Stimuli-Responsive Crystalline Smart Materials: From Rational Design and Fabrication to Applications.

Stimuli-responsive smart materials that can undergo reversible chemical/physical changes under external stimuli such as mechanical stress, heat, light, gas, electricity, and pH, are currently attracting increasing attention in the fields of sensors, actuators, optoelectronic devices, information storage, medical applications, and so forth. The current smart materials mostly concentrate on polymers, carbon materials, crystalline liquids, and hydrogels, which have no or low structural order (i.e., the responsive groups/moieties are disorderly in the structures), inevitably introducing deficiencies such as a relatively low response speeds, energy transformation inefficiencies, and unclear structure-property relationships. Consequently, crystalline materials with well-defined and regular molecular arrays can offer a new opportunity to create novel smart materials with improved stimuli-responsive performance. Crystalline materials include framework materials (e.g., metal-organic frameworks, MOFs; covalent organic frameworks, COFs) and molecular crystals (e.g., organic molecules and molecular cages), which have obvious advantages as smart materials compared to amorphous materials. For example, responsive groups/moieties can be uniformly installed in the skeleton of the crystal materials to form ordered molecular arrays, making energy transfer between external-stimulus signals and responsive sites much faster and more efficiently. Besides that, the well-defined structures facilitate in situ characterization of their structural transformation at the molecular level by means of various techniques and high-tech equipment such as in situ spectra and single-crystal/powder X-ray diffraction, thus benefiting the investigation and understanding of the mechanism behind the stimuli-responsive behaviors and structure-property relationships. Nevertheless, some unsolved challenges remain for crystalline smart materials (CSMs), hampering the fabrication of smart material systems for practical applications. For instance, as the materials' crystallinity increases, their processability and mechanical properties usually decrease, unavoidably hindering their practical application. Moreover, crystalline smart materials mostly exist as micro/nanosized powders, which are difficult to make stimuli-responsive on the macroscale. Thus, developing strategies that can balance the materials' crystallinity and processability and establishing macroscale smart material systems are of great significance for practical applications.In this Account, we mainly summarize the recent research progress achieved by our groups, including (i) the rational design and fabrication of new stimuli-responsive crystalline smart materials, including molecular crystals and framework materials, and an in-depth investigation of their response mechanism and structure-property relationship and (ii) creating chemical/physical modification strategies to improve the processability and mechanical properties for crystalline materials and establishing macroscale smart systems for practical applications. Overall, this Account summarizes the state-of-the-art progress of stimuli-responsive crystalline smart materials and points out the existing challenges and future development directions in the field.

Solvent-Free Synthesis of C=N Linked Two-Dimensional Covalent Organic Frameworks.

Covalent organic frameworks (COFs) are an emerging class of porous crystalline polymers with well-defined structures and tunable functionalities, which have fascinating applications in a wide range of fields. However, the synthetic procedures of COFs are mainly confined to the solvothermal synthesis method which usually requires harsh experimental conditions. In this work, an effective solvent-free synthesis method to construct a series of two-dimensional (2D) COFs including imine-linked, azine-linked, ß-ketoenamine-linked, which avoids the complicated solvent screening process and most of the disadvantages of solvothermal methods is developed. The crystallinity and porosity of these COFs are comparable to those prepared by traditional solvothermal routes. What's more, the advantages of the solvent-free method enable the production of gram-scale of those 2D COFs through a one-pot reaction, demonstrating high industrial application potentials.

Self-Healing Hyper-Cross-Linked Metal-Organic Polyhedra (HCMOPs) Membranes with Antimicrobial Activity and Highly Selective Separation Properties.

Fabrication of hybrid membranes composed of porous materials embedded in polymer matrices is a subject of topical interest. Herein, we introduce a new class of hybrid membranes: hyper-cross-linked metal-organic polyhedra (HCMOPs). These membranes are based upon soluble MOPs that can serve as high-connectivity nodes in hyper-cross-linked polymer networks. HCMOPs spontaneously form macro-scale, defect-free, freestanding membranes, and, thanks to the covalent cross-linking of MOPs, the resulting membranes possess multiple functionalities: strong water permeability; self-healing ability; antimicrobial activity; and better separation and mechanical performance than pristine polyimine membranes. This study introduces a new concept for the design and fabrication of multifunctional membranes and also broadens the applications of MOPs.

Effects of Acetylshikonin on the Infection and Replication of Coxsackievirus A16 in Vitro and in Vivo.

Coxsackievirus A16 (CVA16) is one of the most prevalent enteroviral pathogens associated with hand, foot, and mouth disease. In the present study, we have investigated (1) whether the bioactive compound acetylshikonin (AS) inhibits CVA16 infection in vitro and in vivo and (2) the potential antiviral mechanism(s). The results suggest that AS is nontoxic at concentrations of up to 5 µmol/L and could directly inactivate virus particles at relatively low concentrations (0.08 µmol/L), thereby rendering CVA16 incapable of cellular entry. Correspondingly, the expression of viral RNA in vitro was also reduced 100-fold ( P < 0.05) when compared to infected, untreated controls. Results from a CVA16-infected neonatal mouse model indicate that, in comparison to the virus-infected, untreated group, body weights of the mice in the virus-infected, compound-treated group increased more steadily with less severe clinical symptoms. In addition, viral loads in internal organs significantly decreased in treated animals, concomitantly with both reduced pathology and diminished expression of the proinflammatory cytokines IFN-γ and IL-6. In conclusion, AS exerted an inhibitory effect on CVA16 infection in vitro and in vivo. Our study provides a basis for further investigations of AS-type compounds to develop therapeutics to mitigate CVA-associated disease in children.

A Neonatal Murine Model of Coxsackievirus A6 Infection for Evaluation of Antiviral and Vaccine Efficacy.

Hand, foot, and mouth disease (HFMD) is a global health concern. Family Picornaviridae members, particularly enterovirus A71 (EVA71) and coxsackievirus A16 (CVA16), are the primary etiological agents of HFMD; however, a third enterovirus A species, CVA6, has been recently associated with epidemic outbreaks. Study of the pathogenesis of CVA6 infection and development of antivirals and vaccines are hindered by a lack of appropriate animal models. We have developed and characterized a murine model of CVA6 infection that was employed to evaluate the antiviral activities of different drugs and the protective efficacies of CVA6-inactivated vaccines. Neonatal mice were susceptible to CVA6 infection via intramuscular inoculation, and the susceptibility of mice to CVA6 infection was age and dose dependent. Five-day-old mice infected with 105.5 50% tissue culture infective doses of the CVA6 WF057R strain consistently exhibited clinical signs, including reduced mobility, lower weight gain, and quadriplegia with significant pathology in the brain, hind limb skeletal muscles, and lungs of the infected mice in the moribund state. Immunohistochemical analysis and quantitative reverse transcription-PCR (qRT-PCR) analyses showed high viral loads (11 log10/mg) in skeletal muscle, and elevated levels of interleukin-6 (IL-6; >2,000 pg/ml) were associated with severe viral pneumonia and encephalitis. Ribavirin and gamma interferon administered prophylactically diminished CVA6-associated pathology in vivo, and treatment with IL-6 accelerated the death of neonatal mice. Both specific anti-CVA6 serum and maternal antibody play important roles in controlling CVA6 infection and viral replication. Collectively, these findings indicate that this neonatal murine model will be invaluable in future studies to develop CVA6-specific antivirals and vaccines.IMPORTANCE Although coxsackievirus A6 (CVA6) infections are commonly mild and self-limiting, a small proportion of children may have serious complications, such as encephalitis, acute flaccid paralysis, and neurorespiratory syndrome, leading to fatalities. We have established a mouse model of CVA6 infection by inoculation of neonatal mice with a CVA6 clinical isolate that produced consistent pathological outcomes. Here, using this model of CVA6 infection, we found that high levels of IL-6 were associated with severe viral pneumonia and encephalitis, as in an evaluation of antiviral efficacy in vivo, IL-6 had no protective effect and instead accelerated death in neonatal mice. We demonstrated that, as antiviral drugs, both gamma interferon and ribavirin played important protective roles in the early stages of infection, with increased survival in treated neonatal mice challenged with CVA6. Moreover, active and passive immunization with the inactivated vaccines and anti-CVA6 serum also protected mice against homologous challenge infections.

Protective Efficacies of Formaldehyde-Inactivated Whole-Virus Vaccine and Antivirals in a Murine Model of Coxsackievirus A10 Infection.

Coxsackievirus A10 (CVA10) is one of the major pathogens associated with hand, foot, and mouth disease (HFMD). CVA10 infection can cause herpangina and viral pneumonia, which can be complicated by severe neurological sequelae. The morbidity and mortality of CVA10-associated HFMD have been increasing in recent years, particularly in the pan-Pacific region. There are limited studies, however, on the pathogenesis and immunology of CVA10-associated HFMD infections, and few antiviral drugs or vaccines have been reported. In the present study, a cell-adapted CVA10 strain was employed to inoculate intramuscularly 5-day-old ICR mice, which developed significant clinical signs, including reduced mobility, lower weight gain, and quadriplegia, with significant pathology in the brain, hind limb skeletal muscles, and lungs of infected mice in the moribund state. The severity of illness was associated with abnormally high expression of the proinflammatory cytokine interleukin 6 (IL-6). Antiviral assays demonstrated that ribavirin and gamma interferon administration could significantly inhibit CVA10 replication both in vitro and in vivo In addition, formaldehyde-inactivated CVA10 whole-virus vaccines induced immune responses in adult mice, and maternal neutralizing antibodies could be transmitted to neonatal mice, providing protection against CVA10 clinical strains. Furthermore, high-titer antisera were effective against CVA10 and could relieve early clinical symptoms and improve the survival rates of CVA10-challenged neonatal mice. In summary, we present a novel murine model to study CVA10 pathology that will be extremely useful in developing effective antivirals and vaccines to diminish the burden of HFMD-associated disease.IMPORTANCE Hand, foot, and mouth disease cases in infancy, arising from coxsackievirus A10 (CVA10) infections, are typically benign, resolving without any significant adverse events. Severe disease and fatalities, however, can occur in some children, necessitating the development of vaccines and antiviral therapies. The present study has established a newborn-mouse model of CVA10 that, importantly, recapitulates many aspects of human disease with respect to the neuropathology and skeletal muscle pathology. We found that high levels of the proinflammatory cytokine interleukin 6 correlated with disease severity and that ribavirin and gamma interferon could decrease viral titers in vitro and in vivo Whole-virus vaccines produced immune responses in adult mice, and immunized mothers conferred protection on neonates against challenge from CVA10 clinical strains. Passive immunization with high-titer antisera could also improve survival rates in newborn animals.

Fabrication of Light-Triggered Soft Artificial Muscles via a Mixed-Matrix Membrane Strategy.

Artificial muscles triggered by light are of great importance, especially for the development of non-contact and remotely controlled materials. Common materials for synthesis of photoinduced artificial muscles typically rely on polymer-based photomechanical materials. Herein, we are able to prepare artificial muscles using a mixed-matrix membrane strategy to incorporate photomechanical molecular crystals with connective polymers (e.g. PVDF). The formed hybrid materials inherit not only the advantages of the photomechanical crystals, including faster light response, higher Young's modulus and ordered structure, but also the elastomer properties from polymers. This new type of artificial muscles demonstrates various muscle movements, including lifting objects, grasping objects, crawling and swimming, triggered by light irradiation. These results open a new direction to prepare light-driven artificial muscles based on molecular crystals.

A neonatal murine model of coxsackievirus A4 infection for evaluation of vaccines and antiviral drugs.

Coxsackievirus A4 (CVA4) infection can cause hand, foot and mouth disease (HFMD), an epidemic illness affecting neonatal and paediatric cohorts, which can develop to severe neurological disease with high mortality. In this study, we established the first ICR mouse model of CVA4 infection for the evaluation of inactivated vaccines and antiviral drug screening. The CVA4 YT226R strain was selected to infect the neonatal mice and three infectious factors were optimized to establish the infection model. The 3-day-old neonatal mice exhibited clinical symptoms such as hind limb paralysis and death. The severe inflammatory reactions were closely related to the abnormal expression of the acute phase response proinflammatory cytokine IL-6 and an imbalance in the IFN-γ/IL-4 ratio. Importantly, the inactivated CVA4 whole-virus vaccine induced humoral immune responses in adult females and the maternal antibodies afforded mice complete protection against lethal dose challenges of homologous or heterologous CVA4 strains. Both IFN-α2a and antiserum inhibited the replication of CVA4 and increased the survival rates of neonatal mice during the early stages of infection. This neonatal murine model of CVA4 infection will be useful for the development of prophylactic and therapeutic vaccines and for screening of antiviral drugs targeting CVA4 to decrease morbidity and mortality.

Genome Analysis of Coxsackievirus A4 Isolates From Hand, Foot, and Mouth Disease Cases in Shandong, China.

Coxsackievirus A4 (CVA4) is one of the most prevalent pathogens associated with hand, foot and mouth disease (HFMD), an acute febrile illness in children, and is also associated with acute localized exanthema, myocarditis, hepatitis and pancreatitis. Despite this, limited CVA4 genome sequences are currently available. Herein, complete genome sequences from CVA4 strains (n = 21), isolated from patients with HFMD in Shandong province, China between 2014 and 2016, were determined and phylogenetically characterized. Phylogenetic analysis of the VP1 gene from a larger CVA4 collection (n = 175) showed that CVA4 has evolved into four separable genotypes: A, B, C, and D; and genotype D could be further classified in to two sub-genotypes: D1 and D2. Each of the 21 newly described genomes derived from isolates that segregated with sub-genotype D2. The CVA4 genomes displayed significant intra-genotypic genetic diversity with frequent synonymous substitutions occurring at the third codon positions, particularly within the P2 region. However, VP1 was relatively stable and therefore represents a potential target for molecular diagnostics assays and also for the rational design of vaccine epitopes. The substitution rate of VP1 was estimated to be 5.12 × 10-3 substitutions/site/year, indicative of ongoing CVA4 evolution. Mutations at amino acid residue 169 in VP1 gene may be responsible for differing virulence of CVA4 strains. Bayesian skyline plot analysis showed that the population size of CVA4 has experienced several dynamic fluctuations since 1948. In summary, we describe the phylogenetic and molecular characterization of 21 complete genomes from CVA4 isolates which greatly enriches the known genomic diversity of CVA4 and underscores the need for further surveillance of CVA4 in China.

Characterization of an inactivated whole-virus bivalent vaccine that induces balanced protective immunity against coxsackievirus A6 and A10 in mice.

Coxsackievirus A6 (CVA6) and CVA10 are two of the major pathogens associated with hand, foot and mouth disease (HFMD) in children. The majority of CVA6 and CVA10 infections result in mild, self-limiting episodes (fever and herpangina) in pediatric populations; however, in some cases, can proceed to severe neurological disease and death. Efforts to mitigate viral transmission to decrease the morbidity and mortality associated with infection would be greatly strengthened by the availability of an efficacious CVA6 and CVA10 bivalent vaccine. Here we report the immunogenicity and protective efficacy of a bivalent combination vaccine comprised of formaldehyde-inactivated, whole-virus CVA6 and CVA10. We demonstrate that subcutaneous delivery of the bivalent vaccine can induce antigen-specific systemic immune responses, particularly the induction of polyfunctional T cells, which elicit active immunization to achieve a protection rate of >80% in the infected neonatal mice. Furthermore, passive transfer of the antisera from vaccinated mice potently protected recipient mice against CVA6 and CVA10 challenge. Importantly, the bivalent vaccine could induce high levels of IgG and neutralizing antibodies in adult female mice and the maternal antibody transmitted to the recipient mice played an important role in controlling homotypic and heterotypic CVA6 and CVA10 infections and viral replication in vivo. Collectively, these findings indicate that there is no immunological interference between the two antigens with respect to their ability to induce virus-specific immune responses, and thus provides proof-of-concept for further development of multivalent vaccines for broad protection against HFMD.

Rapid detection of hand, foot and mouth disease enterovirus genotypes by multiplex PCR.

Hand, foot and mouth disease (HFMD) is a pediatric disease associated with infection by enterovirus (EV) genotypes. The major HFMD EV pathogens are enterovirus A71 (EVA71) and coxsackievirus A16 (CVA16); however, recently, coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) have also emerged. EV genotypes cannot be distinguished on clinical grounds and a new methodology for the rapid detection of the four major HFMD EV genotypes is urgently required. In the present study, a multiplex real-time PCR assay was established for the simultaneous detection of CVA6, CVA10, CVA16 and EVA71. The specificity and sensitivity of the assay was determined on a validation panel of clinical samples, comprising cerebrospinal fluid (n = 51), blood (n = 39), feces (n = 58) and throat swabs (n = 29). The results showed that the multiplex real-time PCR exhibited high specificity, no cross-reactivity with other EV genotypes, lower limits of detection for CVA6, CVA10, CVA16 and EVA71 were 4 × 103, 4 × 102, 5 × 102, and 3 × 103 copies/µL, respectively and had comparable sensitivity to singleplex assays testing clinical samples. The multiplex real-time PCR methodology established in this study can be employed for the rapid detection of the four most prevalent HFMD-associated EVs, for epidemiologic surveillance of circulating EV genotypes and for assessing treatment responses and vaccine studies.

[Establishment of a Model of Infection by Enterovirus 71 in ICR Mice].

We aimed to study infections in neonatal ICR mice of different ages infected with Enterovirus 71(EV-A71)through three routes of infection, and to explore the dynamic distribution and infection mechanism of EV-A71 in vivo.Three-,5-and 9-day-old neonatal ICR mice were infected with an EV-A71 strain isolated from a child with severe hand, foot and mouth disease through intramuscular(IM), intraperitoneal (IP)and intracerebral (IC)injection, respectively. Consequently, blood, brain, hind-limb muscle, heart, and intestines of mice were collected at regular intervals. Changes in viral load in organs were measured using real-time polymerase chain reaction. Hematoxylin and eosin staining and immunohistochemical (IHC)analyses were undertaken to detect pathogenic and pathologic changes in infected mice.Five-day-old neonatal mice infected with EV-A71 through IM,IP or IC routes had obvious neurologic symptoms and a high mortality rate. Symptoms were alleviated slightly with increasing age of mice upon injection. However, the pathogenicity associated with IM and IP injections was more severe than that of IC injection. Also, the mortality rates of IM and IP injections were significantly higher than that of IC injection. Compared with the control group, the mean body weight(in g)of 3-day-old neonatal mice at 6days post-infection(dpi)injected by IM,IP and IC routes decreased by 1.54(31.43%),1.31(15.06%)and 2.52(44.28%),respectively. Similarly, the mean body weight(in g)of 5-day-old neonatal mice at 6dpi injected by IM and IP decreased by 0.605(8.95%),0.886(15.51%),whereas that of mice injected by IC increased by 0.904(14.70%).The body weight of all infection groups was significantly lower than that of the control group(P<0.05).All 3-day-old neonatal mice infected with EV-A71 through IM,IP and IC routes died at 9dpi.Survival rates of 5-day-old neonatal mice infected through IM,IP and IC routes at 9dpi and14 dpi were 42.8%,25%,and 87.5%,and 0%,0%,and 25%,respectively.Those of 9-day-old neonatal mice at 14 dpi were 70%,69.23% and 100%,respectively.Pathologic and IHC examination showed that EV-A71 had a strong preference for infecting nervous systems and skeletal muscle, and could also lead to: viremia; necrosis of brain neurons and skeletal muscle; myocardial interstitial edema; inflammatory response of multiple organs. These data suggest that 5-day-old ICR neonatal mice injected through IM or IP routes can establish an ideal model of infection by EV-A71 in mice.