Highly flexible cell membranes are the key to efficient production of lipophilic compounds.

Lipophilic compounds have a variety of positive effects on human physiological functions and exhibit good effects in the prevention and treatment of clinical diseases. This has led to significant interest in the technical applications of synthetic biology for the production of lipophilic compounds. However, the strict selective permeability of the cell membrane and the hydrophobic nature of lipophilic compounds pose significant challenges to their production. During fermentation, lipophilic compounds tend to accumulate within cell membrane compartments rather than being secreted extracellularly. The toxic effects of excessive lipophilic compound accumulation can threaten cell viability, while the limited space within the cell membrane restricts further increases in production yield. Consequently, to achieve efficient production of lipophilic compounds, research is increasingly focused on constructing robust and multifunctional microbial cell factories. Utilizing membrane engineering techniques to construct highly flexible cell membranes is considered an effective strategy to break through the upper limit of lipophilic compound production. Currently, there are two main approaches to cell membrane modification: constructing artificial storage compartments for lipophilic compounds and engineering the cell membrane structure to facilitate product outflow. This review summarizes recent cell membrane engineering strategies applied in microbial cell factories for the production of liposoluble compounds, discussing the challenges and future prospects. These strategies enhance membrane flexibility and effectively promote the production of liposoluble compounds.

Modulation of Tannic Acid on the Cholesteric Structure of Cellulose Nanocrystals.

The chiral nematic phase structure, formed by the self-assembly of cellulose nanocrystals (CNCs) in an aqueous suspension and maintained in a solid film, shows great potential for optical applications. To achieve complex structures in optical devices, it is crucial to subject CNCs to specific shearing processes, such as spinning and printing. Understanding the structural and property changes of the CNC liquid crystal phase in these processes is of utmost importance. In this study, we investigated the effect of adding tannic acid (TA) on the rheological properties and cholesteric phase structures of CNCs/TA mixed suspensions. By calculating the surface site interaction points, we observed that TA can adsorb onto the surface of CNC rods in suspensions through hydrogen bonding. Through characterization techniques, such as polarized optical microscopy, rheology, and synchrotron SAXS, we examined the effects of TA addition on the microstructure and rheological properties of the CNC liquid crystal phase and clarified the change relating to the system composition. Under the same CNC concentration, the volume fraction of the anisotropic phase, the pitch, and the rod spacing of the cholesteric phase were not significantly affected by the addition of TA. However, the system viscosity was significantly reduced with the appropriate amount of TA (2 wt %), in a wide range of CNC concentrations (up to 15 wt % CNCs). The flow indexes (n) in Region I and Region III of steady-state shear curves of CNCs/TA systems (11-15 wt % CNCs) were compared. Moreover, we introduced the well-established theoretical models for liquid crystal polymers to tentatively interpret Region I of the CNCs/TA cholesteric phase and realized that increased numbers of smaller cholesteric-phase domains in the CNCs/TA system and interfacial modification by TA may contribute to the fluidity change. The feature of the domain texture of CNCs/TA systems is verified by polarized optical microscopy observations.

An Integrated Strategy Based on 10-DAB Extraction and In Situ Whole-Cell Biotransformation of Renewable Taxus Needles to Produce Baccatin III.

Baccatin III is a crucial precursor in the biosynthesis pathway of paclitaxel. Its main sources are extraction from Taxus or chemical synthesis using 10-deacetylbaccatin III (10-DAB) as substrate. However, these preparation approaches exhibit serious limitations, including the low content of baccatin III in Taxus and the complicated steps of chemical synthesis. Heterologous expression of 10-deacetylbaccatin III-10-O-acetyltransferase (TcDBAT) in microbial strains for biotransformation of 10-DAB is a promising alternative strategy for baccatin III production. Here, the promotion effects of glycerol supply and slightly acidic conditions with a low-temperature on the catalysis of recombinant TcDBAT strain were clarified using 10-DAB as substrate. Taxus needles is renewable and the content of 10-DAB is relatively high, it can be used as an effective source of the catalytic substrate 10-DAB. Baccatin III was synthesized by integrating the extraction of 10-DAB from renewable Taxus needles and in situ whole-cell catalysis in this study. 40 g/L needles were converted into 20.66 mg/L baccatin III by optimizing and establishing a whole-cell catalytic bioprocess. The method used in this study can shorten the production process of Taxus extraction for baccatin III synthesis and provide a reliable strategy for the efficient production of baccatin III by recombinant strains and the improvement of resource utilization rate of Taxus needles.

Interfacial Engineering of Block Copolymer Nanostructures: Morphology and Solvent Stability.

Interfacial engineering is a critical pathway for modulating the self-assembled nanostructures of block copolymers (BCPs) during solvent exchange. Herein, we demonstrated the generation of different stacked lamellae of polystyrene-block-poly(2-vinyl pyridine) (PS-b-P2VP) nanostructures during solvent exchange by using phosphotungstic acid (PTA) or PTA/NaCl aqueous solution as the nonsolvent. The participation of PTA in the confined microphase separation of PS-b-P2VP in droplets increases the volume fraction of P2VP and decreases the tension at the oil/water interface. Moreover, the addition of NaCl to the PTA solution can further increase the surface coverage of P2VP/PTA on droplets. All factors impact the morphology of assembled BCP nanostructures. In the presence of PTA, ellipsoidal particles composed of alternatively stacked lamellae of PS and P2VP were formed (named BP), whereas, in the coexistence of PTA and NaCl, they changed to stacked disks with PS-core-P2VP-shell (called BPN). The different structures of assembled particles induce their different stabilities in solvents and different dissociation conditions as well. The dissociation of BP particles was easy because PS chains were only entangled together which can be swollen in toluene or chloroform. However, the dissociation of BPN was hard, requiring an organic base in hot ethanol. The structural difference in BP and BPN particles further extended to their dissociated disks, which makes the cargo (like R6G) loaded on these disks to show a different stability in acetone. This study demonstrated that a subtle structural change can greatly affect their properties.

Anti-SARS-CoV-2 IgG levels in relation to disease severity of COVID-19.

The durability of infection-induced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity has crucial implications for reinfection and vaccine effectiveness. However, the relationship between coronavirus disease 2019 (COVID-19) severity and long-term anti-SARS-CoV-2 immunoglobulin G (IgG) antibody level is poorly understood. Here, we measured the longevity of SARS-CoV-2-specific IgG antibodies in survivors who had recovered from COVID-19 1 year previously. In a cohort of 473 survivors with varying disease severity (asymptomatic, mild, moderate, or severe), we observed a positive correlation between virus-specific IgG antibody titers and COVID-19 severity. In particular, the highest virus-specific IgG antibody titers were observed in patients with severe COVID-19. By contrast, 74.4% of recovered asymptomatic carriers had negative anti-SARS-CoV-2 IgG test results, while many others had very low virus-specific IgG antibody titers. Our results demonstrate that SARS-CoV-2-specific IgG persistence and titer depend on COVID-19 severity.

Formation and Transformation of Polystyrene-block-poly(2-vinylpyridine) Hexasomes in the Solvent Exchange.

The generation of inverse micellar nanostructures, especially those with open channels, using commercially available diblock copolymers (BCP), is vital for their wide applications in drug delivery and catalyst templating. However, the rigid requirements for forming inverse morphologies, such as the highly asymmetric molecular structures, the semicrystalline motifs, and concentrated solutions of diblock copolymers, represent obstacles to the development of successful strategies. In this study, the inverse polystyrene-block-poly(2-vinylpyridine) (PS30K-b-P2VP8.5K) micelles, i.e., the hexasomes with p6mm lattice, were generated through a modified solvent exchange via adding d-tartaric acid (d-TA) in the nonsolvent. Various intermediate morphologies have been identified with the change of d-TA concentration. Interestingly, in the high d-TA concentration (∼20 mg/mL), the hexasomes with close-packed hoops changed to mesoporous spheres with regularly packed perpendicular cylindrical channels (VD-TA: VBCP 6:100), and further to the mesoporous spheres with gyri-like open pores (VD-TA: VBCP > 15:100) with the increasing acidity in the mixed solvent. This study presents a simple and economical pathway for fabricating PS30K-b-P2VP8.5K hexasomes and first demonstrates these hexasomes can be modified to the morphology with open channels that will benefit their further applications.

Global and Regional Trends and Projections of Infective Endocarditis-Associated Disease Burden and Attributable Risk Factors from 1990 to 2030.

Objective To forecast the future burden and its attributable risk factors of infective endocarditis (IE). Method We analyzed the disease burden of IE and its risk factors from 1990 to 2019 using the Global Burden of Disease 2019 database and projected the disease burden from 2020 to 2030 using a Bayesian age-period-cohort model. Results By 2030, the incidence of IE will increase uncontrollably on a global scale, with developed countries having the largest number of cases and developing countries experiencing the fastest growth. The affected population will be predominantly males, but the gender gap will narrow. The elderly in high-income countries will bear the greatest burden, with a gradual shift to middle-income countries. The incidence of IE in countries with middle/high-middle social-demographic indicators (SDI) will surpass that of high SDI countries. In China, the incidence rate and the number of IE will reach 18.07 per 100,000 and 451,596 in 2030, respectively. IE-associated deaths and heart failure will continue to impose a significant burden on society, the burden on women will increase and surpass that on men, and the elderly in high-SDI countries will bear the heaviest burden. High systolic blood pressure has become the primary risk factor for IE-related death. Conclusions This study provides comprehensive analyses of the disease burden and risk factors of IE worldwide over the next decade. The IE-associated incidence will increase in the future and the death and heart failure burden will not be appropriately controlled. Gender, age, regional, and country heterogeneity should be taken seriously to facilitate in making effective strategies for lowering the IE disease burden.

Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies.

BACKGROUND: Hydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B12. The introduction of a heterologous de novo vitamin B12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. Although E. coli avoids major limitations that currently faced by industrial producers of vitamin B12, such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B12 production. RESULTS: By designing combinatorial ribosomal binding site libraries of the hemABCD genes in vivo, we found that their optimal relative translational initiation rates are 10:1:1:5. The transcriptional coordination of the uroporphyrinogen III biosynthetic module was realized by promoter engineering of the hemABCD operon. Knockdown of competitive heme and siroheme biosynthesis pathways by RBS engineering enhanced the hydrogenobyrinic acid titer to 20.54 and 15.85 mg L-1, respectively. Combined fine-tuning of the heme and siroheme biosynthetic pathways enhanced the hydrogenobyrinic acid titer to 22.57 mg L-1, representing a remarkable increase of 1356.13% compared with the original strain FH215-HBA. CONCLUSIONS: Through multi-level metabolic engineering strategies, we achieved the metabolic balance of the uroporphyrinogen III biosynthesis pathway, eliminated toxicity due to by-product accumulation, and finally achieved a high HBA titer of 22.57 mg L-1 in E. coli. This lays the foundation for high-yield production of vitamin B12 in E. coli and will hopefully accelerate its industrial production.

Removal of phosphate from wastewater by modified bentonite entrapped in Ca-alginate beads.

Methods of removing phosphate from wastewater with a low phosphate concentration are of great environmental significance. In this study, immobilized beads were prepared by entrapping modified bentonite powder in calcium-alginate (Al-NaBT-CA), and the potential of the beads for phosphate removal from wastewater was investigated. The effects of pH (1-10) and initial phosphate concentration (0.5-50 mg/L) were also examined in batch experiments with Al-NaBT-CA beads. The optimum pH value for phosphate removal by Al-NaBT-CA beads was pH 3. In addition, a high initial phosphate concentration promoted phosphate adsorption. Adsorption kinetics showed that the adsorption of phosphate using beads followed a pseudo-second-order kinetic model (R2 = 0.98-0.99). The adsorption isotherm data was well fitted by the Sips adsorption model. The maximum phosphate adsorption capacity of the Al-NaBT-CA beads was 15.77 mg/g, which was slightly less than that of the modified powder. The specific surface area of the Al-NaBT-CA beads was 17.01 m2/g, and their average pore size was 13.41 nm. Scanning electron microscopy suggested that the high inner porosity and rough outer surface of the beads facilitated phosphate transfer.

Pt-Cu Bimetallic Nanoparticles Loaded in the Lumen of Halloysite Nanotubes.

In this study, we demonstrate that Pt-Cu bimetallic nanoparticles with different compositions (Pt3Cu, PtCu, PtCu3) can be loaded in the lumen of halloysite nanotube (HNT) via a simple one-pot reduction. Increasing the pH of metallic precursor (H2PtCl6 and CuCl2)/HNT solutions enhances the dissociation of H2PtCl6, advancing the association of [PtCl6]2- with the positively charged inner surface (Al-OH) of HNT. Moreover, the shrinkage of bond length from Pt-Cl in [PtCl6]2- to Pt-O in [PtCl4(OH)2]2- due to pH-modulated ligand exchange may also assist Pt(IV) being trapped inside the halloysite. In the meantime, Cu(II) cations may complex with Pt(IV) anions via electrostatic force that would help the formation of Pt-Cu bimetallic nanoparticles inside the halloysite. The obtained PtCu3@HNT system shows a significantly enhanced catalytic performance in the reduction of 4-nitrophenol by sodium borohydride, with a mass activity approximating 60 times higher than that of unloaded Pt nanoparticles. The high catalytic efficiency can be maintained after thermal treatment at 200 or 400 °C.

High-efficiency expression and secretion of human FGF21 in Bacillus subtilis by intercalation of a mini-cistron cassette and combinatorial optimization of cell regulatory components.

BACKGROUND: Recombinant human Fibroblast growth factor 21 (rhFGF21) is an endocrine hormone that has profound effects on treatment of metabolic diseases. However, rhFGF21 is prone to form inclusion body when expressed in bacteria, which results in, the downstream process of purification of bioactive rhFGF21 is time-consuming and labor intensive. The aim of this work is to explore a new method for improving the soluble expression and secretion level of rhFGF21 in B. subtilis. RESULTS: A codon optimized rhFGF21 gene was expressed under the control of a strong inducible promoter PmalA in B. subtilis. A mini-cistron cassette (from gsiB) was located upstream of rhFGF21 in expression vector (pMATEFc5), which could reduce the locally stabilized mRNA secondary structure of transcripts and enhance the efficiency of translation initiation. Then various chaperones were further overexpressed to improve the expression efficiency of rhFGF21. Results showed that overexpression of the chaperone DnaK contributed to the increase of solubility of rhFGF21. Moreover, an extracellular proteases deficient strain B. subtilis Kno6cf was used to accumulate the secreted rhFGF21 solidly. In addition, eleven signal peptides from B. subtilis were evaluated and the SPdacB appeared the highest secretion yield of rhFGF21 in B. subtilis. Finally, the combinatorial optimized strain achieved an about ninefold increase of the soluble rhFGF21 production after 24 h of flask fermentation in comparison with the initial production strain. CONCLUSION: This work provided a comprehensive strategy for secretory expressing the heterologous protein rhFGF21 in B. subtilis. To our knowledge, this is the first report of the highly efficient production of rhFGF21 in B. subtilis and this approach may provide some suggestions for heterologous proteins production in B. subtilis.

Microwave-Assisted Extraction of Multiple Trace Levels of Intermediate Metabolites for Camptothecin Biosynthesis in Camptotheca acuminata and Their Simultaneous Determination by HPLC-LTQ-Orbitrap-MS/MS and HPLC-TSQ-MS.

Camptothecin (CPT) has strong antitumor activity and is used as an anticancer therapeutic agent. To better understand and decipher the pathway of CPT biosynthesis in Camptotheca acuminata, the main purpose here was focused on creating an effective extraction strategy for a rich intermediate metabolite profile. In the present study, a 70% aqueous acetonitrile was verified as an optimal extraction solvent for microwave-assisted extraction (MAE) of metabolites by spiking experiments. Based on multi-objective optimization, the best extraction conditions of a solid-liquid ratio of 1:20, microwave power of 230 W, and a time of 4 min were achieved using a full factorial 34 experimental design. Crude extracts obtained from the shoot apex of C. acuminata using MAE have been qualitatively profiled by high-performance liquid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap-MS/MS) and a HPLC triple quadrupole-MS (HPLC-TSQ-MS) analysis was conducted for their metabolite content in different tissues. CPT, and ten related metabolites and their isomers, including tryptamine, loganic acid, secologanic acid, strictosidinic acid, strictosamide, strictosamide epoxide, strictosamide diol, strictosamide ketolactam, pumiloside, and deoxypumiloside, were detected and tentatively identified. Scanning electron microscopy (SEM) imaging of the shoot apex demonstrated that severe cell disruption was evident after intensified extraction processes. The study showed the difference of metabolite profiles and the enhancement of metabolite content after microwave-pretreated techniques, and the established MAE procedure is an effective methodology to preserve valuable metabolite compounds for analysis.

Polydopamine Generates Hydroxyl Free Radicals under Ultraviolet-Light Illumination.

Polydopamine (PDA) generally demonstrates as an efficient free-radical scavenger. However, its free-radical chemistry under illumination is unclear, which becomes important in view of growing studies of polydopamine applications in photoprotector and photothermal therapy. In this study, for the first time, we reported an experimental investigation of the generation of hydroxyl free radicals from ultraviolet (UV)-illuminated polydopamine in an aqueous environment. By using terephthalic acid as fluorescent probe, we measured hydroxyl radicals generated from UV-illuminated polydopamine with different shapes and sizes. The morphology of PDA shows significant influence on its productions of hydroxyl free radicals. Through characterizations of UV-vis absorption spectroscopy, fluorescence spectroscopy, X-ray photoelectron spectrometry, mass spectrometry, and thermogravimetric analysis, we demonstrated the change of PDA nanomaterials brought by UV-light illumination in composition and thermal stability. We proposed a tentative mechanism for interpreting the relationship between morphology and photostability of PDA nanomaterials. These results reveal underlying complexity of polydopamine chemistry under light illumination that will deepen our understanding and benefit its further application.

Interfacial Interaction in Anodic Aluminum Oxide Templates Modifies Morphology, Surface Area, and Crystallization of Polyamide-6 Nanofibers.

Here, we demonstrated that, when the precipitation process of polyamide-6 (PA6) solution happens in cylindrical channels of an anodized aluminum oxide membrane (AAO), interface interactions between a solid surface, solvent, non-solvent, and PA6 will influence the obtained polymer nanostructures, resulting in complex morphologies, increased surface area, and crystallization changes. With the enhancing interaction of PA6 and the AAO surface, the morphology of PA6 nanostructures changes from solid nanofibers, mesoporous, to bamboo-like, while at the same time, metastable γ-phase domains increase in these PA6 nanostructures. Brunauer-Emmett-Teller (BET) surface areas of solid, bamboo-like, and mesoporous PA6 nanofibers rise from 16, 20.9, to 25 m(2)/g. This study shows that interfacial interaction in AAO template fabrication can be used in manipulating the morphology and crystallization of one-dimensional polymer nanostructures. It also provides us a simple and novel method to create porous PA6 nanofibers with a large surface area.

Characterizations of the Formation of Polydopamine-Coated Halloysite Nanotubes in Various pH Environments.

Recent studies demonstrated that polydopamine (PDA) coating is universal to nearly all substrates, and it endows substrates with biocompatibility, postfunctionality, and other useful properties. Surface chemistry of PDA coating is important for its postmodifications and applications. However, there is less understanding of the formation mechanism and surface functional groups of PDA layers generated in different conditions. Halloysite is a kind of clay mineral with tubular nanostructure. Water-swellable halloysite has unique reactivity. In this study, we have investigated the reaction of dopamine in the presence of water-swellable halloysite. We have tracked the reaction progresses in different pH environments by using UV-vis spectroscopy and surface-enhanced Raman spectroscopy (SERS). The surface properties of PDA on halloysite were clarified by X-ray photoelectron spectroscopy (XPS), SERS, Fourier transform infrared (FTIR) characterizations, zeta potential, surface wettability, and morphological characterizations. We noticed that the interaction between halloysite surface and dopamine strongly influences the surface functionality of coated PDA. In addition, pH condition further modulates surface functional groups, resulting in less content of secondary/aromatic amine in PDA generated in weak acidic environment. This study demonstrates that the formation mechanism of polydopamine becomes complex in the presence of inorganic nanomaterials. Substrate property and reaction condition dominate the functionality of obtained PDA together.

High-level intra- and extra-cellular production of D-psicose 3-epimerase via a modified xylose-inducible expression system in Bacillus subtilis.

D-Psicose 3-epimerase (DPEase) converts D-fructose into D-psicose which exists in nature in limited quantities and has key physiological functions. In this study, RDPE (DPEase from Ruminococcus sp. 5_1_39BFAA) was successfully constitutively expressed in Bacillus subtilis, which is the first report of its kind. Three sugar-inducible promoters were compared, and the xylose-inducible promoter P xylA was proved to be the most efficient for RDPE production. Based on the analysis of the inducer concentration and RDPE expression, we surmised that there was an extremely close correlation between the intracellular RDPE expression and xylose accumulation level. Subsequently, after the metabolic pathway of xylose was blocked by deletion of xylAB, the intra- and extra-cellular RDPE expression was significantly enhanced. Meanwhile, the optimal xylose induction concentration was reduced from 4.0 to 0.5 %. Eventually, the secretion level of RDPE reached 95 U/mL and 2.6 g/L in a 7.5-L fermentor with the fed-batch fermentation, which is the highest production of DPEase by a microbe to date.

Oxidative Self-Polymerization of Dopamine in an Acidic Environment.

A weak alkaline condition (pH > 8) is a general requirement for oxidative self-polymerization of dopamine. Here, we first demonstrated the generation of polydopamine in an acidic environment via a hydrothermal method. The pH scope of self-polymerization of dopamine is extended to pH ∼ 1 in a hydrothermal process. Polydopamine generated via a hydrothermal method shows similar chemical features and radical scavenging activity with that generated in a basic environment.

Folic acid-polydopamine nanofibers show enhanced ordered-stacking via π-π interactions.

Recent research has indicated that polydopamine and synthetic eumelanins are optoelectronic biomaterials in which one-dimensional aggregates composed of ordered-stacking oligomers have been proposed as unique organic semiconductors. However, improving the ordered-stacking of oligomers in polydopamine nanostructures is a big challenge. Herein, we first demonstrate how folic acid molecules influence the morphology and nanostructure of polydopamine via tuning the π-π interactions of oligomers. MALDI-TOF mass spectrometry reveals that porphyrin-like tetramers are characteristic of folic acid-polydopamine (FA-PDA) nanofibers. X-ray diffraction combined with simulation studies indicate that these oligomers favour aggregation into graphite-like ordered nanostructures via strong π-π interactions. High-resolution TEM characterization of carbonized FA-PDA hybrids show that in FA-PDA nanofibers the size of the graphite-like domains is over 100 nm. The addition of folic acid in polydopamine enhances the ordered stacking of oligomers in its nanostructure. Our study steps forward to discover the mystery of the structure-property relationship of FA-PDA hybrids. It paves a way to optimize the properties of PDA through the design and selection of oligomer structures.

Characterization of carbonized polydopamine nanoparticles suggests ordered supramolecular structure of polydopamine.

Polydopamine is not only a multifunctional biopolymer with promising optoelectronic properties but it is also a versatile coating platform for different surfaces. The structure and formation of polydopamine is an active area of research. Some studies have supposed that polydopamine is composed of covalently bonded dihydroxyindole, indoledione, and dopamine units, but others proposed that noncovalent self-assembly contributes to polydopamine formation as well. However, it is difficult to directly find the details of supramolecular structure of polydopamine via self-assembly. In this study, we first report the graphite-like nanostructure observed in the carbonized polydopamine nanoparticles in nitrogen (or argon) environment at 800 °C. Raman characterization, which presents the typical D band and G band, confirmed the existence of graphite-like nanostructures. Our observation provides clear evidence for a layered-stacking supramolecular structure of polydopamine. Particularly, the size of graphite-like domains is similar to that of disk-shaped aggregates hypothesized in previous study about the polymerization of 5,6-dihydroxyindole [ Biomacromolecules 2012 , 13 , 2379 ]. Analysis of the hierarchical structure of polydopamine helps us understand its formation.

Freezing polystyrene-b-poly(2-vinylpyridine) micelle nanoparticles with different nanostructures and sizes.

Herein we report how to control the nanostructures and sizes of polystyrene-b-poly(2-vinylpyridine) (PS-b-P2VP) nanoparticles via manipulating freezing in solvent-exchange. By characterizing and analyzing the distinct structural features of the obtained nanoparticles, we recognized that micelle self-assembly happens in the precipitation of PS-b-P2VP when water is added into the block copolymer (BCP) solution. Solvent properties significantly influence micelle types that are vesicles in acetone/H2O and spherical micelles in tetrahydrofuran/H2O, respectively, thus further inducing different frozen nanostructures of the obtained nanoparticles, onion-like in acetone/H2O and large compound micelles in tetrahydrofuran/H2O. By changing the concentration of the block copolymers and the Vsolvent/VH2O ratio to modify the freezing stage at which block copolymer micelles are frozen, we can further control the size of the nanoparticles. Moreover, small molecules (phosphotungstic acid, pyrene, 1-pyrenebutyric acid) can be trapped into the block copolymer nanoparticles via the freezing process. Their distribution in the nanoparticles relies not only on the solvent property, but also on their interactions with block copolymers. The hybrid nanoparticles with ordered distribution of small molecules can be further changed to partially-void nanoparticles. Our study demonstrated that manipulating the freezing of block copolymers in the solvent exchange process is a simple and controllable fabrication method to generate BCP nanoparticles with different architectures.