Enzyme Carrier Research Articles

Effect of Graphene Oxide Prepared Under Different Conditions on Immobilized α-Amylase

Graphene oxide (GO) has broad application prospects in the field of catalysis, especially as enzyme carrier, owing to its unique intriguing physic-chemical properties. In this paper, the layer structure and oxygen functional group content of GO which may affect the properties of the immobilized enzyme were discussed. GO was prepared from 8000 mesh and nanoscale graphite at different reaction temperatures (40 °C and 95 °C), and used as carriers to immobilize α-amylase by deposition and cross-linking methods, respectively. Several analytical tools, including AFM, FT-IR, UV–Vis, XPS, and Raman, were used to character GOs. The enzyme loading of GO-8000-40 °C-E (of GO-NM-95 °C-GA-E) was 122.1 (49.8) mg/g, which retained 94.7 (82.6) % of the free enzyme activity, the optimum pH, optimum temperature, Km and Vmax were 7.0 (7.0), 70 (75) °C, 18.837 (39.989) mg/mL and 1.584 (1.842) μmol/(mL·min), respectively. Overall results indicated that 8000 mesh graphite was suitable for the preparation of immobilized enzyme by deposition method, while nano-graphite had an advantage in cross-linking immobilized enzyme.

Continuous production of Neisseria meningitidis outer membrane vesicles

Outer membrane vesicles (OMVs) are nanoparticles secreted by Gram-negative bacteria that can be used for diverse biotechnological applications. Interesting applications have been developed, where OMVs are the basis of drug delivery, enzyme carriers, adjuvants, and vaccines. Historically, OMV research has mainly focused on vaccines. Therefore, current OMV production processes have been based on batch processes. The production of OMVs in batch mode is characterized by relatively low yields and high costs. Transition of OMV production processes from batch to continuous processes could increase the volumetric productivity, reduce the production and capital costs, and result in a higher quality product. Here, we study the continuous production of Neisseria meningitidis OMVs to improve volumetric productivity. Continuous cultivation of N. meningitidis resulted in a steady state with similar high OMV concentrations as are reached in current batch processes. The steady state was reproducible and could be maintained for at least 600 h. The volumetric productivity of a continuous culture reached 4.0 × 1014 OMVs per liter culture per day, based on a dilution rate of 1/day. The tested characteristics of the OMVs did not change during the experiments showing feasibility of a continuous production process for the production of OMVs for any application.

Affinity-binding immobilization of D-amino acid oxidase on mesoporous silica by a silica-specific peptide.

Enzyme immobilization is widely used for large-scale industrial applications. However, the weak absorption through physical methods limits the recovery ability. Here, affinity-binding immobilization of enzymes was explored using a silica-specific affinity peptide (SAP) as a fusion tag to intensify the binding force between the enzyme and mesoporous silica (MPS) carrier. D-amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as a model enzyme. The optimal screened SAP (LPHWHPHSHLQP) was selected from a M13 phage display peptide library and fused to the C-terminal of DAAO to obtain fused DAAOs with one, two and three SAP tags, respectively. The activity of DAAO-SAP-MPS was superior comparing with DAAO-2SAP-MPS and DAAO-3SAP-MPS; meanwhile DAAO-SAP-MPS shows 36% higher activity than that of DAAO-MPS. Fusion with one SAP improved the thermal stability with a 10% activity increase for immobilized DAAO-SAP-MPS compared to that of DAAO-MPS at 50°C for 3h. Moreover, the activity recovery of immobilized DAAO-SAP-MPS was 25% higher in operation stability assessment after six-batch conversions of cephalosporin to glutaryl-7-amino cephalosporanic acid than that of DAAO-MPS.

Hollow Prussian Blue nanocubes as peroxidase mimetic and enzyme carriers for colorimetric determination of ethanol.

The peroxidase-like activity of hollow Prussian Blue nanocubes (hPBNCs) is used, in combination with the enzyme alcohol oxidase (AOx), in a colorimetric ethanol assay. Different from other nanozymes, the large cavity structure of the hPBNCs provides a larger surface and more binding sites for AOx to be bound on their surface or in the pores. This extremely enhances the sensitivity of the assay system. In the presence of ethanol, AOx is capable of catalyzing the oxidation of alcohols to aldehydes, accompanied by the generation of hydrogen peroxide (H2O2). The hPBNCs act as peroxidase mimics and then can catalyze the oxidation of 3,3'5,5'-tetramethylbenzidine (TMB) by H2O2, resulting in a color change of the solution from colorless to blue with a strong absorption at 652nm. The lower detection limit for ethanol is 1.41μg∙mL-1. Due to the high catalytic activity of hPBNCs in weakly acidic and neutral solutions, the system was successfully applied to the determination of ethanol in mice blood. This is critically important for studying the alcohol consumption and monitoring the ethanol toxicokinetics. Graphical abstract Schematic representation of hollow Prussian Blue nanocubes (hPBNCs) used as both a peroxidase mimetic and as a carrier for alcohol oxidase. Utilizing hPBNCs along with the ethanol conversion enzyme, a sensitive colorimetric assay for ethanol was developed and applied to blood samples with satisfactory results.

Preparation of chitosan modified magnetic carbon nanotubes and application in immobilized enzymes

ABSTRACTChitosan (CTS) was selected to combine Fe3O4/CNTs with noncovalent bonds to prepare CTS modified magnetic carbon nanotubes (CTS-Fe3O4/CNTs). The chemical structure, morphology, crystal structure, element and magnetic properties were analyzed by means of FTIR, TEM, XRD, XPS and VSM. CTS-Fe3O4/CNTs was used as immobilized enzyme carrier for the degradation of phenolic compounds in sewage. The results of characterizations demonstrated that CTS had successfully modified the surface of Fe3O4/CNTs, amino groups existed simultaneously on the surface of Fe3O4 and CNTs. The surface modification of CTS did not destroy the crystal structure of Fe3O4/CNTs and CTS-Fe3O4/CNTs had super paramagnetic property. The effects of preparation and reaction conditions of CTS-Fe3O4/CNTs immobilized enzyme were investigated. Compared with free enzyme, the carrier CTS-Fe3O4/CNTs immobilized enzyme restricted conformational change of enzyme and stabilized enzyme relative activities, enhanced higher temperature tolerance, wider pH value, repeated using times and storage time under the optimum conditions of immobilized enzyme.

Metabolic Nano-Machines: Extracellular Vesicles Containing Active Enzymes and Their Contribution to Liver Diseases

The field of extracellular vesicles is growing exponentially because of the important role that these extracellular organelles had on cell to cell communication, triggering a large number of review compilations focusing on different aspects of their biology. Although their importance as effectors or potential biomarkers is well covered, the highlight of extracellular vesicles as carriers of active enzymes which have the capability to transform the surrounding media is less covered by bibliographic studies. In the present review, we focus our attention on enzymatic activity carried by vesicles, with special attention on their contribution to liver conditions. Extracellular vesicles are circulating membrane-bound entities, characterized by a specific cargo. This cargo depends on the parental cell and the stimulus that triggers their release. Interestingly, the cargo includes active enzymes which had the ability of transforming the extracellular environment. Among them, extracellular vesicles derived from hepatocytes harbor specific liver enzymes that may cause an impact in the surrounds and target cells. In this review, we summarize different active enzymes described in extracellular vesicles and we focus on enzymatic activities associated to liver damage. Since their release increases under liver damage conditions, their activity impact could play a role in the pathogenesis of liver and liver-associated diseases. Numerous examples in different liver conditions provided evidence of the potential of extracellular vesicles as therapeutic targets.

Biocatalyst Immobilization by Anchor Peptides on an Additively Manufacturable Material

Additive manufacturing refers to manufacturing methods, which are being used to build up three-dimensional (3D) structures by adding a certain material stepwise onto a support. Nowadays, these manufacturing methods can be more material- and cost-efficient compared to conventional methods and allowing the defined production of a wide variety of 3D structures using computer aided design (CAD). A broad range of materials can be additively manufactured (AM) resulting in specific properties and highly diverse structures making them promising matrices for the utilization as enzyme carriers. The variety of materials offers the possibility to select materials with properties needed for particular biocatalytic processes. This is especially true for hybrid reactor concepts where multiple operations, including catalytic reactions and downstream processes, are combined into a single apparatus. For the enzymatic decarboxylation of ferulic acid, polyethylene terephthalate (PET) has been chosen as an additively manufacturable carrier material for the immobilization of phenolic acid decarboxylase (PAD) from Mycobacterium columbiense. The genetic fusion of PAD with anchor peptides enabled the adsorptive immobilization on PET. Starting from an immobilizate activity of 0.39 ± 0.19 U m–2 and a conversion of 19.2 ± 3.7% after 2 h the optimization of the peptide and spacer sequence between anchor peptide and PAD resulted in immobilizates with activities up to 1.80 ± 0.41 U m–2 and conversions of 59.9 ± 3.9% after 2 h. Moreover, within this study integrating an in situ product removal, enabled by an extraction with n-heptane, the altering of surface hydrophobicity of PET and a conversion of 88.0 ± 3.8% after 2 h could be observed.

Preparation of immobilized lipase on magnetic nanoparticles dialdehyde starch

Dialdehyde starch (DAS) is a kind of modified starch which contains many active aldehyde groups and has good biocompatibility. In this study, magnetic dialdehyde starch nanoparticles were successfully used to immobilize lipase. The lipase was immobilized onto magnetic nanoparticles by using DAS instead of glutaraldehyde as a crosslinker. The parameters like DAS dosage, enzyme concentration and immobilization time were optimized. Enzymatic properties studies exhibited that after DAS cross-linking, the storage stability of the immobilized enzyme reached 82.5%, and the recycling rate reached 53.6%, whereas in case of glutaraldehyde cross linker, it was 79.4% and 46.8%, the former also exhibited better stability and durability. Compared with the free enzyme, the immobilized enzyme indicated higher acid-base tolerance and thermal stability, and had good enzymatic properties. Magnetic dialdehyde starch nanoparticles may have application prospects as an excellent enzyme carrier, which provides a reference for the preparation of other immobilized enzymes with excellent performance.

Fabrication of short peptide cages by interfacial self-assembly on CaCO3 templates

Short peptide cages have great potential applications in drug encapsulation and release because of excellent biocompatibility, biodegradability and multi-functionality originated from their abundant sequences. However, constructing short peptide cages are still a challenge, which is caused by the rapid kinetic and unlimited self-assembly behavior of short peptide in a macroscopical media. Herein, we developed smart procedures to produce short peptide cages, in which porous CaCO3 microparticles were used as enzyme (thermolysin) carriers and applied as catalyzers to promote the condensation reaction of Fmoc-Y and L-NH2 in confined space of water/n-hexane emulsion which limited the self-assembly behavior of Fmoc-YL-NH2 and allowed the peptide to just self-assemble on CaCO3 surface. Intact short peptide cages would be produced after the removing the templates.

Encapsulated functionalized stereocomplex PLA particles: An effective system to support mucolytic enzymes

In this work, the preparation of a novel enzyme carrier based on a polymer multicomponent system was assessed. Indeed, the design of the above system considered several issues that are the need of applying a biodegradable polymer carrier, characterized by a nanometric dimension, thus suitable to diffuse into the dense mucus structure, with functionalities capable of interacting/reacting with enzymes but resistant to enzymatic degradation. The particles were prepared from solutions containing equimolar amount of high-molecular-weight poly(L-lactide) (PLLA) and poly(D-lactide) (PDLA) and by applying the nanoprecipitation method. Dynamic Light Scattering (DLS) measurements allowed to establish the optimal preparation conditions to obtain polymer particles characterized by diameters lower than 1 μm, which dimensions were confirmed by Field Emission Scanning Electron Microscope (FE-SEM) analysis. In order to produce surface functionalization, necessary for anchoring enzymes, the stereocomplexed particles, whose structuration was confirmed by Differential Scanning Calorimetry (DSC) measurements, underwent an amminolysis reaction by using a diamine as reactant. The treated particles were characterized by means of FE-SEM, Fourier-Transform Infrared Spectroscopy (FTIR), DLS and zeta potential measurements and their characteristics were compared with those of the neat PLLA/PDLA particles. The degree of functionalization turned out to depend on the applied conditions, it increasing by enhancing the reaction time. The activity of enzymes, i.e. papain and alginate lyase, anchored to the particles, was evaluated by Quartz Crystal Microbalance (QCM) and UV measurements. Moreover, with the aim at exploiting the material for an inhalation administration, a method to encapsulate the enzyme-particles systems was assessed. Conversely to free enzymes, the developed systems were found to be capable of diminishing the viscosity of two hydrogels, ad hoc prepared and based on the main constituents of the real mucus.

Fabrication of paper-based enzyme immobilized microarray by 3D-printing technique for screening \u03b1-glucosidase inhibitors in mulberry leaves and lotus leaves

BackgroundThe discovery of bioactive compounds in traditional Chinese medicine (TCM) has become an important field in TCM modernization. Ligand fishing is a suitable method for discovery of bioactive compounds in complex mixtures such as TCM with high selectivity. Because of unique advantage of low cost and convenience, paper-based microdevices can be good carriers for enzyme immobilized ligand fishing.MethodsAs an important enzyme for glucose metabolism, α-glucosidase was immobilized on polycaprolactone–chitosan-modified paper to prepare the microdevice with unique microfluid structure generated by 3D printing technology, which can be easily applied to screen active compounds in herbal extracts. The preparation conditions of the paper microarray were optimized. The activity of immobilized α-glucosidase was verified by colorimetric reactions which can be easily monitored by cellphone. The paper microarray with α-glucosidase immobilized was used to screen active compounds in the water extracts of mulberry leaves and lotus leaves.ResultsSeveral key parameters including Na2CO3 solution concentration, Na2CO3 solution volume, glutaraldehyde concentration, crosslinking time of glutaraldehyde and time of α-glucosidase immobilization were optimized. The proposed paper-based microarray was successfully applied in screening active compounds in two herbal extracts. Four compounds including chlorogenic acid, quercetin-3-O-glucuronide, isoquercetin, and quercetin were identified as α-glucosidase inhibitors. The compounds with significant non-specific adsorption caused by chitosan, such as isoquercitrin, astragalin, quercetin, were also found to be active compounds.ConclusionsAn enzyme immobilized paper microarray was designed and fabricated in this work. Polycaprolactone and chitosan were used to modify filter paper to prepare paper microarrays. Parameters of paper device preparation were optimized. Our findings suggested that 3D-printing paper-based microarrays can be a simple and low-cost approach for discovery of active compounds of TCM.

Facile Fabrication of a Novel and Reusable 3D Laccase Reactor for Efficient Removal of Pollutants from Wastewater

An ultra simple 3D bioreactor was prepared via Fe3+ linking to primary amines of melamine sponge (MeS) for cooperative complex formation with enzyme. Laccase worked as a model of enzyme and no chemical modification was employed during the material preparation. The 3D structure of MeS can enhance the enzyme loading amount up to 180 µg/mg, albeit MeS as monolithic material for enzyme immobilization is rather unexplored. The 3D bioreactor (MeS@Fe3+@laccase) also improved the catalytic efficiency and showed excellent reusability, stability, and reproducibility. In this study, the as-prepared bioreactor (10 mg) was successfully applied to the degradation of dyes and micropollutants, which has shown higher efficiency than free laccase in the large-scale decolorization of dyes and showed up to almost 100% removal of 2,4,6-trichlorophenol (2,4,6-TCP) and sulfisoxazole. Therefore, the MeS@Fe3+ with low cost, good stability and easy separation can be employed as a new enzyme carrier such as trypsin and lipase in biotechnological applications.

Surface Microstructure Regulation of Porous Polymer Microspheres by Volume Contraction of Phase Separation Process in Traditional Suspension Polymerization System.

In the present work, the suspension polymerization method is used for the preparation of porous polymer microspheres with different surface morphology, and the preparation mechanism is systematically expounded. The morphology results show that the smooth, convex, and wrinkled microspheres could be controlled by adjusting the ratio of monomer to porogens. The micelles forming the framework support the "Eggshell," and its size and shape directly affect the morphology of "Eggshell." The addition of monomers (GMA), whose polymer has low glass transition temperature (Tg ), is important for the formation of wrinkled morphology. The amount of toluene and polydimethylsiloxane also affects the surface morphology of microspheres. In addition, the effect of polydimethylsiloxane is also more significant. The preparation process of the wrinkled P(GMA-St-EGDA) microspheres with abundant epoxy groups can be amplified. The morphology of the material prepared in the 100 L reactor is well maintained, and the yield in the size range of 80-160 µm is more than 80%. The surface wrinkled porous polymer microspheres have potential applications in the fields of enzyme carrier, separation and purification, and light scattering.

Valued Applications of Lignin Nanoparticles

Technical lignins from the biorefinery, pulp, and paper industries are largely underutilized, even though this aromatic and randomly structured biopolymer could be an interesting raw material for advanced applications in addition to bulk daily goods. Recently, colloidal lignin particles (CLPs) have gained much of the research interests due to the attractive multi-functional properties of the biopolymer. Utilization of lignin in nanoparticulate morphology resolves most of the drawbacks when using lignin (heterogeneity and low solubility). Stable lignin nanodispersions in different formulations is an attractive method to prepare tailored nanobiomaterials. Potential value-added applications include adhesives for wound sealing, edible coatings for foods, fiber modification for textiles to improve adhesion, hydrophobicity, antimicrobial and anti-oxidative properties of the material, as recently shown using chamois and nanocellulose model matrixes. Moreover, CLPs could be used as carriers for enzymes, emulsifiers for colloids, adsorbents in water purification and controlled-release vectors for drugs and pesticides. In this contribution the recent advances are highlighted.

Effects of Environmental Conditions on High-Yield Magnetosome Production by Magnetospirillum gryphiswaldense MSR-1

Background:Magnetotactic bacteria are a heterogeneous group of Gram-negative prokaryote cells that produce linear chains of magnetic particles called magnetosomes, intracellular organelles composed of magnetic iron particles. Many important applications have been defined for magnetic nanoparticles in biotechnology, such as cell separation applications, as well as acting as carriers of enzymes, antibodies, or anti-cancer drugs. Since the bacterial growth is difficult and the yield of magnetosome production is low, the application of magnetosome has not been developed on a commercial scale.Methods:Magnetospirillum gryphiswaldense strain MSR-1 was used in a modified current culture medium supplemented by different concentrations of oxygen, iron, carbon, and nitrogen, to increase the yield of magnetosomes.Results:Our improved MSR-1 culture medium increased magnetosome yield, magnetosome number per bacterial cell, magnetic response, and bacterial cell growth yield significantly. The yield of magnetosome increased approximately four times. The optimized culture medium containing 25 mM of Na-pyruvate, 40 mM of NaNO3, 200 µM of ferrous sulfate, and 5-10 ppm of dissolved oxygen (DO) resulted in 186.67 mg of magnetosome per liter of culture medium. The iron uptake increased significantly, and the magnetic response of the bacteria to the magnetic field was higher than threefold as compared to the previously reported procedures.Conclusion:This technique not only decreases the cultivation time but also reduces the production cost. In this modified method, the iron and DO are the major factors affecting the production of magnetosome by M. gryphiswaldense strain MSR-1. However, refining this technique will enable a further yield of magnetosome and cell density.

Core–Shell Magnetic Mesoporous Silica Microspheres with Large Mesopores for Enzyme Immobilization in Biocatalysis

Magnetic mesoporous silica microspheres with core-shell structure and large pores are highly desired in macromolecules delivery and biocatalysis, biospeparation, and adsorption. In this work, a controllable solvent evaporation induced solution-phase interface co-assembly approach was developed to synthesize core-shell structural magnetic mesoporous silica microspheres with ultralarge mesopore size (denoted as LP-MMS). The synthesis was achieved by employing large-molecular-weight amphiphilic block copolymers poly(ethylene oxide)- block-poly(methyl methacrylate) (PEO- b-PMMA) and small surfactant cetyltrimethylammonium bromide as co-templates, which can co-assemble with silica source in tetrahydrofuran/water solutions. The obtained LP-MMS microspheres possess uniform rasberry-like morphology with a diameter of 600 nm, large primary spherical mesopores (ca. 36 nm), large specific surface area (348 m2/g), high specific pore volume (0.59 cm3/g), and fast magnetic responsivity with high magnetization (15.9 emu/g). The mesopore morphology can be transformed from spherical to cylindrical through introducing a shearing force during the interfacial co-assembly in the synthesis system. The designed LP-MMS microspheres turn out to be good carriers for enzyme (trypsin) immobilization with a high loading capacity of 80 μg/mg and demonstrate excellent biocatalysis efficiency up to 99.1% for protein digestion within 30 min and good recycling stability with negligible decay in digestion efficiency after reuse for five times.

Metallic Nanoparticles Obtained via “Green” Synthesis as a Platform for Biosensor Construction

Novel nanomaterials, including metallic nanoparticles obtained via green synthesis (gNPs), have a great potential for application in biotechnology, industry and medicine. The special role of gNPs is related to antibacterial agents, fluorescent markers and carriers for drug delivery. However, application of gNPs for construction of amperometric biosensors (ABSs) is not well documented. The aim of the current research was to study potential advantages of using gNPs in biosensorics. The extracellular metabolites of the yeast Ogataea polymorpha were used as reducing agents for obtaining gNPs from the corresponding inorganic ions. Several gNPs were synthesized, characterized and tested as enzyme carriers on the surface of graphite electrodes (GEs). The most effective were Pd-based gNPs (gPdNPs), and these were studied further and applied for construction of laccase- and alcohol oxidase (AO)-based ABSs. AO/GE, AO-gPdNPs/GE, laccase/GE and laccase-gPdNPs/GE were obtained, and their analytical characteristics were studied. Both gPdNPs-modified ABSs were found to have broader linear ranges and higher storage stabilities than control electrodes, although they are less sensitive toward corresponding substrates. We thus conclude that gPdNPs may be promising for construction of ABSs for enzymes with very high affinities to their substrates.

Lipid Sponge-Phase Nanoparticles as Enzyme Carriers - Structure and Intermolecular Interaction Controlling the Enzyme Inclusion

Lipid Sponge-Phase Nanoparticles as Enzyme Carriers - Structure and Intermolecular Interaction Controlling the Enzyme Inclusion

Preparation and anti-tumor efficiency evaluation of bacterial magnetosome-anti-4-1BB antibody complex: Bacterial magnetosome as antibody carriers isolated from Magnetospirillum gryphiswaldense.

Bacterial magnetosomes (BMs) are used as carriers for antibodies, enzymes, and nucleic acids. This study aimed to demonstrate the clinical utility of BMs derived from Magnetospirillum gryphiswaldense for use in anti-tumor immunotherapy. Bis(sulfosuccinimidyl) suberate (BS3) was used to prepare BM-anti-4-1BB antibody complexes. We used syngeneic TC-1 mouse models of cancer to investigate whether BMs combined with an anti-4-1BB agonistic antibodies could enhance the therapeutic effects of anti-4-1BB antibodies in localized disease settings. Anti-4-1BB antibodies were combined with purified BMs at a concentration of 168 mg antibody per milligram BM (mg IgG/mg BM) using BS3. The anti-4-1BB antibody-coupled BMs (BM-Ab complexes) and control BMs displayed similar morphologies and measurements when examined by transmission electron microscope (TEM). In a mouse tumor model, intravenous injection of BM-Abs combined with magnetic treatment resulted in greater tumor protection than did other treatment methods (P < 0.05). These results demonstrate the in vivo anti-tumor properties of BM-Abs complexes. The coupling of anti-4-1BB antibodies to magnetosomes may have potential for clinical application to antitumor antibody therapy.

Critical Features for Mesoporous Silica Nanoparticles Encapsulated into Erythrocytes.

Mesoporous silica nanoparticles (MSNs) hold great potential as a versatile platform for biomedical applications, especially drug delivery. However, evidence shows that MSNs even when PEGylated are rapidly cleared from the bloodstream by the monocyte phagocytic system. Erythrocytes, also called red blood cells (RBCs), can serve as biocompatible carriers of various bioactive substances, including drugs, enzymes, and peptides. In this work, we synthesize a series of fluorescent PEGylated MSNs with different synthetic diameters ranging from 10 to 200 nm and investigate the size effect on their encapsulation in human RBCs (hRBCs) by a hypotonic dialysis-based method. According to fluorescence images and flow cytometry analyses, we demonstrated that a hydrodynamic diameter below 30 nm is critical for efficient MSN encapsulation. Confocal microscopy and scanning electron microscopy images further confirmed that PEGylated MSNs were successfully embedded inside RBC. PEGylation serves an important role not only for stabilizing MSNs in biological milieu but also for reducing significant hemolysis caused by bare MSNs and thus for successful encapsulation. In addition to PEGylation, we further introduce positively charged functional groups onto the MSNs to show that nanoparticle-encapsulated hRBCs could serve as depots for delivering biological molecules through electrostatic attraction or chemical conjugation with MSNs. Also, we verify the existence of CD47 membrane protein, a marker of self, on the nanoparticle-encapsulated hRBCs and assess its ability of circulation in the blood, which could act as a circulation reservoir for delivering pharmacological substances through an osmosis-based method with MSNs.