In situ bio-mineralized Mn nanoadjuvant enhances anti-influenza immunity of recombinant virus-like particle vaccines.

Virus like particles (VLPs) have been well recognized as one of the most important vaccine platforms due to their structural similarity to natural viruses to induce effective humoral and cellular immune responses. Nevertheless, lack of viral nucleic acids in VLPs usually leads the vaccine candidates less efficient in provoking innate immune against viral infection. Here, we constructed a biomimetic dual antigen hybrid influenza nanovaccines THM-HA@Mn with robust immunogenicity via in situ synthesizing a stimulator of interferon genes (STING) agonist Mn3O4 inside the cavity of a recombinant Hepatitis B core antigen VLP (HBc VLP) having fused SpyTag and influenza M2e antigen peptides (Tag-HBc-M2e, THM for short), followed by conjugating a recombinant hemagglutinin (rHA) antigen on the surface of the nanoparticles through SpyTag/SpyCatcher ligating. Such inside Mn3O4 immunostimulator-outside rHA antigen design, together with the chimeric M2e antigen on the HBc skeleton, enabled the synthesized hybrid nanovaccines THM-HA@Mn to well imitate the spatial distribution of M2e/HA antigens and immunostimulant in natural influenza virus. In vitro cellular experiments indicated that compared with the THM-HA antigen without Mn3O4 and a mixture vaccine consisting of THM-HA + MnOx, the THM-HA@Mn hybrid nanovaccines showed the highest efficacies in dendritic cells uptake and in promoting BMDC maturation, as well as inducing expression of TNF-α and type I interferon IFN-ß. The THM-HA@Mn also displayed the most sustained antigen release at the injection site, the highest efficacies in promoting the DC maturation in lymph nodes and germinal center B cells activation in the spleen of the immunized mice. The co-delivery of immunostimulant and antigens enabled the THM-HA@Mn nanovaccines to induce the highest systemic antigen-specific antibody responses and cellular immunogenicity in mice. Together with the excellent colloid dispersion stability, low cytotoxicity, as well as good biosafety, the synthetic hybrid nanovaccines presented in this study offers a promising strategy to design VLP-based vaccine with robust natural and adaptive immunogenicity against emerging viral pathogens.

On-column capping of poly dT media-tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase.

The messenger RNA (mRNA) 5'-cap structure is indispensable for mRNA translation initiation and stability. Despite its importance, large-scale production of capped mRNA through in vitro transcription (IVT) synthesis using vaccinia capping enzyme (VCE) is challenging, due to the requirement of tedious and multiple pre-and-post separation steps causing mRNA loss and degradation. Here in the present study, we found that the VCE together with 2'-O-methyltransferase can efficiently catalyze the capping of poly dT media-tethered mRNA to produce mRNA with cap-1 structure under an optimized condition. We have therefore designed an integrated purification and solid-based capping protocol, which involved capturing the mRNA from the IVT system by using poly dT media through its affinity binding for 3'-end poly-A in mRNA, in situ capping of mRNA 5'-end by supplying the enzymes, and subsequent eluting of the capped mRNA from the poly dT media. Using mRNA encoding the enhanced green fluorescent protein as a model system, we have demonstrated that the new strategy greatly simplified the mRNA manufacturing process and improved its overall recovery without sacrificing the capping efficiency, as compared with the conventional process, which involved at least mRNA preseparation from IVT, solution-based capping, and post-separation and recovering steps. Specifically, the new process accomplished a 1.76-fold (84.21% over 47.79%) increase in mRNA overall recovery, a twofold decrease in operation time (70 vs. 140 min), and similar high capping efficiency (both close to 100%). Furthermore, the solid-based capping process greatly improved mRNA stability, such that the integrity of the mRNA could be well kept during the capping process even in the presence of exogenously added RNase; in contrast, mRNA in the solution-based capping process degraded almost completely. Meanwhile, we showed that such a strategy can be operated both in a batch mode and in an on-column continuous mode. The results presented in this work demonstrated that the new on-column capping process developed here can accomplish high capping efficiency, enhanced mRNA recovery, and improved stability against RNase; therefore, can act as a simple, efficient, and cost-effective platform technology suitable for large-scale production of capped mRNA.

Study of self-assembling properties of HBc-VLP derivatives aided by molecular dynamic simulations from a thermodynamic perspective.

Self-assembling protein nanoparticles showed promise for vaccine design due to efficient antigen presentations and safety. However, the unpredictable formations of epitopes-fused protein assemblies remain challenging in the upstream design. This study suggests employing molecular dynamic (MD) simulations to investigate the assembly properties of Hepatitis B core protein (HBc) from thermodynamic perspectives. Eight HBc derivatives were expressed in E. coli, with their self-assembly properties characterised by high-performance liquid chromatography and transmission electron microscopy. MD simulations on the dimers, based on AlphaFold-predicted 3D structures, analysed the derivative at the atomic level. Results revealed that HBc derivatives can form dissociative polymers or large multi-subunit structures due to assembly failures. The instability of the dimer in aqueous solvents or inappropriate intradimer distances could cause major assembly failures. Polar solvation energies played a vital role too in forming assemble-incompetent dimers. Importantly, our study demonstrated that MD simulations on dimers can provide preliminary predictions on the assembly properties of HBc derivatives, thus aiding vaccine design by lowering the risk of self-assembling failures in engineered proteins.Communicated by Ramaswamy H. Sarma.

[Quantification of complete viral particles in inactivated avian influenza virus antigen by high performance size exclusion chromatography coupled with multi-angle laser light scattering].

We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R2=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.

Messenger RNA chromatographic purification: advances and challenges.

Messenger RNA (mRNA) technologies have shown great potential in prophylactic vaccines and therapeutic medicines due to their adaptability, rapidity, efficacy, and safety. The purity of mRNA determines the efficacy and safety of mRNA drugs. Though chromatographic technologies are currently employed in mRNA purification, they are facing challenges, mainly arising from the large size, relatively simple chemical composition, instability, and high resemblance of by-products to the target mRNA. In this review, we will first make a comprehensive analysis of physiochemical properties differences between mRNA and proteins, then the major challenges facing in mRNA purification and general considerations are highlighted. A detailed summary of the state-of-arts in mRNA chromatographic purification will be provided, which are mainly classified into physicochemical property-based (size, charge, and hydrophobicity) and chemical structure-based (phosphate backbone, bases, cap structure, and poly A tail) technologies. Efforts in eliminating dsRNA byproducts via post in vitro transcript (IVT) purification and by manipulating the IVT process to reduce the generation of dsRNA are highlighted. Finally, a brief summary of the current status of chromatographic purification of the emerging circular mRNA (circRNA) is provided. We hope this review will provide some useful guidance for the Quality by Design (QbD) of mRNA downstream process development.

The position of Spy Tag/Catcher system in hepatitis B core protein particles affects the immunogenicity and stability of the synthetic vaccine.

Presenting exogenous antigens on virus-like particles (VLPs) through "plug-and-display" decoration strategies based on SpyTag/SpyCatcher isopeptide bonding have emerged as attractive technology for vaccine synthesis. However, whether the position of ligation site in VLPs will impose effects on immunogenicity and physiochemical properties of the synthetic vaccine remains rarely investigated. Here in the present work, the well-established hepatitis B core (HBc) protein was used as chassis to construct dual-antigen influenza nanovaccines, with the conserved epitope peptides derived from extracellular domain of matrix protein M2 (M2e) and hemagglutinin (HA) as target antigens. The M2e antigen was genetically fused to the HBc in the MIR region, together with the SpyTag peptide, which was fused either in the MIR region or at the N-terminal of the protein, so that a recombinant HA antigen (rHA) linked to SpyCatcher can be displayed on it, at two different localizations. Both synthetic nanovaccines showed ability in inducing strong M2e and rHA-specific antibodies and cellular immunogenicity; nevertheless, the one in which rHA was conjugated by N-terminal Tag ligation, was superior to another one synthesized by linking the rHA to MIR region SpyTagged-HBc in all aspects, including higher antigen-specific immunogenicity responses, lower anti-HBc carrier antibody, as well as better dispersion stability. Surface charge and hydrophobicity properties of the two synthetic nanovaccines were analyzed, results revealed that linking the rHA to MIR region SpyTagged-HBc lead to more significant and disadvantageous alteration in physiochemical properties of the HBc chassis. This study will expand our knowledge on "plug-and-display" decoration strategies and provide helpful guidance for the rational design of HBc-VLPs based modular vaccines by using SpyTag/Catcher synthesis.

Thermal-triggered loading and GSH-responsive releasing property of HBc particles for drug delivery.

Hepatitis B core protein virus-like particles (HBc VLPs) have attracted wide attentions using as drug delivery vehicles, due to its excellent stability and easy in large scale production. Here in the present work, we report unique thermal-triggered loading and glutathione-responsive releasing property of the HBc particles for anticancer drug delivery. Through reversible temperature-dependent hole gating of the HBc particle capsid, about 4248 doxorubicin (DOX) were successfully encapsulated inside nanocage of a single nanoparticle at high HBc recovery of 83.2%, by simply incubating the DOX with HBc at 70 °C for 90 min. The new strategy was significantly superior to the disassembly-reassembly methods, which can only yield 3556 DOX loading at 52.3% HBc recovery. The thermal-sensitive drug entry channel in HBc was analyzed by molecular dynamic simulations, and the G113, G117 and R127 were identified as the key amino acid residues that are not conducive to the entrance of DOX but sensitive to temperature. Especially, the ΔGbind of R127 become even higher at high temperature, mutation of the R127 would be the first choice to make the drug entry thermodynamically easier. Due to plenty of disulfide bonds linking the HBc subunits, the HBc particles loaded with DOX exhibited intrinsic glutathione (GSH) responsivity for efficient controlled release in tumor sites. To further increase the tumor-targeting effect of the drug, Cyclo(Arg-Gly-Asp-d-Tyr-Lys) peptide was conjugated to the surface of HBc through a PEG linker. The prepared HBc-based anticancer drug showed significantly improved stability, tumor specificity, and in vivo anticancer activity on MCF7-bearing Balb/c-nu mice. Overall, our work demonstrated that the HBc VLPs can be an ideal drug carrier to fulfill requirement of the intelligent loading and "on demand" release of the therapeutic agents for efficient cancer therapy with minimal adverse effects.

Synthesis of a crystalline zeolitic imidazole framework-8 nano-coating on single environment-sensitive viral particles for enhanced immune responses.

Encapsulating antigens with zeolitic imidazole framework-8 (ZIF-8) exhibits many advantages in vaccine development. However, most viral antigens with complex particulate structures are sensitive to pH or ionic strength, which cannot tolerate harsh synthesis conditions of ZIF-8. Balancing the viral integrity and the growth of ZIF-8 crystals is crucial for the successful encapsulation of these environment-sensitive antigens in ZIF-8. Here, we explored the synthesis of ZIF-8 on inactivated foot and mouth disease virus (known as 146S), which is easily disassociated into no immunogenic subunits under the existing ZIF-8 synthesis conditions. Our results showed that intact 146S could be encapsulated into ZIF-8 with high embedding efficiency by lowering the pH of the 2-MIM solution to 9.0. The size and morphology of 146S@ZIF-8 could be further optimized by increasing the amount of Zn2+ or adding cetyltrimethylammonium bromide (CTAB). 146S@ZIF-8 with a uniform diameter of about 49 nm could be synthesized by adding 0.01% CTAB, which was speculated to be composed of single 146S armored with nanometer-scale ZIF-8 crystal networks. Plenty of histidine on the 146S surface forms a unique His-Zn-MIM coordination in the near vicinity of 146S particles, which greatly increases the thermostability of 146S by about 5 °C, and the nano-scale ZIF-8 crystal coating exhibited extraordinary stability to resist EDTE-treatment. More importantly, the well-controlled size and morphology enabled 146S@ZIF-8(0.01% CTAB) to facilitate antigen uptake. The immunization of 146S@ZIF-8(4×Zn2+) or 146S@ZIF-8(0.01% CTAB) significantly enhanced the specific antibody titers and promoted the differentiation of memory T cells without adding another immunopotentiator. This study reported for the first time the strategy of the synthesis of crystalline ZIF-8 on an environment-sensitive antigen and demonstrated that the nano-size and appropriate morphology of ZIF-8 are crucial to exert adjuvant effects, thus expanding the application of MOFs in vaccine delivery.

Mechanism and inhibition of abnormal chromatographic behavior of serotype type a inactivated foot and mouth disease virus in high-performance size-exclusion chromatography.

High-performance size-exclusion chromatography (HPSEC) has been developed for the rapid and quantitative analysis of inactivated foot and mouth disease virus (FMDV) and adopted by regulatory agencies and vaccine manufacturers. However, strong non-specific adsorption of type A/AKT III FMDV was found on some batches of TSK G4000 SWXL column, which significantly affected the analysis accuracy. The adsorption mechanism was studied by investigating the charge and hydrophobicity of A/AKT III FMDV and another serotype O/Mya 98, as well as several model proteins, by zeta potential and hydrophobic interaction chromatography analysis. Adsorption was related to both the FMDV strain and column lots. Some specific amino acids residues on the A/AKT III FMDV surface may strongly interact with the column if the silica-based stationary phase was not completely diol-modified. Several amino acids and chaotropic salts were screened as additives in the mobile phase to suppress the non-specific adsorption of AKT III FMDV in HPSEC analysis. Results showed that adding 0.4 M of arginine (Arg), lysine (Lys), NaClO4, or NaSCN achieved 100% FMDV recovery and normal retention time. Suppression of interaction between FMDV and the backbone of the silica matrix through competitive binding with residues of FMDV or the matrix is considered as the main mechanism by which these four additives act as suppressors. The addition of Arg, NaClO4, or NaSCN led to an apparent decrease in the thermal dissociation temperature Tm of FMDV, whereas Lys slightly increased viral stability. Finally, the mobile phase comprising 0.4 M Lys was screened as optimum that allowed accurate quantification of both two serotypes of FMDV according to method validation; particularly, a relative standard deviation (RSD) < 5% was achieved for AKT III FMDV using three different lots of columns.

Oil-in-ionic liquid nanoemulsion-based adjuvant simultaneously enhances the stability and immune responses of inactivated foot-and-mouth disease virus.

Maintaining structural integrity and enhancing stability of inactivated foot-and-mouth disease virus (iFMDV) antigen in adjuvants is crucial to ensure the vaccine potency. Unfortunately, formulation with most reported adjuvants leads to the accelerated dissociation of iFMDV into inactive pentamers. Here, an ionic liquid, i.e., choline and niacin ([Cho][Nic]), which was found to stabilize iFMDV against the acid- and thermo- induced dissociation in buffer solution, was applied to construct a novel oil-in-ionic liquid (o/IL) nanoemulsion adjuvant composed of [Cho][Nic], squalene, and Tween 80. The o/IL nanoemulsion formulated with iFMDV has a monodisperse diameter of 135.8 ± 40.4 nm. The thermostability and long-term stability of iFMDV were remarkably enhanced in o/IL nanoemulsion compared with that in the o/w emulsion without [Cho][Nic] and in the commercial Montanide ISA 206 adjuvant. The o/IL nanoemulsion exerted its adjuvant effects by improving the humoral immune responses. Immunization of o/IL nanoemulsion adjuvanted iFMDV induced specific IgG titers similar to that adjuvanted by Montanide ISA 206 and about 4-fold higher than the un-adjuvanted iFMDV, also promoted the activation of B lymphocytes and the secretion of interleukin-4 in the mice model. This [Cho][Nic]-based o/IL nanoemulsion can serve as a promising adjuvant platform for the foot-and-mouth disease vaccine.

[Characterization of the antigens in inactivated porcine circovirus type 2 vaccines and virus-like particle vaccines by high-performance size-exclusion chromatography coupled with multi-angle laser light scattering].

This paper aims to detect the antigens in porcine circovirus type 2 (PCV2) vaccines by high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS). With purified inactivated PCV2 and PCV2 virus-like particles (VLP) as references, two inactivated vaccines (a and b) and two VLP vaccines (c and d) for PCV2 from four manufacturers were analyzed by HPSEC-MALLS after demulsification. The antigen peaks in HPSEC-MALLS were identified by PCV2 antigen test strips, Western blotting and transmission electron microscope (TEM). The repeatability and linearity of the method were investigated. The results showed the virus antigens in the two inactivated vaccines were eluted at about 13.3 min in HPSEC. The molecular weight of these antigens was 2.61×106 (±4.34%) Da and 2.40×106 (±2.51%) Da, respectively, as calculated by MALLS. The antigen peaks of the two VLP vaccines also appeared at 13.3 min and the molecular weight was 2.09×106 (±2.94%) Da and 2.88×106 (±11.85%) Da, respectively, which was close to the theoretical molecular weight of PCV2. Moreover, an antigen peak of VLP vaccine c was observed at 11.4 min and the molecular weight was 4.37×106 (±0.42%) Da. The antigen was verified to be the dimer of VLP by TEM. Vaccine d and purified Cap VLP antigens were tested repeatedly, and the RSD of the peak area (n=3) was all < 1.5%, indicating that the method was repeatable. The purified VLP were diluted in serial and tested for linearity. The result suggested good linear relationship between the peak area of VLP or VLP aggregates and the protein concentration of the sample with R2 of 0.999 and 0.997, respectively. Thus, the method met the requirement for quantification and aggregate analysis. This method is accurate and efficient in in vitro quality evaluation and improvement of PCV2 vaccine.

Artificial photosynthesis systems for solar energy conversion and storage: platforms and their realities.

In natural photosynthesis, photosynthetic organisms such as green plants realize efficient solar energy conversion and storage by integrating photosynthetic components on the thylakoid membrane of chloroplasts. Inspired by natural photosynthesis, researchers have developed many artificial photosynthesis systems (APS's) that integrate various photocatalysts and biocatalysts to convert and store solar energy in the fields of resource, environment, food, and energy. To improve the system efficiency and reduce the operation cost, reaction platforms are introduced in APS's since they allow for great stability and continuous processing. A systematic understanding of how a reaction platform affects the performance of artificial photosynthesis is conducive for designing an APS with superb solar energy utilization. In this review, we discuss the recent APS's researches, especially those confined on/in platforms. The importance of different platforms and their influences on APS's performance are emphasized. Generally, confined platforms can enhance the stability and repeatability of both photocatalysts and biocatalysts in APS's as well as improve the photosynthetic performance due to the proximity effect. For functional platforms that can participate in the artificial photosynthesis reactions as active parts, a high integration of APS's components on/in these platforms can lead to efficient electron transfer, enhanced light-harvesting, or synergistic catalysis, resulting in superior photosynthesis performance. Therefore, the integration of APS's components is beneficial for the transfer of substrates and photoexcited electrons in artificial photosynthesis. We finally summarize the current challenges of APS's development and further efforts on the improvement of APS's.

Size exclusion chromatography using large pore size media induces adverse conformational changes of inactivated foot-and-mouth disease virus particles.

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 CaCl2 in mobile phase of SEC separation was proposed and proved to efficiently maintain the conformation of the FMDV.

Oil-in-ionic liquid nanoemulsion-based intranasal delivery system for influenza split-virus vaccine.

Effective antigen delivery and immune stimulation in nasal mucosa determine the success of mucosal immunity. Here, an oil-in-ionic liquid (o/IL) nanoemulsion formulated with choline and niacin IL ([Cho][Nic]), squalene, and Tween 80 surfactant is explored as a vaccine delivery system for intranasal mucosal immunization. Compared to the o/w emulsion counterpart without the ILs, the o/IL manoemulsion showed a reduced and more uniform size of approximately 168 nm and significantly improved stability. Studies in mice model showed that when was used as an intranasal vaccine delivery system for influenza split-virus antigens, the antigens in the o/IL nanoemulsion induced strong mucosal immune responses with secretory IgA titers 25- and 5.8-fold higher than those of naked and commercial MF59-adjuvanted antigens, respectively. The o/IL nanoemulsion system also induced stronger systemic humoral responses. The excellent mucosal adjuvant effects of the o/IL nanoemulsion mainly benefited from the prolonged retention of antigens in the nasal cavity, enhanced antigen permeation into the submucosa, and the consequently promoted proliferation of CD11b cells and CD4+ T cells in nasal mucosa-associated lymphoid tissue. Moreover, when used as an injection adjuvant, the o/IL nanoemulsion also induced stronger humoral immune responses than MF59. Thus, the [Cho][Nic]-based o/IL nanoemulsion vaccine delivery system can serve as a promising adjuvant platform.

Synthesis of red/black phosphorus-based composite nanosheets with a Z-scheme heterostructure for high-performance cancer phototherapy.

Two dimensional black phosphorus nanosheets (BP NSs) have attracted plenty of attention in the research field of cancer photonic therapy. However, the poor stability and relatively low efficiency of reactive oxygen species (ROS) generation of BP NSs limit their practical application. To address these drawbacks, herein we report a red/black phosphorus (RP/BP) composite nanosheet, M-RP/BP@ZnFe2O4, which was synthesized by (1) partially converting red phosphorus (RP) to black phosphorus (BP) followed by liquid-phase ultrasonic exfoliation to form RP/BP NSs, (2) in situ synthesis of ZnFe2O4 nanoparticles on the surface of RP/BP NSs, (3) and wrapping with the MCF-7 cell membrane. Due to the presence of RP, BP, ZnFe2O4 and the cell membrane, the M-RP/BP@ZnFe2O4 NSs exhibited high performance in cancer phototherapy with the following features: (i) a Z-scheme heterojunction structure was formed between RP/BP NSs thus enabling high separation efficiency of the photogenerated electrons and holes; (ii) the photoexcitation holes in the valence band of RP can break the tumor microenvironment by oxidizing glutathione; (iii) the NSs could decompose water to produce H2O2 and O2, which can be further converted to toxic ˙OH through the ZnFe2O4 catalyzed Fenton reaction and 1O2 through energy transfer, respectively; and (iv) the cell membrane wrapping improved the targeting of the composite NSs at the tumor site and photonic therapy can be finally triggered by a 660 nm laser to convert O2 to ˙O2- and 1O2. The in vitro cytotoxicity experiments showed that more than 90% cells were killed after photodynamic therapy (PDT) at 0.3 mg mL-1 M-RP/BP@ZnFe2O4 NSs, and the animal experiments with xenograft tumor model mice indicated that tumor growth was completely inhibited and the highest survival rate of 83.3% at 60 days post PDT was obtained.

Thermal-triggered packing of lipophilic NIR dye IR780 in hepatitis B core at critical ionic strength and cargo-host ratio for improved stability and enhanced cancer phototherapy.

Virus-like particles (VLPs) holding internal cavity with diameter from tens up to one hundred nanometers are attractive platform for drug delivery. Nevertheless, the packing of drugs in the nanocage mainly relies on complicated disassembly-reassembly process. In this study, hepatitis B core protein (HBc) VLPs which can withstand temperature up to 90 °C was employed as carrier to load a lipophilic near infrared dye IR780. It was found that an attaching-dis-atching-diffusing process was involved for the entering of IR780 in the cavity of HBc. The first two steps were associated with the electrostatic interactions between oppositely charged HBc and IR780, which was critically manipulated by ionic strength and HBc/IR780 mass ratio at which they were mixed; while the diffusion of IR780 across the shell of HBc showed a temperature-dependent manner that can be triggered by thermal induced pore-opening of the HBc capsid. At optimized condition, about 1055 IR780 molecules were encapsulated in each HBc by simply mixing them for 10 min at 60 °C. Compared with free IR780, the HBc-IR780 particles showed significantly improved aqueous and photostability, as well as enhanced photothermal and photodynamic performance for cancer therapy. This study provides a novel drug loading strategy and nanomemedicine for cancer phototherapies.

[Advances in quantification of lentiviral vectors].

Immunotherapy is becoming an effective and less invasive strategy that can be applied to the treatment of various malignancies. Lentiviral vectors (LVs) have shown great potential in immunotherapy as they can stably integrate relatively large foreign DNA, and effectively transduce dividing and non-dividing cells. Clinical application needs high quality LVs, and therefore strict quality control of the final products is necessary to ensure their purity, efficacy and safety. The quantitative detection of LVs is among the key parts of product development and quality control. In this paper, the existing methods for quantitative detection of LVs are summarized, including fluorescence activated cell sorter (FACS), P24 enzyme-linked immuno sorbent assay (P24 ELISA), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing(TRPS) and virus counter(VC).Their advantages and disadvantages are listed, and future development and challenges are discussed.

Possible Action of Transition Divalent Metal Ions at the Inter-Pentameric Interface of Inactivated Foot-and-Mouth Disease Virus Provide A Simple but Effective Approach to Enhance Stability.

The structural instability of inactivated foot-and-mouth disease virus (FMDV) hinders the development of vaccine industry. Here we found that some transition metal ions like Cu2+ and Ni2+ could specifically bind to FMDV capsids at capacities about 7089 and 3448 metal ions per capsid, respectively. These values are about 33- and 16-folds of the binding capacity of non-transition metal ion Ca2+ (about 214 per capsid). Further thermodynamic studies indicated that all these three metal ions bound to the capsids in spontaneous enthalpy driving manners (ΔG<0, ΔH<0, ΔS<0), and the Cu2+ binding had the highest affinity. The binding of Cu2+ and Ni2+ could enhance both the thermostability and acid-resistant stability of capsids, while the binding of Ca2+ was helpful only to the thermostability of the capsids. Animal experiments showed that the immunization of FMDV bound with Cu2+ induced the highest specific antibody titers in mice. Coincidently, the FMDV bound with Cu2+ exhibited significantly enhanced affinities to integrin ß6 and heparin sulfate, both of which are important cell surface receptors for FMDV attaching. Finally, the specific interaction between capsids and Cu2+ or Ni2+ was applied to direct purification of FMDV from crude cell culture feedstock by the immobilized metal affinity chromatography. Based on our new findings and structural analysis of the FMDV capsid, a "transition metal ion bridges" mechanism that describes linkage between adjacent histidine and other amino acids at the inter-pentameric interface of the capsids by transition metal ions coordination action was proposed to explain their stabilizing effect imposed on the capsid.IMPORTANCE How to stabilize the inactivated FMDV without affecting virus infectivity and immunogenicity is a big challenge in vaccine industry. The electrostatic repulsion induced by protonation of a large amount of histidine residues at the inter-pentameric interface of viral capsids is one of the major mechanisms causing the dissociation of capsids. In the present work, this structural disadvantage inspired us to stabilize the capsids through coordinating transition metal ions with the adjacent histidine residues in FMDV capsid, instead of removing or substituting them. This approach was proved effective to enhance not only the stability of FMDV, but also enhance the specific antibody responses; thus, providing a new guideline for designing an easy-to-use strategy suitable for large-scale production of FMDV vaccine antigen.

Performance of agarose and gigaporous chromatographic media as function of pore-to-adsorbate size ratio over wide span from ovalbumin to virus like particles.

Two commercially available agarose ion exchange media, DEAE-Capto and DEAE-Sepharose FF (DEAE-FF), and two gigaporous media DEAE -AP-120 nm and DEAE-AP-280 nm were evaluated for their applicability in adsorption of five proteins with large span of radius ranges from 2.9 nm to 14.1 nm, which include ovalbumin, bovine serum albumin (BSA), haptoglobin, thyroglobulin and hepatitis B surface antigen (HBsAg) virus like particle. The average pore radius of the four media was determined to be 6.9 nm, 18.5 nm, 59.4 nm and 139.3 nm, respectively, which was obtained by log normal distribution for DEAE-Capto and DEAE-FF and by bimodal Gaussian distribution for the two DEAE-AP media. The performance of these four media including phase ratio, static and dynamic binding capacity, and transport properties for the adsorption of these five model proteins as function of pore-to-adsorbate size ratio were investigated and compared. The best ratio of pore-to-adsorbate size was found dependent on the protein size. For protein with radius from 2.9 nm (ovalbumin) to 5.4 nm (BSA), the agarose media was superior to gigaporous media. Both the static and dynamic adsorption capacities reduced with the increase of pore size, and the highest values were obtained at the smallest pore-to-adsorbate size of about 2 times in this study, although the highest accessible surface area was obtained at pore-to-adsorbate size ratio about 16 to 20. For proteins with radius of 5.4 nm or larger than that, their adsorption capacities decreased firstly and then increased with the increase of ratio of pore-to-adsorbate size, and the highest values were obtained on the gigaporous media DEAE-AP-280 nm, which could provide faster diffusivity and larger accessible surface area. However, protein with radius of 14.1 nm (HBsAg) had much lower capacities compared to other proteins at the same pore-to-adsorbate size ratio, implying large protein needs greater pore-to-adsorbate size ratio to achieve a satisfactory capacity. For all the five tested proteins, the DEAE-Capto media having the smallest pore radius and branched dextran chains, was found superior to DEAE-FF in terms of both higher adsorption capacities and uptake kinetics, which suggested that the "chain delivery effect" took place on proteins over large size span from ovalbumin to HBsAg, though the effect on the larger proteins was much less significant than that on the smaller ones. Results from the present work provided more information on how do the relationships of pore size of chromatography media and adsorbate size interactively affect the chromatography behaviors, thus will provide general guidance for selection of suitable adsorbent for biologics of a given size.

Effect of pore structure on protein adsorption mechanism on ion exchange media: A preliminary study using low field nuclear magnetic resonance.

The adsorption process of bovine serum albumin (BSA), ovalbumin (OVA) and human immunoglobulin G (IgG) on agarose ion-exchange media Q Sepharose FF and two dextran-grafted agarose media including Q Sepharose XL and Capto Q were studied using low field nuclear magnetic resonance (NMR). The T2 relaxation time was found directly proportional to the pore size and diminished after protein adsorbed, therefore, a theoretical model describing the relationship between protein binding amount and T2 relaxation signals was established. The model parameters, a, which reflects the contact area between the adsorbed protein and media surface, and the δ, which defined as the ratio of the protein volume to the pore volume after adsorption, were found to describe the pore occupation states of proteins in media with different pore structures very well. For small proteins, such as BSA and OVA, monolayer adsorption occurred on Q Sepharose FF, which has no dextran chains. Therefore, the adsorbed protein only occupied 49.05% of the pore volume for BSA and 25.51% for OVA, and contact area of each protein on the media were also low, suggesting mostly monolayer adsorption occurred. In the contrast, their adsorption to Q Sepharose XL and Capto Q with dextran chains tended to form multilayer adsorption, thus higher contact area was obtained and the pore volumes were almost 100% occupied. For large protein, such as IgG, the adsorption to all these three media was similar and about 30% of the pore volume were occupied, probably due to the similar restriction for IgG to entering the media pore. Results of this study will help to elucidate the relationship between protein adsorption and pore size variation, which present the significance of low field NMR in understanding protein adsorption mechanism.