafted microspheres, the functionalized microspheres presented excellent permeability and high capacity to rapid loading hIgG by maintaining a stable level of dynamic binding capacity at fast flow rate above 110 column volume per hour (cv/h) and very short residence time below 0.5 min. Such functionalized microspheres provide a facile and broadly applicable strategy to develop the attractive candidate for rapid and cost-reduced purification of antibody.

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.

Trivalent arsenic (AsIII) is an effective agent for treating patients with acute promyelocytic leukaemia, but its ionic nature leads to several major limitations like low effective concentrations in leukaemia cells and substantial off-target cytotoxicity, which limits its general application to other types of leukaemia. Here, building from our clinical discovery that cancerous cells from patients with different leukaemia forms featured stable and strong expression of CD71, we designed a ferritin-based As nanomedicine, As@Fn, that bound to leukaemia cells with very high affinity, and efficiently delivered cytotoxic AsIII into a large diversity of leukaemia cell lines and patient cells. Moreover, As@Fn exerted strong anti-leukaemia effects in diverse cell-line-derived xenograft models, as well as in a patient-derived xenograft model, in which it consistently outperformed the gold standard, showing its potential as a precision treatment for a variety of leukaemias. Trivalent arsenic (AsIII) is a clinically approved treatment agent for patients with promyelocytic leukaemia, but cannot be used for other types of leukaemia due to its toxicity. Here the authors show that different patient-derived leukaemia cells express CD71 and design a ferritin-based nanoparticle for specific delivery of AsIII to these cells, demonstrating substantially improved efficacy towards different leukaemia types in animal models, with reduced side effects.

The aggregative instinct of platelets was exploited as a biomimetic strategy for synergistic combined therapy against cancer., To address long-standing issues with tumor penetration and targeting among cancer therapeutics, we developed an anticancer platelet-based biomimetic formulation (N+R@PLTs), integrating photothermal nanoparticles (N) and immunostimulator (R) into platelets (PLTs). Exploiting the aggregative properties of platelets and high photothermal capacity, N+R@PLTs functioned as an arsenal by targeting defective tumor vascular endothelial cells, accumulating in a positive feedback aggregation cascade at sites of acute vascular damage induced by N-generated local hyperthermia, and subsequently secreting nanosized proplatelets (nPLTs) to transport active components to deep tumor tissue. The immunostimulator augmented the immunogenicity of antigens released from ablated tumors, inducing a stronger immunological response to attack residual, metastatic, and recurrent tumors. Following activation by low-power near-infrared light irradiation, the photothermal and immunological components synergistically provide exceptionally high therapeutic efficacy across nine murine models that mimicked a range of clinical requirements, and, most notably, a sophisticated model based on humanized mouse and patient-derived tumor xenograft.

To generate effective immunity post-vaccination, antigens need to be effectively captured and taken up by antigen-presenting cells (APCs) as a prerequisite. Biomimetic designs that mimic natural pathogen-like properties have provided platforms for antigen delivery. However, the structural dynamic properties of pathogens leading to their efficient internalization have been neglected in most platforms. Herein, we redesigned a special multiple emulsion with chitosan hydrogel nanoparticles inside, mimicking the configurational flexibility and deformational flexibility of pathogens. With the assistance of chitosan-antigen particles, the novel emulsion exhibited amplified deformability and the vaccine-cell contact zone was increased. Additionally, its configurational transitions, which offered sustained exposure of sheltered uptake signals including antigens and stimulator chitosan during endocytosis, resulted in efficient antigen delivery to APCs. Prolonged antigen depot effect, versatile antigen presentation, multiple immunocyte activation, and marked adjuvant-sparing effects were achieved as compared with those in the control groups. As a result, the intracellular emulsion formulation robustly induced both humoral and cellular immunity, especially CTL response, against the foot-and-mouth disease virus (FMDV) with improved biosafety. Our study highlights the positive impact of biomimetic structural dynamic properties on robust vaccine-cell interactions and provides a promising FMDV vaccine candidate.

Local anesthetic drugs are widely used in postoperative analgesia, local nerve block in surgery, and in the therapy of chronic pain. Due to their short half-time, a single administration of local anesthetics merely maintains the nerve block for several hours, which cannot meet the long-term analgesia effect needed in clinical treatment. In order to break through this limitation of local anesthetics, research work regarding controlled drug release from microspheres has attracted broad and current interest. This review introduces the recent research and development in local anesthetic-loaded microspheres for future clinical applications, where for an efficient microsphere formulation, the most critical aspects are an optimum preparation method, a high loading efficiency, and an ideal release rate. This review first summarizes the recent preparation methods of local anesthetic-loaded microspheres, which includes emulsion-solvent evaporation, spray-drying, microfluidic droplets, and premix membrane emulsification technology. Next, strategies for increasing the loading efficiency of the drug in microspheres are reviewed based on the solidification conditions, polymer properties, microsphere formulation, including the external water phase, pH, and polymer concentration. Finally, the effects of the preparation conditions, material characteristics, particle characteristics, and hydrogel/microsphere composite systems on the controlled release behavior are summarized.

For rapid response against the prevailing COVID-19 (coronavirus disease 19), it is a global imperative to exploit the immunogenicity of existing formulations for safe and efficient vaccines. As the most accessible adjuvant, aluminum hydroxide (alum) is still the sole employed adjuvant in most countries. However, alum tends to attach on the membrane rather than entering the dendritic cells (DCs), leading to the absence of intracellular transfer and process of the antigens, and thus limits T-cell-mediated immunity. To address this, alum is packed on the squalene/water interphase is packed, forming an alum-stabilized Pickering emulsion (PAPE). "Inheriting" from alum and squalene, PAPE demonstrates a good biosafety profile. Intriguingly, with the dense array of alum on the oil/water interphase, PAPE not only adsorbs large quantities of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) antigens, but also harbors a higher affinity for DC uptake, which provokes the uptake and cross-presentation of the delivered antigens. Compared with alum-treated groups, more than six times higher antigen-specific antibody titer and three-fold more IFN-γ-secreting T cells are induced, indicating the potent humoral and cellular immune activations. Collectively, the data suggest that PAPE may provide potential insights toward a safe and efficient adjuvant platform for the enhanced COVID-19 vaccinations.

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 Zn

Here we present a biomimetic strategy towards an influenza vaccine design based on hepatitis B virus core virus-like particles (HBc VLP). To this end, a temperature-shift based encapsulation process based on analysis of the unique thermal-associated structural flexibility of HBc VLP nanocages was proposed and proved efficient for encapsulation of antigen inside the VLP. By displaying a matrix protein 2 ectodomain (M2e) antigen on the exterior of HBc VLP through genetic fusion, and encapsulate a conserved internal nucleoprotein (NP) antigen peptide inside the VLP, a biomimetic dual-antigen influenza vaccine with interior NP/exterior M2e was constructed. For comparison, another non-biomimetic dual-antigen vaccine with interior M2e/exterior NP, and other four VLP-based single-antigen vaccines with NP or M2e either being encapsulated inside or genetically displayed outside the VLP were also constructed. Upon intraperitoneal immunization in mice, the dual-antigen VLP influenza vaccine elicited both NP and M2e-specific antibodies, which were stronger than those elicited by the single-antigen vaccines. Most importantly, after a lethal challenge of H1N1 virus, the biomimetic dual-antigen vaccine conferred the mice 100% protection without noticeable body weight loss in the absence of any adjuvant. While the protective efficacy conferred by the non-biomimetic one was only 62.5%, accompanying 12.5% body weight loss in the immunized mice. Besides the high level of antigen-specific antibodies, more efficient formation of total germinal center (GC) B cells and a higher level of effector memory CD8+ T cell population were observed in the biomimetic vaccine group, as compared with the non-biomimetic one. All these results demonstrate that VLP assembly and display of antigens in a biomimetic manner making this a promising strategy for the production of efficient universal vaccines to influenza and other rapidly emerging pathogens.

Recent years have seen enormous advances in nanovaccines for both prophylactic and therapeutic applications, but most of these technologies employ chemical or hybrid semi-biosynthetic production methods. Thus, production of nanovaccines has to date failed to exploit biology-only processes like complex sequential post-translational biochemical modifications and scalability, limiting the realization of the initial promise for offering major performance advantages and improved therapeutic outcomes over conventional vaccines. A Nano-B5 platform for in vivo production of fully protein-based, self-assembling, stable nanovaccines bearing diverse antigens including peptides and polysaccharides is presented here. Combined with the self-assembly capacities of pentamer domains from the bacterial AB5 toxin and unnatural trimer peptides, diverse nanovaccine structures can be produced in common Escherichia coli strains and in attenuated pathogenic strains. Notably, the chassis of these nanovaccines functions as an immunostimulant. After showing excellent lymph node targeting and immunoresponse elicitation and safety performance in both mouse and monkey models, the strong prophylactic effects of these nanovaccines against infection, as well as their efficient therapeutic effects against tumors are further demonstrated. Thus, the Nano-B5 platform can efficiently combine diverse modular components and antigen cargos to efficiently generate a potentially very large diversity of nanovaccine structures using many bacterial species.

At present, AIDS drugs are typical inhibitors that cannot achieve permanent effects. Therefore, the research of blocking HIV infection is essential. Especially for people in the high‐risk environment, long‐term prevention is important, because HIV can easily infect cells once the drug is interrupted. However, there is still no long‐acting AIDS prevention drug approved. Hence, the purpose of this study is to prepare a fusion inhibitor loaded poly(d, l‐lactic‐co‐glycolic acid) (PLGA) microspheres as a sustained‐release system for long‐term AIDS prevention. As the HIV membrane fusion inhibitor (LP‐98) used in this research is amphiphilic lipopeptide, W1/O/W2 double‐emulsion method was chosen, and premix membrane emulsification technique was used for controlling the uniformity of particle size. Several process parameters that can impact drug loading efficiency were summarized: the concentration of LP‐98 and PLGA, and the preparation condition of primary emulsion. Finally, the microspheres with high loading efficiency (>8%) and encapsulation efficiency (>90%) were successfully prepared under optimum conditions. Pharmacokinetic studies showed that LP‐98‐loaded microspheres were capable to continuously release for 24 days in rats. This research can promote the application of sustained‐release microspheres in AIDS prevention, and the embedding technique used in this study can also provide references for the loading of other amphipathic drugs.

Recruiting positively charged armed nanoparticles into the ocular fundus was achieved by utilizing the natural intraocular electrical field. Their subsequent penetration into the fundus sub-layers was also tuned by optimizing the surface charge density. In addition to the sustained release behavior, such a precise delivery fulfilled various intraocular delivery requirements for different chorioretinal diseases.

Polymeric microparticles are promising adjuvants and they exhibit various physicochemical characteristics that can regulate the immune response, including hydrodynamic size, morphology, and surface properties, among others. Surface hydrophobicity is also a key microparticle characteristic, but how it affects microparticle adjuvanticity remains unknown. To study the correlation between microparticle hydrophobicity and adjuvanticity in-depth, we prepared poly(d,l-lactic acid) (PLA)-, poly(d,l-lactic-co-glycolic acid) (PLGA)-, and poly(monomethoxypolyethylene glycol-co-d,l-lactide) (mPEG-PLA, PELA)-based microparticles by premix membrane emulsification, which were similar in size and morphology but differed in surface hydrophobicity. We then systematically evaluated their ability to induce immune responses in vitro and in vivo. Increased surface hydrophobicity on PLA-based microparticles greatly promoted antigen internalization into dendritic cells (DCs) as well as MHC II and CD86 expression on DCs in vitro. Similarly, in vivo studies showed that increased microparticle surface hydrophobicity significantly elevated cytokine secretion levels by splenocytes harvested from vaccinated mice. Adhesion force measurements confirmed that increased surface hydrophobicity enhanced the physical interaction between microparticles and cell membranes, a condition favorable for promoting microparticle internalization into cells. Taken together, these results indicated that microparticle hydrophobicity is an important factor that determines the magnitude of immune responses elicited by vaccination with different particulate systems.

Highly efficient immobilization of multi-enzyme systems involving cofactor regeneration represents one of the greatest challenges in bioprocessing. Particulate artificial cells with enzymes and cofactors encapsulated within microcapsules have long been the major type of multi-enzyme biocatalysts. In the present work, a novel hollow nanofiber-based artificial cell that performs multi-step reactions involving efficient coenzyme regeneration was fabricated in situ by a facile co-axial electrospinning process. To that end, a mixture of glycerol and water containing the dissolved multi-enzyme system for the bile acid assay, which included 3α-hydroxysteroid dehydrogenase (3α-HSD), diaphorase (DP) and NADH was fed as the core phase solution, and a N,N-dimethylacetylamide solution of 30 wt% polyurethane was fed as the shell phase solution during the co-axial electrospinning. The relationship between the structures of the hollow nanofibers and the activity and stability of the encapsulated enzymes was studied. At core and shell phase electrospinning solution flow rates of 0.07 and 0.5 mL h−1, activity recoveries as high as 76% and 82% were obtained for the encapsulated 3α-HSD and DP. The hollow nanofiber-based artificial cells were successfully used for the bile acid assay, yielding good linearity for bile acid concentrations ranging from 0–200 μM. Compared with the solution-based multi-enzyme system, the hollow nanofiber-based multi-enzyme system presented a lumped activity recovery of 75%. In addition, the hollow nanofiber provided the multi-enzyme system confined inside the nano-domain of the hollow fibers with a unique stabilizing mechanism, such that more than a 170-fold increase in half-life at 25 °C was obtained for the encapsulated 3α-HSD and DP. This study is expected to greatly promote and broaden the application of multi-enzyme systems in industry, biosensor, biomedical, and many other related research fields.

The role of a biophysical signal on cell response has excited tremendous interest recently. Herein, we exploited an “intake method” together with uniform autofluorescent cell-sized particles to investigate macrophage responses against different particle burdens. Our work not only revealed an insatiable macrophage uptake of cell-sized microparticles (MPs) but also widened the theoretical size and volume range for particle entry. MPs outperform NPs in the utmost volume of intracellular particles, indicating a converse size-associated event than originally anticipated. Such a superior volume burden (2.64-fold of the cell volume) for MPs is highly correlated to less membrane loss (19-fold below that for NPs) and more evident deformation of the cell membrane/nucleus. In addition, the cells with a high burden of MPs exhibit moderate migration activity, which is in line with mild cytokine release. These results highlight the indispensable role of physical burden on the regulation of macrophage functions, providing important views for desired biological outcomes and minimal side effects.

The highly immunosuppressive tumor microenvironment (TME) in solid tumors often dampens the efficacy of immunotherapy. In this study, bacterial outer membrane vesicles (OMVs) are demonstrated as powerful immunostimulants for TME reprogramming. To overcome the obstacles of antibody-dependent clearance and high toxicity induced by OMVs upon intravenous injection (a classic clinically relevant delivery mode), calcium phosphate (CaP) shells are employed to cover the surface of OMVs, which enables potent OMV-based TME reprograming without side effects. Meanwhile, the pH-sensitive CaP shells facilitate the neutralization of acidic TME, leading to highly beneficial M2-to-M1 polarization of macrophages for improved antitumor effect. Moreover, the outer shells can be integrated with functional components like folic acid or photosensitizer agents, which facilitates the use of the OMV-based platform in combination therapies for a synergic therapeutic effect.

The goal of this study was to explore the regulatory mechanisms of phenyllactic acid (PLA) overaccumulation in

Low encapsulation efficiency of the drug usually exist in hydrophilic drug which was embedded by hydrophobic materials directly in traditional method. In order to solve this problem, a novel preparation strategy which called "post-loading mode" was innovatively designed in this study: ropivacaine hydrochloride (ROP), a hydrophilic drug used in the field of anesthesia and analgesia, was encapsulated into the pre-prepared porous Poly (lactic-co-glycolic acid) (PLGA) microspheres; the porous PLGA microspheres (PLGA-Ms) with self-healing characteristic were used to obtain ROP-PLGA-Ms (with particle size around were 38 µm), in which drug loading (DL) was 8.72%. A rat sciatic nerve block model was established to evaluate the efficacy of ROP-PLGA-Ms. Exparel®, a bupivacaine liposome suspension approved by the FDA, was defined as reference agents in this study. The results showed that the injection of ROP, Exparel®, and ROP-PLGA-Ms were injected to the peripheral sciatic nerve could lead to motor dysfunction and sensory nerve block unanimously, and the onset time was less than 10 min for all cases. In addition, in comparison with ROP injection and Exparel®, the nerve block time of ROP-PLGA-Ms was significantly prolonged (P 0.05). Effective analgesia duration of ROP-PLGA-Ms was about 5 h, 2.5 and 1.7 folds longer than that of ROP injection and Exparel®, respectively. The rats in each group could recover eventually within 8 h after administration. HE showed that no inflammatory reaction was observed at the injection location. Analysis of blood biochemistry showed an insignificant difference between the microsphere experimental group and the negative group, which further indicated the safety of microsphere bioformulation.

Hepatitis B core virus-like particle (HBc-VLP) has become a carrier for expression and presentation of foreign epitopes as vaccine candidates. Efficient purification is necessary for preparation of HBc-VLP and its derivatives with various foreign epitopes. In previous reports, HBc-VLP was mainly purified with ion exchange chromatography. In this study, we developed a platform purification technique based on hydrophobic interaction chromatography (HIC). The underlying principle is the strong hydrophobicity on the surface of HBc-VLP, which was found by mathematical calculation and experimental measurement. Based on HIC, a complete downstream process, from feedstock supernatant to the final pure product, was developed involving a heat pretreatment, an HIC, an ultrafiltration concentration and a size exclusion chromatography. The total recovery of HBc-VLP was 41.92% with nearly 100% purity. HIC was also applicable to three HBc-based vaccine candidates displaying epitope from nuclear protein (NP) and matrix protein 2 (M2e) of the influenza A virus, as well as from ovalbumin (OVA). Optimal HIC condition for these three recombinant VLPs was rationally designed by analysis on their surface hydrophobicity, which was influenced upon insertion of the foreign epitope. By applying the same process developed for HBc-VLP, satisfactory results were achieved for product recovery, purity and host cell DNA removal, thus it is possible to become a platform technique for various HBc-VLP based vaccine candidates.

The direct connection between a photosensitizer and an electron mediator, achieved by a precise arrangement based on chlorosome, provides photosynthetic bacteria the maximum efficiency in solar energy conversion. Herein, this study reports the fabrication of a biomimicking chlorosome for biocatalyzed artificial photosynthesis through self-assembly of simple molecules to functional systems. TCPP/EYx/Rh8–x macromolecules, synthesized through a sequential amidation reaction of porphyrin (TCPP), eosin Y (EY), and [Cp*RhCl2]2, were found to self-assemble into chlorosome-mimicking supramolecular assemblies through noncovalent interactions. Intra- and intermolecular dual channels for enhancing electron transfer were constructed in TCPP/EYx/Rh8–x supramolecular assemblies. In addition, the energy band structure of TCPP/EY4/Rh4 supramolecular assemblies also made a perfect coordination with oxidation and reduction potentials of an electron donor and NAD+, which led to a fast and oriented electron transfer along th...

Phagocytosis is one of the earliest cellular functions, developing approximately 2 billion years ago. Although FcR-based phagocytic signaling is well-studied, how it originated from ancient phagocytosis is unknown. Lipid redistribution upregulates a phagocytic program recapitulating FcR-based phagocytosis with complete dependence on Src family kinases, Syk, and phosphoinositide 3-kinases (PI3K). Here we show that in phagocytes, an atypical ITAM sequence in the ancient membrane anchor protein Moesin transduces signal without receptor activation. Plasma membrane deformation created by solid structure binding generates phosphatidylinositol 4,5-bisphosphate (PIP2) accumulation at the contact site, which binds the Moesin FERM domain and relocalizes Syk to the membrane via the ITAM motif. Phylogenic analysis traces this signaling using PI3K and Syk to 0.8 billion years ago, earlier than immune receptor signaling. The proposed general model of solid structure phagocytosis implies a preexisting lipid redistribution-based activation platform collecting intracellular signaling components for the emergence of immune receptors., The origin of Fc receptor-based phagocytosis is unknown, although plasma lipid redistribution after solid structure binding induces similar phagocytic signaling. Here, Mu et al. show that PIP2 accumulation from plasma membrane deformation recruits Moesin to trigger receptor-independent phagocytosis.

There is a complex interplay existing between the tumor microenvironment and cellular function, while the influence of tumor biophysical signals on the macrophage function remain unclear. Herein, we constructed a three dimensional hydrogel model to study the symphysial effect of physical signal (stiffness) and biological signal (tumor cells) on macrophage polarization in different stage of tumor progression. In the absence of biological signal, the macrophage response was slightly changed with different stiffness (100 Pa, 300 Pa, and 900 Pa). Once co-incubated with the tumor cell, macrophage induced more significant cancer facilitated cytokines (e.g. IL-10, MMP-9) and chemokines (MCP-1). In contrast to the education role from the co-cultured cancer cells, a stiffness around the normal tissue (100 Pa, CO-L group) provided a physical cue for cancer cell regression. This cue triggered the active role of M1 type macrophages (e.g. IL-12), indicating the interacting role of stiffness in the presence of cancer cell. The stiffness around the premalignant tissue (M, 300 Pa) induced much higher level of cytokines (e.g. IL-6, IL-10, IL-12, CD86, and CD206), while the immune activation cytokines (M1 type) was largely attenuated at high stiffness (900 Pa). This study revealed the pleiotropic tuning of macrophage function under different biophysical signals, which provided important knowledge for cancer progress and may benefit the design of cancer-related therapies.

Protein A chromatography has been a popular method for purification of therapeutic monoclonal antibodies (mAb). Protein A chromatographic media using alkali-resistant rProtein A ligands from site-directed coupling method have been pursued for both high dynamic capacities and excellent stabilities. However, the mechanism of rProtein A leaking under cleaning-in-place (CIP) conditions is not very clear and difficulties have been commonly encountered when improving the media's chromatographic performance. We investigated the chromatographic performance of site-directed coupled rProtein A chromatographic media during CIP procedure. Trace amount of ligands leaked during the chromatographic media's incubation in 0.5 M NaOH was detected, explaining for the decline of chromatographic media's CIP performance. Decrease of rProtein A's concentration in 0.5 M NaOH was consistent with chromatographic media's binding capacity. A novel rProtein A chromatographic media were prepared by site-directed coupling a newly-constructed alkali-resistant rProtein A to highly cross-linked agarose-based matrix. The media had a dynamic binding capacity of 63.2 mg hIgG/mL higher than 48.1 mg hIgG/mL of the commercial one, and the CIP performance was improved greatly with the remained dynamic binding capacity increased from 86% to 95% of the initial value after 40 CIP cycles.

Poly(D,L-lactic-co-glycolic acid) (PLGA)/poly (lactic acid) (PLA) microspheres/nanoparticles are one of the most successful drug delivery systems (DDS) in lab and clinic. Because of good biocompatibility and biodegradability, they can be used in various areas, such as long-term release system, vaccine adjuvant, tissue engineering, etc. There have been 15 products available on the US market, but the system still has many problems during development and manufacturing, such as wide size distribution, drug stability issues, and so on. Recently, many new and modified methods have been developed to overcome the above problems. Some of the methods are easy to scale up, and have been available on the market to achieve pilot scale or even industrial production scale. Furthermore, the relevant FDA guidance on the DDS is still incomplete, especially for abbreviated new drug application. In this review, we present some recent achievement of the PLGA/PLA microspheres/nanoparticles, and discuss some promising manufacturing methods. Finally, we focus on the current FDA guidance on the DDS. The review provides an overview on the development of the system in pharmaceutical industry.

High resolution has been constantly pursued in both preparative and analytical chromatography. Chromatographic media are a key factor during the entire separation process. Tailor-made chromatographic media have gained more attention because of their adjustable structure appropriate for application. Uniform polysaccharide composite microspheres were prepared with a mixture of agarose and dextran solution by membrane emulsification technique for the first time. Their pore structure was deliberately regulated by adjusting both the polysaccharide composition and the molecular weight of dextran. Compared with pure agarose microspheres, polysaccharide composite microspheres had a higher separation resolution and their separation range was controllable. By increasing agarose concentration and decreasing dextran concentration at the same time during the preparation of composite microspheres, the mean pore size increased first and then decreased later, and also the pore size distribution became narrower. By increasing the molecular weight of dextran, the pores became smaller with a narrower pore size distribution. Microspheres with a composition of 10% agarose/2% dextran T40 or 8% agarose/4% dextran T150 showed a higher separation resolution for proteins within range of low molecular weight. Furthermore, the mechanical strength of this composite microsphere was improved by adjusting its composition. Atomic force microscope (AFM) results showed that pores were distributed evenly on both the surface and the inner part of microspheres, beneficial for the passage of biomolecules. These novel uniform polysaccharide composite microspheres have great potential for high-resolution bioseparation. Graphical abstract ᅟ.

The central solenoid model coil (CSMC) project of China fusion engineering test reactor (CFETR) began in 2014 and the purpose is to develop and verify the manufacture technology of the large-scale superconducting magnet. The Nb 3Sn cable-in-conduit-conductor (CICC) is chosen for CFETR CSMC as the best conductor type because of 12 T field and 1.5 m inner diameter design requirement. The CFETR CSMC consists of two coaxial solenoid coils named Nb3 Sn coil in the high field region and NbTi coil in the low field region to optimize the manufacture cost effectively. The maximum field of the Nb3Sn and NbTi coil can reach to 12 T and 6.1 T, respectively, when the operating current is 47.65 kA. In this paper, first, the basic design aim and operation requirements of CFETR CSMC are summarized. Second, the characteristics of Nb3Sn superconducting strands and CICCs are described in detailed. Subsequently, the basic structure design of CFETR CSMC is presented in the third part. The current sharing temperature and coupling loss measurement of Nb3Sn CICC sample, as the most important stage assessment, are performed in SUTLAN test facility and some meaningful conclusions are.

A novel photocatalyst/biocatalyst integrated artificial photosynthesis system (APS) based on polyurethane hollow nanofibers doped with graphene oxide (GO) and poly(allylamine hydrochloride) (PAH) was developed and employed for selective methanol conversion from CO2. The biocatalysts, including formate, formaldehyde, and alcohol dehydrogenases, as well as NAD+, were in situ coencapsulated inside the lumen of the GO-PAH-doped PU nanofibers (G-Fiber) by simply predissolving them in the core-phase solution for coaxial electrospinning, while the precise assembling of the photocatalyst parts involving visible light active photosensitizer (PS) and electron mediator (M) on the surface of the G-Fiber was realized by their π–π interactions with the GO doped in the shell of fibers. By using this highly integrated APS, about 10-times higher methanol yield was accomplished as compared with the solution-based system. The significantly enhanced reaction efficiency of the G-Fiber-based APS is considered predominately due...