Critical Features for Mesoporous Silica Nanoparticles Encapsulated

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Biological and Medical Applications of Materials and Interfaces

Critical Features for Mesoporous Silica Nanoparticles Encapsulated into Erythrocytes Zih An Chen, Sihan Wu, Peilin Chen, Yi-Ping Chen, and Chung-Yuan Mou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18434 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 15, 2019

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ACS Applied Materials & Interfaces

Critical Features for Mesoporous Silica Nanoparticles Encapsulated into Erythrocytes Zih-An Chen 1, Si-Han Wu 2,3, Peilin Chen4, Yi-Ping Chen*,2,3 and Chung-Yuan Mou*,1,2 1Department

of Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Road,

Taipei 10617, Taiwan 2Graduate Institute of Nanomedicine and Medical Engineering, Taipei Medical University, No. 250, Wu Xinyi Street, Taipei 11031, Taiwan. 3International

PhD Program in Biomedical Engineering, College of Biomedical

Engineering, Taipei Medical University, Taipei 11031, Taiwan 4Research Center for Applied Sciences, Academia Sinica, 128 Sec. 2, Academia Rd., Nankang, Taipei 11529, Taiwan. E-mail: [email protected] KEYWORDS Mesoporous silica nanoparticles, PEGylated nanoparticle encapsulation, red blood cells, hypotonic dialysis based method

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ABSTRACT Mesoporous silica nanoparticles (MSNs) hold great potential as a versatile platform for biomedical applications, especially drug delivery. However, evidence shows 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 nm 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 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 allow 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 assessed the ability of circulation in the blood, which could act as a circulation reservoir for delivering pharmacological substances through osmosis-based method with MSNs.


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1. INTRODUCTION

This paper reports on a combination of nanoparticle and red blood cell (RBC) as a biomedical carrier/delivery system. The nanoparticles are mesoporous silica nanoparticle (MSN) system which has been studied extensively in recent years as a biomedical carrier. In previous reports on nanomaterials, mesoporous silica nanoparticles (MSNs) have shown potential for controlled release, drug delivery, and biomedical sensing applications1-2. However, injected MSNs suffer from rapid clearance by the mononuclear phagocytic system (MPS) located primarily in the liver and spleen, thereby limiting the dose reaching the disease site3. Even with careful surface functionalization of stealth agent, such as PEG, their blood circulation time is often no more than 24 hours. Human red blood cells (hRBCs), with a long blood circulation lifetime (up to 120 days4), are seen as a favorable alternative to other widely used delivery systems to extend drug circulation time4-6. RBC can be used to improve vascular and systemic delivery of pharmacological substances either encapsulated within the RBC, or on its surface7.

RBC-hitchhiking, in which nanoparticles adsorbed onto the RBCs to, improves delivery for a wide range and provides target-organ specificity8. However, the hitchhiking approach has the problem of rapidly losing the carried nanoparticle upon squeezing of RBC through capillary vessels. Hence its circulation time is still limited. For example, it was demonstrated that polymeric particles attached to mouse RBCs via noncovalent adhesion remained in circulation for about only 10 h11, 12. In another approach, nanoparticles coated with natural RBC membranes also exhibited a long-circulating cargo delivery9-10. Despite considerable interest in their biomedical applications, relatively few


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studies have been published on coating MSNs with natural erythrocyte membranes, including both membrane lipids and associated membrane proteins for long-circulating cargo delivery. This is partly due to the complicated and tedious preparation procedure. More fundamentally, nanoparticle’s very small external surface area limits the number of surface proteins and receptors that can be carrier (often less than 1) on each particle. Recently, it has been demonstrated that RBCs can be successfully utilized to encapsulate magnetic nanoparticles, which suggested that a preferential entrapment of nanoparticles with hydrodynamic diameter below 60 nm occurs by size-selection through the osmosis-based method11. Also in previous article reports the encapsulation of different magnetic nanoparticles into human erythrocytes to increase their blood circulation time12. Thus, we propose encapsulating MSNs into the interior of the RBCs would be a better approach than those previously proposed where nanoparticles are adhered onto the RBC through non-covalent attachment when circulating through the blood vessels13-14. Several methods have been reported to encapsulate drugs or other agents into erythrocytes4. Of these methods, hypotonic dialysis based method was often used to load enzymes and lipids into RBCs via an osmotic gradient that leads to opening transient pores in the membrane to exchange intracellular hemoglobin for the extracellular components4,

15.

This method of hypotonic treatment allowed efficient

encapsulation with nanoparticles into the RBCs. We may thus have the advantages of extended half-lives, sustained drug release, and limited immunogenicity and cytotoxicity in a nanoparticle/RBC encapsulation system.

In this work we synthesize a series of fluorescent PEGylated MSNs with different synthetic diameters and investigate the size effect on their encapsulation in RBCs by


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hypotonic dialysis. We demonstrated that a hydrodynamic diameter below 30 nm is critical for sufficient RBC encapsulation, which successfully embedded into RBCs. PEGylation serves important roles not only for stabilizing MSNs in biological milieu but also reducing significant hemolysis caused by bare MSNs. Also, we confirm that existence of CD47 membrane protein on the engineered MSNs-encapsulated RBCs which increased the ability of circulation in the blood. The engineered MSNs-encapsulated RBCs could be a reservoir for delivering pharmacological substances through osmosisbased method with MSNs.


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2. EXPERIMENTAL SECTION 2.1 Chemicals and Reagents All chemicals were used without additional purification. Fluorescein isothiocyanatedextran, molecular weight, 70 kDa (FITC-dextran), rhodamine B isothiocyanate (RITC), adenine 99+%, inosine 99%, adenosine 5’-triphosphate (ATP) disodium salt dihydrate and sodium pyruvate were purchased from Sigma-Aldrich (Milwaukee, WI). Cetyltrimethylammonium bromide (CTAB, 99+%) and tetraethyl orthosilicate (TEOS, 98%), tetramethyl orthosilicate (TMOS, 98%), ammonium hydroxide (NH4OH, 28-30 wt% as NH3), hydrochloric acid (for analysis, fuming, 37% solution in water), HEPES (99%, for biochemistry), D(+)-Glucose, sodium chloride (99.5%, for analysis), potassium chloride (99+%) and magnesium chloride hexahydroxy (99+%, ACS reagent) were obtained

from

ACROS

9propyl]trimethoxysilane,

tech-90

Organics™. (PEG-silane,

Trimethoxysilylpropyl-N,N,N-trimethylammonium

2-[Methoxy(polyethyleneoxy)6M.W. chloride

460-590

g/mol),

(TA-silane,

50%

Nin

methanol) and trimethoxysilylpropyl modified (polyethylenimine) (PEI-silane, 50% in isopropanol) were acquired from Gelest (Morrisville, PA). Ammonium hydroxide (NH4OH, 35 wt% as NH3), sodium bicarbonate (powder/certified ACS) and sodium phosphate monobasic dihydrate (crystalline/certified) were purchased from Fisher Scientific. DiD (1, 1'-Dioctadecyl-3, 3, 3', 3'-tetramethylindodicarbocyanine, 4chlorobenzenesulfonate salt) was obtained from Thermo Fisher Scientific. 99.5% and 95% ethanol were purchased from Choneye Pure Chemicals. Dulbecco’s phosphatebuffered saline (PBS) was obtained from Invitrogen. Anti-CD47 antibody [B6H12], prediluted (FITC) obtained from Abcam.


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2.2 Synthesis of PEGylated Fluorescent MSNs PEGylated MSNs incorporated with fluorescent dye (RITC) were synthesized and particle size (25, 50 and 200 nm) controlled by adjusting ammonia concentration at the desired temperature in a previous study16-18. The obtained particle solution (~50 mL) underwent a hydrothermal treatment (70℃ and 90℃) for two days. Hydrochloric acid (37%) was used to remove the surfactant templates. The samples collected by centrifugation and stored in 99.5% ethanol (denoted as RMSN-PEG). 2.3 Synthesis of Ultra-Small Size RMSN-PEG (RMSN-PEG-10) As detailed in previously reported19-20, the ultra-small diameter particle synthesis with TMOS as silica source in aqueous solution in the presence of CTAB. PEG-silane (0.21 mmol) was added directly into the synthesis batch to quench particle formation. Otherwise, the removing surfactant procedure was similar to RMSN-PEG. 2.4 Surface Modification of RMSN-PEG-10 The synthesis procedure was similar to RMSN-PEG-10. For ultra-small size RMSNPEG-TA and RMSN-PEG-PEI synthesis, the only difference was adding TA-silane (0.1 mmol) and PEI-silane (0.002 mmol), respectively. After addition of surface modification, the surfactant templates of particles removed by hydrochloric acid and collected by centrifugation and stored in 99.5% ethanol. 2.5 Hemolysis Assay The hemolysis method described in a previous study13-14, 16. Human RBCs were obtained from a volunteer and isolated by centrifugation from removing excess plasma for several times. The diluted RBC suspension was added to MSN solutions at a sequence of concentrations (from 6.25 to 8000 μg/mL). After placing in the dark at room temperature


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for 3 h, the samples were centrifuged and the supernatant was taken for absorbance measurement (Abs 540 nm). The percent hemolysis of the RBCs was calculated using the formula in previous study13-14, 16. 2.6 Loading Procedure with Hypotonic Dialysis Based Method All these procedures came from previous reports11-12. Human RBCs (hematocrit 33%) were dialyzed in the presence of 8 mg RMSN-PEG for 2 h at 4°C using a tube with a 12– 14 kDa cut-off in a dialysis buffer (the osmolarity of dialysis buffer was 100 mOsm). Resealing of the RBCs was obtained by adding PIGPA-NaCl buffer for 1 h at 25°C. The resealed cells were recovered by centrifugation and washed several times with HEPES buffer to remove unbound particles, which stored at 4℃ in PBS (designed as the engineered RMSN-RBCs). 2.7 Red Blood Cell Indices To evaluate the size and hemoglobin content of the native RBCs as well as engineered RMSN-RBCs, mean corpuscular volume (MCV), mean hemoglobin concentration (MCH) and mean corpuscular hemoglobin concentration (MCHC) were measured with an automated hemocytometer (IDEXX ProCyte Dx). 2.8 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Analysis The amount of silica in the engineered RMSN-RBCs (108 cells) was analyzed by ICP-MS (NexION 2000, Perkin Elmer). The intensity of this emission is a measure for the amount of Si present. Calibration was performed by comparison with intensities produced by standard solutions. 2.9 Fluorescent Imaging and Flow Cytometry Analysis


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The fluorescence images of the engineered RMSN-RBCs observed in 24-well plates by using fluorescence microscope (Olympus) and quantitative data were analysis with ﬂow cytometry (BD Biosciences). The distribution of cell populations with particles were assessed using their fluorescence intensity and mean fluorescence intensity in the PE-A (RITC, λem = 578 nm) and FITC-A (FITC, λem = 518 nm) gate. 2.10 Confocal Imaging Examination For confirming the truth of RMSN-PEG distributed inside the RBC, labeling membrane with lipophilic tracers dye reagent (DiD λem = 665 nm), the engineered RMSN-RBCs dispersed in 1 mL of PBS containing 1 μL of DiD stock solution (2.5 mg/mL) stirring for 1 h at 37°C and washed several times to remove unbinding dye reagent. The DiD labeling RMSN-RBCs were fixed on poly-L-lysine-coated coverslips for 2 h. After being washed, the sample placed in 6-well plates for observation. The fluorescent imaging obtained with a confocal laser scanning microscope (LSM 880, Zeiss). 2.11 Scanning Electron Microscopy (SEM) Image Observation All these procedures followed from previous reports11-12, 14. The engineered RMSN-RBCs fixed by incubating a 2.5% glutaraldehyde solution on poly-L-lysine-coated coverslips at 4°C for 12 h. Following post-fixation with 1% osmium tetroxide in PBS for 1 h at 4°C, the samples were dehydrated in increasing concentrations of ethanol (50, 70, 95, 99.5 and 100%) for 15 min each. After alcohol dehydration was performed, specimens were coated with Au before viewing under scanning electron microscope (S-4800 Field Emission). 2.12 Identification CD47 on the Engineered RMSN-RBCs The distribution of CD47 retained on the engineered RMSN-RBCs (106 cells) was accomplished via the labeling with commercial antibody, anti-CD47-FITC ([B6H12],


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Abcam) in the dark for 1 h at 4℃. Non-internalized RBCs were washed away with PBS. The engineered RBCs were then scraped off the tubes and analyzed with flow cytometry. 2.13 Circulation Imaging of the Engineered RMSN-RBCs in NOD/SCID Mice To study the systemic circulation time, the engineered RMSN-RBCs were injected intravenously (108 cells) into NOD/SCID mice (BioLASCO Taiwan). The actual dynamic images were obtained from earlobe of mice with multi-photon laser scanning microscope (FVMPE-RS, Olympus) at various time points (0.5, 1, 2 and 3 h). 2.14 Characterization Transmission electron microscopy (TEM) images were taken on a JEOL 1200 EXII with a 120 kV voltage. TEM samples were prepared by air-dried drop (10 μL) of nanoparticle solution (99.5% ethanol) on copper grids. Nanoparticle sizes counted in the micrographs using Sigma Scan Pro 5.0 software (Ashburn, VA). The samples for dynamic light scattering (DLS) measurements were suspended in various solvents (DI water, PBS, and HEPES buffer) for each sample to get at least three runs by a Nano ZS90 laser particle analyzer (Malvern Instruments, UK). For zeta potential, each sample was measured in HEPES buffer (pH 7.4). X-ray powder diffraction was measured on a X' Pert PRO (PANalytical) powder using Cu Kα1 radiation (λ=1.54 Å) and interplanar spacing calculated from Bragg formulation. The N2 adsorption-desorption isotherms of RMSNPEG obtained from on a Micrometrics ASAP 2020 (Norcross, GA). The surface area and pore size were calculated using the Brunauer-Emmet-Teller (BET) equation and standard Barrett-Joyer-Halenda (BJH) method. Thermogravimetric analysis (TGA) was recorded from 150 to 800℃ on a thermal analyzer heating rate of 10℃/min in an air purge of 40 mL/min.


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3. RESULTS and DISCUSSION 3.1 Characterization of RMSN-PEG We synthesized PEGylated MSNs of four distinct diameters and their mean particle sizes were counted for each size population (12.7 ± 1.5, 23.2 ± 5.2, 38.8 ± 6.7 and 172.5 ± 15 nm) observed in TEM images (Fig. 1a). Dynamic light scattering (DLS) shows that the number distribution of RMSN-PEG in aqueous solutions with various sizes (Fig. 1c) correspond to the mean particle sizes distributions (Fig. 1b) observed from the TEM images. The functionalization of PEG to RMSN can prevent aggregation of MSNs in solution. It also can minimize nonspecific binding on the surface of RBCs3. The zeta potential reveals weakly negative-charged RMSN in HEPES buffer (Table S2) due to deprotonation of the silanol group. To further compare the structural properties of RMSN-PEG of various sizes, we performed powder XRD, N2 adsorption-desorption isotherms measurements and thermogravimetric (TGA) analysis (Fig. S1 a-c and Table S1). The XRD patterns for RMSN-PEG with various sizes show that a single broad (100) peak and analogous inter-planar spacing (except for RMSN-PEG-10) which is characteristic of 2D hexagonally ordered structure21 (Fig S1a). The N2 adsorptiondesorption isotherms exhibited a type IV isotherm according to IUPAC classification, which had a total surface area ranging from 610 to 887 m2/g were calculated using the Brunauer-Emmet-Teller (BET) equation and similar pore size distribution curves with a pore size of about 2.0 nm by the Barrett–Joyner–Halenda (BJH) method (textural data shown in Table S1). It is worth mentioning that RMSN-PEG-10 without plasma treatment did not appear to have any accessible structural order and surface area, which we attribute to the PEG chains blocking on surface of particles. Thus its porosity was


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measured after calcination19-20,

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. Instead of RMSN-PEG-10, we employ the N2

adsorption-desorption curve of the bare-RMSN (without PEGylation) in Fig. S1b. The TGA analysis reveals similar curves from 150 to 800ºC of the RMSN-PEG in various sizes (Fig. S1c)

Figure 1. (a) TEM images of RMSN-PEG with various diameters: (i) RMSN-PEG-10, (ii) RMSN-PEG-25, (iii) RMSN-PEG-50 and (iv) RMSN-PEG-200. (b) Particle size distributions of RMSN-PEG with various sizes counting from TEM micrographs. (c) The size distribution of RMSN-PEG with various sizes measured in dynamic light scattering.


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3.2 Hemolytic Activity of MSNs The hemolysis assay was applied to assess the cytotoxic effect of nanoparticles on human RBC since silica materials might cause membrane damage to RBCs14, 16, 23. Owing to the optical absorption (λmax = 540 nm) of hemoglobin in the RBCs overlapping with RITC fluorescent dye (λmax = 543 nm), mesoporous silica nanoparticles synthesized without the RITC dye (denoted as MSNs) were employed for hemolysis study. The PEGylated MSNs exhibited similar morphology and hydrodynamic diameters to the fluorescent MSN counterparts (Fig. S2 and Table S2). As shown in Fig. 2, bare-MSNs (without PEGylation) caused higher hemolytic activity than PEGylated MSNs. This result demonstrates that PEGylation played important roles not only for enhancing the MSN stability but also for reducing significant hemolysis.


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Figure 2. (a-b) Percentage and photographs of hemolysis of RBCs in the presence of bare-MSN and MSN-PEG with four sizes at different concentrations ranging from 6.25 to 8000 μg/mL for 3 h. Data represent the mean from at least three independent experiments. The presence of red hemoglobin in the supernatant indicates damaged RBCs. D.I. water (+) and PBS (-) act as positive and negative controls, respectively. The percent hemolysis of the RBCs was calculated using the following formula: (sample absorbance-negative control absorbance) / (positive control absorbance-negative control absorbance) x 100%.


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3.3 Optical and Fluorescent Imaging of the Engineered RMSN-RBCs The method of encapsulation of nanoparticles into RBCs is hypotonic dialysis in which transiently opening of pores on the cell membrane results in the exchange of intracellular hemoglobin for the extracellular components4-5,

11-12.

Hypotonic dialysis

based on the exposure of RBCs to the substance to be encapsulated contained in a dialysis bag to the hypotonic buffer, so that nanoparticles can enter RBCs and the pores were resealed in an isotonic buffer24-25. To confirm the integrity of the hypotonic dialysis method in this study, FITC-dextran acts as a positive control26 which shows the emission of green fluorescence on the RBCs (denoted as dextran@RBCs) in Fig. S4. RMSN-PEG10 exhibit successful incorporation into the RBCs compared to the larger sizes of particles (25, 50 and 200 nm), which indicated RBCs membrane pores allow a preferential embedding of nanoparticles with smaller sizes (Fig. 3). Actually RMSNPEG-10 suspended in HEPES buffer have a Z-average = 26.4 ± 0.75 nm due to hydration and some slight aggregation26-27 (Table S2), which meant the selection of the particle sizes of RMSN for encapsulation in RBCs

by hypotonic treatment requires a

hydrodynamic diameter below about 30 nm. To verify the structural integrity of the engineered RMSN-RBCs, the interior of the cells were examined via confocal and scanning electron microscopy (SEM) imaging. DiD dye reagent was used as a lipophilic tracer used to fluorescently label cell membranes to visualize the morphology of RBC. The dextran@RBCs show typical annular shape of green fluorescence in a section view of the upper edge on the RBC, which illustrates FITC-dextran internalized into the RBC (Fig. S5); similarly Fig. 4 exhibits co-localization fluorescence (orange) of RMSN-PEG10 (yellow) and RBCs (red), which demonstrates RMSN-PEG-10 indeed internalized into


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the RBCs. On the contrary, RMSN-PEG with larger sizes (25, 50 and 200 nm) reveal sporadic fluorescence on the RBC, which indicated large particles cannot be encapsulation into the interior of RBCs. These resulting imaging confirm that hydrodynamic diameter plays the role of encapsulation inside the RBC. To investigate the relationship of RMSN-PEG with the surface of the RBCs, observed the surface morphology of engineered RMSN-RBCs via using SEM imaging shows that RMSNPEG-10 encapsulation into RBCs have normal cell morphology and no significant differences with respect to native RBCs and dextran@RBCs and no particles on the smooth surface of the cell differ to particles with large sizes (Fig. 5 and Fig. S6), which indicated RMSN-PEG with large sizes, might be aggregation and attachment on the surface of RBC. From optical and fluorescent imaging, RMSN-PEG-10 is performed superior encapsulation into the RBC which denoted as RMSN-PEG@RBCs.


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Figure 3. Optical and fluorescent imaging of the engineered RMSN-RBCs, which is (a) native RBCs in HEPES buffer and processed with RMSN-PEG with diameter (b) 10 nm, (c) 25 nm, (d) 50 nm and (e) 200 nm by using the hypotonic method. From top to bottom of imaging channel, BF: bright field, RITC: detecting the RBCs containing RITC dyecontaining particles and RITC/BF: merge BF and RITC imaging. All of the images were taken at 40× original magnification.


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Figure 4.

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Confocal imaging of the engineered RMSN-RBCs. DiD was used as a

fluroscent membrane dye and RITC was used to visualize MSNs. Confocal images of (a) native RBCs, RMSN-PEG with diameter (b) 10 nm, (c) 25 nm, (d) 50 nm and (e) 200 nm encapsulation into RBCs by hypotonic dialysis based method. DiD: fluroscent membrane of RBCs, RITC: detecting the RBCs containing RITC dye-containing particles and RITC/DiD: merge BF and DiD imaging.

Figure 5. SEM imaging of the engineered RMSN-RBCs, from left to right represents (a) native RBCs, RMSN-PEG with diameter (b) 10 nm, (c) 25 nm, (d) 50 nm and (e) 200 nm encapsulation into RBCs by hypotonic method. The features highlighted with white squares indicate particles attached on the surface of RBCs.


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3.4 Optical and Fluorescent Imaging of the modified RMSN-PEG@RBCs To explore other surface-modified nanoparticles for RBC encapsulation, RMSNPEG-10 was functionalized with TA-silane containing quaternary ammonium (RMSNPEG-TA) and PEI-silane composed of primary, secondary and tertiary amines (RMSNPEG-PEI). These two types of surface modification have different interactions with cells and tissues due to relative exposure of surface amines, as identified in previous studies1718, 28.

Both the modified RMSN-PEG@RBCs exhibit superior encapsulation and maintain

normal concave shape of RBCs (Fig. 6), which demonstrated surface-modified nanoparticles have no effect on encapsulation into the RBCs. Although modified RMSNPEG-10 show multi-peaks in DLS study and a larger hydrodynamic diameter due to small degree of aggregation29 (Fig. S3a and Table S2), the number distributions emphasize that the smaller particles in the distribution that indicated modified RMSNPEG-10 could still pass though RBCs’ membrane pores

allowing a preferential

embedding of nanoparticles (Fig. S3b). Regardless, functionalized RMSN-PEG-10 is also performed superiorly for encapsulation through the osmosis-based process.


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Figure 6. Optical and fluorescent imaging of the surface-modified RMSN-PEG@RBCs, which is (a) native RBCs treated with (b) bare-RMSN, (c) RMSN-PEG, (d) RMSN-PEGPEI and (e) RMSN-PEG-TA with ultra-small size 10 nm by using the hypotonic dialysis based method. BF: bright field, RITC: fluorescent MSNs and RITC/BF: merge BF and RITC imaging. All of the images were taken at 40× original magnification.


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3.5 Cell Integrity of the Engineered RBCs In order to evaluate the cell integrity of the engineered RBCs, red blood cell indices including MCV, MCH and MCHC were measured with an automated hemocytometer (Table S3). After the osmosis-based procedure of encapsulation, all cell recovery percentage of the engineered RBCs and native RBCs(with or without RMSN) are similar and ranging between 70% and 80%, indicating the compromised cell integrity was mainly due to the osmotic pressure change but not the presence of RMSN. The information of RBC size and granularity parameters were according to the forward and side scattering of light (FSC, SSC, respectively) on the flow cytometry (Fig S7). The FSC parameter on the x-axis of the light scattering plot the RBCs size can be measured at around 6-8 μm. Along the y-axis, the SSC parameter represents a measurement of the amount of the laser beam that bounces off of granularity inside of the RBCs. The RBCs with hypotonic treatment (denoted as dialyzed RBCs) show a shift on FSC/SSC plot compared to the native RBCs. These results analyzed by hemocytometer were consistent with that obtained by flow cytometry, where FSC/SSC population shifted compared to its native RBC, indicating that the contents of the RBCs have been decreased after the osmosis-based procedure of encapsulation. 3.6 Determination of the Amount of RMSNs in the Engineered RBCs Silica concentrations of the engineered RBCs measured by ICP-MS are shown in Table S4. The results show the mass of silica uptake in RMSN-RBC-10 (0.085 pg/cell), as determined by subtracting silicon content of the native RBC, was approximately 2 times that of RMSN-RBC-25, 2.5 times that of RMSN-RBC-50. However, ICP-MS


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analysis of RMSN-RBC-200 was not applicable because of the silica encapsulation was too low. 3.7 Flow Cytometry of the Engineered RMSN-RBCs Analysis Flow cytometry was used to analyze the fluorescence intensity of encapsulated RMSN. The cell counts on the basis of the engineered RBCs (Fig. 7a) perform that RMSN-PEG-10 also exhibits a higher percentage (84.2 ± 5.8%) and mean of fluorescence intensity than larger sizes (25, 50 and 200 nm), which is corresponding to that RBC membrane pores allow a preferential encapsulation to require hydrodynamic diameter below 30 nm. RMSN-PEG-TA and RMSN-PEG-PEI with 10 nm show higher percentages (PEI is 98.6 ± 0.2% and TA is 99.6 ± 0.3%) which demonstrated surfacemodified RMSN-PEG have no effect on encapsulation into RBCs and are performed higher mean of fluorescence intensity than RMSN-PEG-10 owing to particles with positive charged group can increase the expose of RMSN-PEG to surface which negative is of RBCs (Fig. 7b). Flow cytometry analysis not only quantifies the engineered RMSNRBCs but also corresponds to above described optical and fluorescent imaging (Fig. 3 and 6).


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Figure 7. Flow cytometry of the engineered RMSN-RBCs analysis. Fluorescence intensity (%) and mean fluorescence intensity (a.u.) of (a) RMSN-PEG with various sizes and (b) the modified RMSN-PEG@RBCs. The data represented as fluorescence intensity (%) and mean fluorescence intensity (a.u.) are calculated from the PE-A channel of the flow cytometer used to detect RBCs containing RITC dye containing particles.


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3.8 Verification of CD47 Existence on the RMSN-PEG@RBCs CD47 is an integral membrane protein embedded in RBC membranes, exhibiting an extracellular domain that prevented its elimination by binding to the inhibitory receptor signal regulatory protein alpha (SIRPα)30-31. To study the expression of CD47 on the RBCs was evaluated with anti-CD47-FITC staining and quantified by flow cytometry (Fig. 8). All three types of RBCs, including of native RBCs (red), dialyzed RBCs (green) and RMSN-PEG@RBCs (blue), appeared overlapping peak which meant expressed the same level of CD47 on the RBCs (Fig. 8a). Quantitative identification of CD47 on the RMSN-PEG@RBCs analyzed by mean fluorescence intensity, which performs higher intensity more than native RBCs (black bar) in Fig. 8b. Our results clearly confirm the existence of CD47 membrane protein on the RBCs for encapsulating MSN through the osmosis-based process.


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Figure 8. Flow cytometry analysis of membrane protein CD47 on RBC surface. (a) The areas represent native RBCs (gray), RBCs (red) labeling anti-CD47, dialyzed RBCs (green) labeling anti-CD47 and RMSN-PEG@RBC (blue) labeling anti-CD47. (b) The profile of mean fluorescence intensity obtained from (a).


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3.9 Circulation Imaging of RMSN-PEG@RBCs in NOD/SCID mice To study the systemic circulation time of RMSN-PEG@RBCs, we injected our RBCs intravenously into NOD/SCID mice, which are known to lack functional T, B, and NK cells32-33, and observed dynamic imaging in the earlobe of mice via multiphoton microscopy (Fig. 9). The time-lapsed images, obtained from corresponding video (Video S1), which reveal that RMSN-PEG@RBCs persisted 3 hours in circulating time in the blood. The resulting imaging further verifies RMSNPEG@RBCs assessed the ability of circulation in the blood. Nonetheless, the blood circulation of RMSN-PEG@RBCs is still relatively short because of the trans-species nature of the hRBC. At this moment, we do not have permission of performing human experiment to inject the modified hRBCs into human. This requires future clinical experiment to prove it.

It would have been more rational to perform blood

circulation experiments on same animal species with animal RBC. We have attempted similar hypotonic dialysis-based method in encapsulating PEG-MSN in mouse RBC (mRBC). However, as shown in the Fig. S8, it results in extensive hemolysis of mRBC. Thus a parallel experiment of studying PEG-MSN encapsulated in mRBC was not executed. We should comment here about the differences between mRBC and hRBC. It has been known, compared to hRBCs, mRBCs have decreased deformability, membrane rigidity, aggregability, and microvesiculation after various manufacturing process34. Mouse RBCs show higher hemolysis compared to human RBCs after hypotonic treatment.


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Figure 9. In vivo two-photon time-lapse images, taken from Video S1, representing the movement of RMSN@RBCs (108 cells) were intravenously injected into NOD/SCID mice at the indicated times (0.5, 1, 2 and 3 h). The blood vessels (green) were visualized with FITC-dextran (molecular size, 70 kDa). White arrows indicate redfluorescent

RBC

moving

within

the

blood

vessels

(dotted

line).


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4. CONCLUSIONS In this work, we studied critical features for PEGlyated fluorescent MSNs (RMSN-PEG) encapsulation into RBCs. According to fluorescence images and flow cytometry analyses, it performed superior encapsulation into the RBCs with RMSN-PEG of a hydrodynamic diameter below 30 nm. Confocal microscopy and scanning electron imaging confirmed that RMSN-PEG were successfully embedded inside RBCs and that smaller particles exhibited a higher degree of encapsulation. Additionally, surface PEG modification on the RMSN-PEG was critical not only for stabilizing the nanoparticles in biological milieu but also to reduce significant hemolysis otherwise caused by bareMSNs. Also, we further verify the existence of CD47 membrane protein on the RMSNPEG@RBCs and assessed the ability of circulation in the blood. The designed RMSNPEG@RBCs with small size offers a circulation depot for delivering pharmacological substances through osmosis-based method with RMSN-PEG. Moreover, the established safety of blood transfusions gives confidence that these nanoparticle encapsulated within hRBCs

indeed

will

find

use

in

humans.


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ASSOCIATED CONTENT Supporting Information. XRD, BET, TGA analysis, optical imaging, flow cytometry, cell integity, ICP-MS and video

ACKNOWLEDGMENT This work was supported by Ministry of Science and Technology (MOST 105-2119-M002-024). The authors thank Ms. Sabiha Runa for her assistance with editing article and Mr. Shun-Min Yang for his assistance with animal experiment. Thanks to the Institute of Biomedical Engineering and Nanomedicine (National Health Research Institutes) for ICP-MS analysis. Thanks to the national laboratory animal center (National Applied Research Laboratories) for the cell integity analysis. Thanks to Ms. C.-Y. Chien of Ministry of Science and Technology (National Taiwan University) for the assistance in TEM experiments


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Table of contents (TOC) graphic:


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Critical Features for Mesoporous Silica Nanoparticles Encapsulated

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