Impacts of Cross-Linkers on Biological Effects of Mesoporous Silica

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Impacts of Crosslinkers on Biological Effects of Mesoporous Silica Nanoparticles Yi-Ping Chen, Si-Han Wu, I-Chih Chen, and Chien-Tsu Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00240 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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ACS Applied Materials & Interfaces

Impacts of Crosslinkers on Biological Effects of Mesoporous Silica Nanoparticles Yi-Ping Chen,,,# Si-Han Wu,,,# I-Chih Chen§, and Chien-Tsu Chen*,§

†

Graduate Institute of Nanomedicine and Medical Engineering and ‡International Ph.D.

Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei 110, Taiwan §

Department of Biochemistry and Molecular Cell Biology, College of Medicine, Taipei

Medical University, Taipei 110, Taiwan E-mail: [email protected] KEYWORDS crosslinker, mesoporous silica, surface effect, cell response, reactive oxygen species

ACS Paragon Plus Environment


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Abstract Chemically synthesized crosslinkers play decisive roles in variable cargos attached to nanoparticles (NPs). Previous studies reported that surface properties, such as the size, charge, and surface chemistry, are particularly important determinants influencing the biological fate and actions of NPs and cells. Recent studies also focused on the relationship of serum proteins with the surface properties of NPs (also called the protein corona), which is recognized as a key factor in determining the cytotoxicity and biodistribution. However, there is concern that crosslinkers conjugated onto NPs might induce undesirable biological effects. Cell responses induced by crosslinkers have not yet been precisely elucidated. Herein, using mesoporous silica nanoparticles (MSNs) the surfaces of which were separately conjugated with four popular heterobifunctional crosslinkers,

i.e.,

N-[α-maleimidoacetoxy]

maleimidobenzoyl-N-hydroxysuccinimide

succinimide

ester

(MBS),

ester

(AMAS),

succinimidyl

m4-N-

maleimidomethyl cyclohexane-1-carboxylate (SMCC), and maleimide polyethylene glycol succinimidyl carboxymethyl ester (MAL-PEG-SCM), we investigated crosslinkerconjugated MSNs to determine whether they can cause cytotoxicity, or enhance reactive oxygen species (ROS) generation, nuclear factor (NF)-κB activation, and p-p38 or p21 protein expressions in RAW264.7 macrophage cells. Furthermore, we also separately conjugated two biomolecules containing TAT peptides and bovine serum albumin (BSA) as model systems to study their cell responses in detail. Finally, in vivo mice studies evaluated the biodistribution and blood assays (biochemistry and complete blood count) of PEG-derivative NPs, and results suggested that TAT peptides caused significant white blood cell (WBC)-related cell and platelet abnormalities, as well as liver and kidney


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dysfunction compared to BSA when conjugated onto MSNs. The results showed that attention to crosslinkers should be considered an issue in the surface modification of NPs. We anticipate that our results could be helpful in developing biosafety nanomaterials.

Introduction The innovative development of nanomaterials has contributed versatile benefits to biotechnology with health impacts. Various types of engineered nanoparticles (NPs) containing organic and inorganic constituents are popularly used for drug or biomolecule delivery, bioimaging, and therapeutics or disease diagnostics. Because NPs have shown great potential and excellent applications for use in the fields of biomedical and life sciences, the multiple properties of NPs, especially concerning their potential nanohazards, need to be carefully assessed and comprehensively understood before preclinical studies are carried out. Recently, the biocompatibility of NPs in terms of reducing cell toxicity or causing an immune response remains an issue of great concern. Hence, many reports began to explore the biosafety of various types of NPs that have very valuable potential in future medicine.1 In the past, most functionalized NPs were evaluated by cell viability or the survival rate as a cytotoxicity index.2 He et al. reported that the cytotoxicity of NPs was associated with the particle size in human breast cancer cells and monkey kidney cells.3 ViveroEscoto et al. demonstrated that synthesized interior grafted phenanthridium aminopropyl mesoporous silica NPs (MSNs; AP-PAP-MSNs) exhibited significantly lower cytotoxicity upon exposure of HeLa cells.4 Although researchers understand that biocompatibility or cytotoxicity is considered an important issue in developing NPs for



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use in biomedicine, most experiments have only been investigated by an MTT or WST-1 assay. Few reports have noted that the potentially adverse effects of NPs to cells may be shown because of interference with components of signaling pathways. Cell signaling is part of a complex system of communication that governs basic cellular activities and coordinates cell actions to perceive and correctly respond to their microenvironment for the basis of development, tissue repair, and immunity as well as normal tissue homeostasis. Errors in cellular information processing are well understood to result in diseases, such as cancer, autoimmunity, diabetes, and so on.5-6 In recent research, the cytotoxic assay has been expanded to analyze cell responses by introducing many molecular biological technologies. Results indicated that the toxicity of NPs toward cells is frequently dependent on their size, shape, and surface properties that can lead to the generation of reactive oxygen species (ROS).7-10 Excess ROS generation induces undesirable biological effects, including DNA damage, inflammation, oxidative stress, apoptosis, and genotoxicity, and is known to activate mitogen-activated protein kinase (MAPK) pathways which are important mediators of signaling transduction involved in a variety of fundamental cellular processes such as proliferation, differentiation, motility, stress response, apoptosis, and survival.11 MAPK pathways, a group consisting of three components including extracellular signal-regulated kinase 1 and 2 (Erk1/2 or p44/42), c-Jun N-terminal kinases 1-3 (JNK1-3) and p38, are activated in response to oxidative stress and play a role in nuclear factor (NF)-κB activation through IκB-α phosphorylation and degradation.12-13 Activation of the NF-κB transcription factor leads to specific physiological responses, for example, inflammatory, immune, and proliferative responses.14 To date, research on NP-induced cytotoxicity


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have clearly focused on ROS, the MAPK pathway, and NF-κB activation. Recent studies showed that silica NPs or titanium dioxide NPs directly induce cell dysfunction and damage through ROS production and activation of the MAPK and NF-κB pathways.15-19 Nickel oxide NPs and amorphous nanosilica NPs were reported to induce oxidative stress, genotoxicity, and inflammation both in vitro and in vivo.9, 20 Nano-hazards were also observed in gold or silver NP-induced NF-κB activation and functionalized quantum dot-triggered proinflammatory response.21-23 MSNs as one nanomaterial have attracted much attention in biochemical and pharmaceutical applications due to their tunable particle size, high pore volume, physicochemical stability, surface functionalization, and biocompatibility.24 Nowadays, MSNs are considered a promising nano-carrier system that can be used to load or to carry multiple molecules such as medicinal compounds, enzymes, antibodies, and genetic materials.25-29 Thus, functionalized MSNs provide opportunities for bioapplications in a wide variety of fields in drug/protein/gene delivery, imaging, biosensing, tracking, and targeting.30-31 Silica is generally considered to be non-cytotoxic.32 However, development of MSNs for biomedical applications requires close attention to biosafety issues. According to a previous study, MSNs with high porosity induced a reduction of in vitro cytotoxicity and inflammation compared to non-porous silica NPs, colloidal silica.33 Their results showed that MSNs exhibited favorable biocompatibility both in vitro and in vivo, and could play a key role in various intracellular processes for many future biomedical applications. Recently, the biocompatibility of MSNs was also reported in relation to their size, shape, charge, and surface properties in vitro as well as the biodistribution in vivo.



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Regarding the particle size, size-controlled multifunctional Fe3O4@MSNs were reported to have good biocompatibility, which did not affect the cell viability of HeLa, PC-12, or HCT-116 cell lines or the hemolysis of blood cells.34 Effects of size and surface properties on interactions of MSNs with human red blood cell membranes showed no harm to the cell morphology.35-36 Regarding the surface area of particles, Di Pasqua et al. found that most toxic MSNs are those with the largest BET surface area.37 In spite of encouraging data from in vitro studies, current safety concerns with MSN were reported in vivo. In vivo human cancer xenograft investigations showed that fluorescent MSNs preferably accumulated in tumor cells containing low hepatic toxicity.38 Not causing any significant toxicity in vivo in terms of hematology, serum biochemistry, and histopathology illustrates that two differently shaped (short rod and long rod) fluorescent MSNs were biocompatible; however, they showed potential induction of biliary excretion and glomerular filtration dysfunction.39 To date, synthesizing biocompatible MSNs suitable for biomedical applications can be carried out. However, surface modification is an important aspect of NPs designed to synthesize multifunctional NPs. Chemical functionalization of MSNs can be carried out through a variety of techniques such as one-pot synthesis, post-synthetic grafting methods, and imprint coating.40 In order to functionalize with peptides, oligonucleotides, and other active molecules, several chemically synthesized crosslinkers are widely used to conjugate onto MSN surfaces. Chemically synthesized crosslinkers can be classified into homobifunctional or heterobifunctional in accordance with the reactive groups at both ends, such as amines or thiols. It was envisioned that the synthesis of functionalized NPs would leave unoccupied crosslinkers which probably give rise to concerns of nano-


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hazard generation. However, to develop more-biocompatible NPs for use in vivo, these chemically synthesized crosslinkers with unique chemical structural characteristics or functional groups should be a critical issue in the physiological response of cells, when they are used for surface modification. However, research is lacking on whether crosslinkers conjugated on NPs induce undesirable biological effects. We propose that crosslinker conjugation could induce cell responses through: (1) the unconjugated free ends of the crosslinkers, (2) the functional groups of the crosslinkers themselves, or (3) enriched crosslinkers on the surface of each NP. We herein emphasize that using a cell viability assay to evaluate crosslinker-induced cytotoxicity is not exact when conjugated onto NPs. It is worth noting if the above three parameters cause undesirable cellular responses or nano-hazards to cells by influencing cell signaling pathways. To the best of our knowledge, no relevant paper has been published evaluating the biosafety of a series of chemically synthesized crosslinkers when conjugated onto NPs. Consequently, it is essential to know the cytotoxicity information and biological effects of these crosslinkers for biomedical use. In this study, we mainly aimed to investigate cell responses by focusing on whether chemically synthesized crosslinkers on MSN surfaces lead to nano-hazards. We therefore selected four popular heterobifunctional crosslinkers,41-44

i.e.,

N-[α-maleimidoacetoxy]

maleimidobenzoyl-N-hydroxysuccinimide

ester

succinimide (MBS),

ester

(AMAS),

succinimidyl

m-

4-[N-

maleimidomethyl] cyclohexane-1-carboxylate (SMCC), and maleimide polyethylene glycol succinimidyl carboxymethyl (MAL-PEG-SCM), to conjugate onto MSN surfaces to examine the cell uptake efficiency, ROS generation, NF-κB activation, and p-p38 and p21 protein expressions in RAW264.7 microphage cells. In addition, TAT peptides and



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bovine serum albumin (BSA) were conjugated onto the MSN surface to further explore cell responses to unconjugated free ends and functional groups of crosslinkers, respectively. Finally, in vivo mouse studies evaluated blood assays (biochemistry and complete blood count) and biodistribution of PEG derivatives (MSN-APTMS-PEG, MSN-APTMS-PEG-TAT, and MSN-APTMS-PEG-BSA). In brief, our results suggested that crosslinkers induced nano-hazards through cell signaling pathways, thus emphasizing the importance of crosslinker properties when considering surface modifications of NPs.

EXPERIMENTAL SECTION Chemicals and Reagents All chemicals were obtained from commercial suppliers and used without further purification. Ammonium hydroxide (ACS reagent, 28-30% solution in water), cetyltrimethylammonium bromide (CTAB, 99%+), tetraethyl orthosilicate (TEOS, 98%), 3-aminopropyltrimethoxysilane (APTMS, 95%), fluorescein isothiocyanate isomer (FITC), and tris (2-carboxyethyl) phosphine (TCEP) were purchased from Acros. Bovine serum albumin (BSA, ≥98%) was purchased from Sigma-Aldrich. Absolute ethanol (99.5%) was purchased from Shimakyu’s Pure Chemicals. Distilled water (resistivity, 18.2 MQ) was taken from a Millipore Milli-Q Plus system. AMAS, MBS, and SMCC were purchased from Pierce. MAL-PEG-SCM (MW 3.4K) was purchased from the Creative PEGWorks Biotechnology Company. Dihydroethidium (DHE) was purchased from Invitrogen. The WST-1 cell proliferation reagent was purchased from Clontech. Synthesis of Green Fluorescent MSNs and Amine-functionalized MSNs (MSNAPTMS)


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The synthesis of green fluorescent MSNs were performed according to our previous study.26 The surface of green fluorescent MSNs was functionalized with amine groups by treatment with APTMS. Typically, 200 mg of MSNs with addition of 500 µL of APTMS were dispersed in 50 mL of 99.5% ethanol and refluxed for 18 h. After that, aminefunctionalized MSNs were collected by centrifugation and washed with 99.5% ethanol, followed by surfactant extraction in acidic ethanol (1 g of HCl in 50 mL of 99.5% ethanol) under reflux for 24 h. After centrifugation and washing with 95% ethanol, amine-functionalized MSNs (MSN-APTMS) were dispersed and stored in 99.5% ethanol. Conjugation of Different Crosslinkers with MSN-APTMS (MSN-APTMS-linker Synthesis) The heterobifunctional crosslinkers AMAS, MBS, SMCC, and MAL-PEG-SCM were covalently conjugated onto MSN-APTMS through the NHS group by reacting with the amine group on MSN-APTMS. AMAS (5.25 mg), MBS (6.54 mg), SMCC (9.08 mg), and MAL-PEG-SCM (70.72 mg) crosslinkers were separately dissolved in a DMSO solution (9 mL) followed by the addition of a DMSO solution (3 mL) which contained MSN-APTMS (30 mg). Subsequently, PBS solution (0.3 mL) was added to the above solution, and the mixture was stirred for 24 h at room temperature. The products, called MSN-AMAS,

MSN-MBS,

MSN-SMCC,

and

MSN-PEG,

were

collected

by

centrifugation and washed with ethanol several times to remove unreacted AMAS, MBS, SMCC, and MAL-PEG-SCM crosslinkers. Finally, these MSN-APTMS-linkers were dispersed in 99.5% ethanol and stored at room temperature. Synthesis of TAT Functionalized MSN-APTMS-linkers (MSN-APTMS-linkers-TAT Synthesis)



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The Cys-TAT peptide (Cys-YGRKKRRQRRR) was covalently conjugated onto MSNAPYMS-linkers through the sulfhydryl group. In this work, 5.7 mg of cys-TAT was dissolved in DMSO (0.85 ml) followed by the addition of 5 mg of MSN-APTMS-linkers in DMSO (1 ml). Subsequently, 7 mg of TCEP in DMSO (0.7 mL) was added to the above solution. The mixture was then stirred at 4 °C for 18 h. Subsequently, excess TAT was removed by repeatedly washing the NPs with 95% ethanol several times. Finally, MSN-APTMS-linkers-TAT were dispersed in 99.5% ethanol and stored at 4 °C. Immobilization of the BSA Protein on MSN-APTMS-linkers (MSN-APTMS-linkersBSA Synthesis) The BSA protein (21 mg) was mixed and incubated with MSN-APTMS-linkers (1 mg) in 5 mL of conjugation buffer (0.1x PBS) at 4 °C for 18 h. After that, the BSA-conjugated MSN-APTMS-linkers (MSN-APTMS-linkers-BSA) were isolated by centrifugation and washed several times with 0.1x PBS. Afterward, the MSN-APTMS-linkers-BSA were suspended and stored in 0.1x PBS at 4 °C. Cell Culture and Cell Viability Assay RAW264.7 cells (a mouse macrophage cell line) were purchased from the American Type Culture Collection. RAW264.7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Gibco, Life Technology) at 37 °C and 5% CO2 in a humidified atmosphere. For proliferation assays, RAW264.7 cells were seeded in 24-well plates at the density of 3×105 cells per well. Next, various amounts of NP suspensions were added into cells under serum-free or serum-containing DMEM. After 4 h incubation, cells were rinsed twice with culture medium, and incubated with the


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WST-1 cell proliferation reagent (Clontech) at 37 °C. After 2 h incubation, cell viability was assessed by measuring the absorbance at 450 nm and a reference wavelength of 620 nm using a microplate reader (Bio-Rad, model 680). Western Blot Analysis For a given experimental condition, 30 µg of protein extracts were separated on a 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. Next, a blocking step was performed in TBST buffer (Tris-buffered saline with Tween 20) with nonfat dry milk (5% w/v) for 1 h at room temperature. After that, membranes were incubated with primary antibodies at 4 °C overnight. Phospho-p38 was diluted with 5% w/v BSA in TBST (Cell Signaling Technology at 1: 500 dilution), p21 (Santa Cruz Biotechnology at 1:300 dilution) and α-tubulin (Santa Cruz Biotechnology at 1:15,000 dilution) were diluted with 5% w/v milk in TBST. Finally, TVDF membranes were washed followed by incubation with a horseradish peroxide-conjugated secondary immunoglobulin G antibody (Santa Cruz Biotechnology at 1: 2000 dilution) for 1 h at room temperature. Immunoreactive bands were revealed using an enhanced chemiluminescence substrate kit (Amersham Pharmacia Biotech). Superoxide Detection To investigate the in situ production of superoxide anion in cells, 5 µM DHE was used. For the positive control, the rise of superoxide anion was confirmed by an increased intensity of DHE-derived fluorescence at 570 nm upon paraquat (a superoxide anion generator) stimulation. For a given experimental condition, fluorescence cell images were



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obtained under an inverted fluorescence microscope (IX-71, Olympus). Quantitative measurements were carried out from ﬂow cytometry. Immunofluorescence and Confocal Laser Scanning Microscopy For a given experimental condition, RAW264.7 cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, followed by rinsing briefly in PBS. Next, cells were blocked for 1 h in TBST buffer (1x Tris-buffered saline, 0.1% Tween 20) with 5% w/v nonfat dry milk at room temperature, and incubated with an NF-κB p65 primary antibody (Santa Cruz Biotechnology at 1:50 dilution) in blocking buffer at 4 °C for another 18 h. After that, cells were extensively washed followed by incubation with an Alexa Fluor 546-conjugated secondary antibody (1:200 dilution) in blocking buffer. After 2 h incubation, cellular nuclei were stained with DAPI (4',6diamidino-2-phenylindole). Finally, immunofluorescence analyses of stained cells were performed with a Leica TCS SP5 confocal microscope. Characterization Transmission electron microscopic (TEM) images were obtained on a JEOL JSM-1200 EX II at an accelerating voltage of 100 kV. The hydrodynamic diameters and zeta potentials of NP were determined using Malvern Zetasizer Nano ZS (Malvern, UK). Cellular uptake of NP was quantified by flow cytometric analysis (Becton Dickinson, FACS CantoTM II). Fluorescent images were obtained with a with a Leica TCS SP5 confocal microscope. Elemental analyses were performed with an elemental analyzer (TCD, Elementar vario EL cube). Animals


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All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University. Six- or 7-week-old female ICR mice were used as the experimental animals and were purchased from BioLASCO Taiwan (Taipei, Taiwan). Animals were maintained in a temperature- and humidity-controlled facility with a 12-h light/dark cycle. Biodistribution and Blood Assays (Complete Blood Count and Biochemistry) For animal studies, rhodamine B isothiocyanate (RITC) fluorescent MSNs were synthesized based on the above method. Mice were intravenously injected with various NPs at a dosage of 50 mg/kg via the tail vein. Treated mice were then sacrificed, and the major organs were harvested at 4 and 24 h post-injection for individual organ imaging. The fluorescence signals of NPs (excitation at 500~550 nm and emission at 575~650 nm) were collected with the Xenogen IVIS-200 System. At 24 h post-injection, whole blood was collected in an anticoagulant EDTA-coated tube. Hematological analysis was performed using the IDEXX ProCyte Dx® Hematology Analyzer. For the biochemical analysis, blood was preserved in a 1.5-mL Eppendorf tube, and serum was aliquoted to measure blood chemistry levels using a Fuji Dri-chem 4000i Analyzer. All blood assays were measured by Taiwan Mouse Clinic (National Comprehensive Mouse Phenotyping and Drug Testing Center). Statistical Analysis Data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed by Student's t test. * p < 0.05 and ** p < 0.01 were considered statistically significant, and extreme significance, respectively.



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Results Characterization of MSN-APTMS-linkers Several crosslinkers, such as AMAS, MBS, SMCC, and MAL-PEG-SCM, were mainly used for surface functionalization in previous research.41-44 To study relationships of these crosslinkers conjugated onto NP surfaces with cells, we aimed to synthesize MSNs and conjugate them with these four crosslinkers. The strategy for chemical conjugation of MSNs and crosslinkers is presented in Scheme 1. MSNs were first synthesized according to our previously reported methods. For the purpose of tracing the location of NPs in cells and the quantitative analysis, FITC was introduced into MSNs while it was as-synthesized via co-condensation. For further crosslinker conjugation, amino groups (APTMS) were adapted onto the external surface of MSN to form MSNAPTMS by a post-modification method.45 Through a solvent extraction method in an acidic condition, surfactants of both MSNs and MSN-APTMS were individually removed. Subsequently, the N-hydroxysuccinimide (NHS) of crosslinkers, i.e., AMAS, MBS, SMCC, and MAL-PEG-SCM, were conjugated with the amino group of MSNAPTMS to form the MSN-APTMS-linkers, i.e., MSN-APTMS-AMAS, MSN-APTMSMBS, MSN-APTMS-SMCC, and MSN-APTMS-PEG. The nitrogen adsorption and desorption isotherms of the MSNs were measured, and results indicated a mesopore structural profile (Figure S1). The physical properties of MSN-APTMS-linkers were characterized using transmission electron microscopy (TEM), X-ray powder diffraction (XRD), zeta potential, DLS, etc. The TEM micrographs showed that all of the MSN-APTMS-linkers displayed well-ordered mesoporous structure with a size of 50 nm (Figure 1), which would cause higher cell uptake in


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accordance with our previous study.46 The diffractometric analysis showed an intense XRD peak and relatively two weaker peaks, which are characteristic of a 2D hexagonally ordered structure (Figure S2). These data illustrated that the surface modification did not affect the size, shape, or mesoporous structure of the MSNs. As shown in Table 1, the zeta potential analysis showed the value of the MSNs to be -13.6 mV. After reacting with APTMS, MSN-APTMS exhibited a relatively more-positive value of -1.53 mV, because the amino group of APTMS could shelter the negative charges of the MSNs. Similarly, conjugating the NHS of crosslinkers via reacting with the amino groups of MSN-APTMS led them to become a little negative at -6.5 to -8.31 mV. The DLS revealed that the sizes of these NPs in DMEM were 260~363 nm, and values of the polydispersity index (PDI) were between 0.259 and 0.439. These indicate a small degree of aggregation of NPs, but they were still well-suspended in media. An elemental analysis was used to investigate the amount of crosslinkers on MSNs calculated in Table S1. All of the crosslinkers had little different amounts on the MSN surfaces especially for MAL-PEG-SCM, suggesting that the conjugation efficiency could be correlated with the molecular weight or the length of different crosslinkers. Biological effects of MSN-APTMS-linkers on cells We then evaluated the biological characterization of MSN-APTMS-linkers by focusing on cell survival and cell responses. First, the WST-1 assay in RAW264.7 macrophages treated with four different concentrations (50, 100, 250, and 500 µg/mL) of NPs in serum-free DMEM was used to determine the cell viability for 4 and 24 h. There was no obvious cell death in the treatment of MSN-APTMS-linkers (Figure 2a, s3). To assay the cellular uptake ability, flow cytometry was carried out to quantify the percentage of FITC



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fluorescence-containing cells (Figure 2b). After 100 µg/mL of NPs was delivered for 2 and 4 h, MSN-APTMS had the best uptake because the more-positive surface charge led to fast attachment and internalization with RAW264.7 cells. In comparison, both MSNAPTMS-AMAS and MSN-APTMS-MBS had similar cellular uptake abilities and were also better than MSN-APTMS-SMCC. MSN-APTMS-PEG was not taken up by cells. Considering all the MSN-APTMS-linkers had similar zeta potentials which would effect cellular uptake, we therefore proposed that differences in surface properties caused by crosslinkers play a critical role in cellular uptake. Although the cell viability assay demonstrated that no cell death occurred, we wondered whether any unclear cell responses containing cell signaling could have occurred in cells treated with these MSNAPTMS-linkers. According to previous reports, ROS are involved in NP-induced cell cytotoxicity.47 ROS, such as free radicals and peroxides, lead to oxidative damage to cells or tissues. In order to examine whether MSN-APTMS-linkers caused oxidative stress to RAW264.7 cells, generated ROS were stained with DHE (5 µM),48 an ROS dye, and detected using flow cytometry. Results, as shown in Figure 2c, indicated that after cell delivery of 100 µg/mL of NPs for 4 h, levels of ROS production by MSNs and MSNAPTMS were similar to the cell control. In contrast, all of the MSN-APTMS-linkers induced significant cellular ROS generation. We were surprised that MSN-APTMS-PEG without obvious cellular uptake stimulated ROS production. Because intracellular ROS can induce indiscriminate damage and turn on different signal pathways, for example, JNK, p38, IKK, or NF-κB, we wondered if the p-p38 and p21 protein expression levels could be upregulated in RAW264.7 cells treated with MSN-APTMS-linkers (100 µg/mL) for 4 h. As a stress-activated MAPK, p-p38 protein is


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an important biomarker to detect and respond to intracellular oxidative stress.49 p-p38 is also involved in the NF-κB signaling pathway for subsequent cellular regulation. From the Western blot results of Figure 2d, we found that the expression level of p-p38 in RAW264.7 cells could be upregulated after treatment with MSN-APTMS-AMAS, MSNAPTMS-MBS, MSN-APTMS-SMCC, and the LPS control (1µg/mL), but not MSNAPTMS-PEG. Additionally, the protein expression level of p21 (CIP1/WAF1),50 a cell cycle arrest and cellular senescence biomarker, showed an obvious increase after all NP treatments in RAW264.7 cells (Figure 2d). Concurrently, inflammatory signaling in cells via ROS production was accompanied by activation of NF-κB p65 nuclear translocation. We further examined NF-κB p65 activation by confocal microscopy. Images in Figure 3 show that nuclear NF-κB p65 expression (p65: red, nucleus: blue) induced by MSNs or MSN-APTMS was low, and NF-κB p65 was mainly found in the cytosol of cells. LPS was used as a positive control to activate NF-κB p65 nuclear translocation, as seen in the overlapping blue and red (purple to pink) images in the “merged” column in the nucleus. After treatment with MSN-APTMS-AMAS, MSN-APTMS-MBS, and MSN-APTMS-SMCC, as well as LPS treatment, NF-κB p65 expression was significantly enhanced in the cytosol and nucleus, indicating that NF-κB p65 was activated and translocated from the cytosol to the nucleus. In addition, MSN-APTMS-MBS-induced activation was stronger than that of MSNAPTMS-AMAS and MSN-APTMS-SMCC. We found that MSN-APTMS-PEG caused no significant NF-κB p65 activation. In brief, MSN-APTMS-AMAS, MSN-APTMS-MBS, and MSN-APTMS-SMCC not only induced ROS production but turned on p-p38 expression, followed by NF-κB p65



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activation. However, MSN-APTMS-PEG did not activate the p-p38 or NF-κB p65 protein even though it induced ROS production. However, all of the MSN-APTMSlinkers significantly activated p21 expression. Cell responses of MSN-APTMS-linkers-TAT and MSN-APTMS-linkers-BSA As carriers, most NPs are often functionalized with biomolecules for biomedical applications. In order to further explore the crosslinker effects on cells after biomolecule conjugation, we synthesized MSN-APTMS-linker-TAT as a model system to study cell responses in detail. The TAT peptide (AYGRKKRRQRRR), a well-known cellpenetrating peptide containing the core region of the transactivating transcriptional activator protein from HIV-1, was shown to freely travel across biological barriers, including cellular and nucleic membranes and the blood-brain barrier (BBB).51-52 Currently, conjugating TAT onto NPs could overcome multidrug resistance (MDR), enhance NP delivery and cross the BBB.53-54 To prepare MSN-APTMS-linkers-TAT, MSN-APTMS-linkers were conjugated with the thiol group of Cys-TAT via covalent binding (Scheme 1). Subsequently, physical characteristics (zeta potential and DLS) and the cell viability of MSN-APTMS-linkers-TAT were evaluated. As shown in Table 2 and Figure S4, after being conjugated with TAT peptides, there were no significantly changes except for values of the zeta potential with an obvious positive shift, which was probably due to the positive charge of the arginine-rich domain of the TAT peptide. The zeta potentials of MSN-APTMS-AMAS-TAT, MSN-APTMS-MBS-TAT, MSN-APTMSSMCC-TAT, and MSN-APTMS-PEG-TAT were between 1.65 and -0.42 mV. The WST1 assay showed that all kinds of MSN-APTMS-linkers-TAT still retained full cell viability for 4 and 24 h. Flow cytometry-evaluated MSN-APTMS-linkers-TAT had better


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cellular uptake ability than MSN-APTMS-linkers in RAW264.7 cells for 2 and 4 h owing to the positive charge from TAT functionalization, which helps electrostatic interactions with negatively charged cell membranes, leading to enhanced NP delivery (Figure 4a). Also, the efficiency of MSN-APTMS-PEG without cellular uptake was raised when conjugated with TAT peptides. Compared to MSN-APTMS-linkers, Western blotting revealed that cell responses at the protein expression level of p-p38 and p21 had similar expression profiles in RAW264.7 cells treated with MSN-APTMS-linkers-TAT (100 µg/mL) for 4 h. MSN-APTMS-linkers-TAT treatment activated the p-p38 and p21 proteins except for MSN-APTMS-PEG-TAT, which could not activate p-p38 (Figure 4b). TAT is a short peptide that fills in most of the free maleimide groups on crosslinkers when conjugated to MSN-APTMS-linkers. However, conjugation of large biomolecules (antibodies or proteins/enzymes) leaves many free unconjugated maleimide groups due to their large size. The effects of these free unconjugated maleimide groups on cells needs to be further examined. Therefore, to imitate large biomolecule conjugation we used BSA, instead of TAT, to conjugate with MSN-APTMS-linkers to form MSN-APTMSlinkers-BSA (Scheme1). We found that the cell viability, cellular uptake, and p-p38 and p-21 expressions after treatment with MSN-APTMS-linkers-BSA were the same as those with MSN-APTMS-linker treatment (Figure 4c, d, S5). Surprisingly, MSN-APTMSPEG-BSA treatment obviously caused p-p38 activation, compared to MSN-APTMS-PEG or MSN-APTMS-PEG-TAT treatment. Biological effects of PEG derivatives in vivo Although in vitro cellular studies provided an easy and fast way to evaluate the interaction or responses between NPs and cells, understanding the fate of NPs in vivo is



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crucial in exploring nano-hazards. Because PEG is a better crosslinker than the others, we next attempted to assess the biodistribution and blood assays (biochemistry and complete blood count) of PEG derivatives (MSN-APTMS-PEG, MSN-APTMS-PEG-TAT, and MSN-APTMS-PEG-BSA) in a mouse model. Mice were injected with 50 mg/kg of each PEG derivative via the tail vein, and fluorescence signals were captured at 4 and 24 h using an IVIS analysis. As shown in Figure 5, the three PEG derivatives had accumulated in the liver and lungs at 4 and 24 h, except for MSN-APTMS-PEG-BSA in the liver, and the majority of MSN-APTMS-PEG-TAT and MSN-APTMS-PEG-BSA strongly remained in the lungs for 4 h (Figure 5a). At 24 h after the injection, the fluorescence signals of MSN-APTMS-PEG and MSN-APTMS-PEG-TAT in the lungs had diminished due to clearance or elimination from the body. However, MSN-APTMS-PEG-BSA still showed an abundant distribution in the lungs compared to the other PEG derivatives (Figure 5b). To reveal potential side effects of mice treated with PEG derivatives, blood hematology and biochemistry were further analyzed (Figure 5c, Table S2). There was no obvious statistical difference between MSN-APTMS-PEG-treated mice and control mice. However, neutrophil (NEU), monocyte (MONO), glutamic-oxaloacetic transaminase (GOT), blood urea nitrogen (BUN), creatine (CRE), and total bilirubin (TBIL) of mice treated with MSN-APTMS-PEG-TAT were significantly elevated compared to those of control mice (p < 0.05; BUN: p < 0.01). The same mice also showed decreased levels of lymphocyte (LYM), platelet (PLT) count, procalcitonin (PCT) % value, and alkaline phosphatase (ALP) (p < 0.05). In addition, these parameters remained within the normal range after treatment with MSN-APTMS-PEG-BSA except for the level of MONO (p < 0.05).


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Discussion An ideal NP needs to have not only a good therapeutic effect, but also excellent biosafety. The biosafety of NPs involving biocompatibility or cytotoxicity is therefore causing lots of concern. MSNs are a good biocompatible nanomaterial without significant cytotoxicity or cell responses. However, to prepare an effective NP for bioapplications, functionalization of different kinds of biomolecules such as small interfering (si)RNA, peptides, and proteins is necessary. For functionalization, crosslinkers are often conjugated onto NPs for surface modifications to further carry out the linkage between NPs and biomolecules. Hence, whether crosslinkers cause cytotoxicity or cell responses remains an issue of great concern after conjugation, which is an important problem in nanomedical applications. In order to respond to changes in the immediate environment, cells must be able to receive and process signals by cell signaling transduction to promote the survival, proliferation, differentiation, death, and gene regulation of cells. In general, cell signaling can be activated through two routes: (1) stimulation of a specific receptor at the surface of cell membranes (cell surface receptors) or inside the cell (cytoplasmic and nuclear receptors); and (2) interactions with intracellular proteins that initiate a signaling cascade. For example, the epidermal growth factor (EGF) receptor on the cell surface binds with its ligand, EGF, which activates tyrosine protein kinase to further catalyze intracellular reactions.55 In the cytosol, caspase family members or the p53 protein interact with their downstream proteins for apoptosis regulation.56-57 In brief, our results demonstrated that MSN-APTMS-PEG did not activate p-p38 expression (Figure 2d) or NF-κB p65 nuclear



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translocation (Figure 3); but it slightly induced ROS production (Figure 2c). Compared to other crosslinkers, PEG should be a better crosslinker for conjugating with NPs because it had fewer cell responses. In addition, as shown in Figure 2b, MSN-APTMS-PEG was not taken up by RAW264.7 cells after delivery for 24 h, meaning that MSN-APTMS-PEGinduced intracellular ROS production must be from stimulation of cell surface receptors through cell signaling transduction. In contrast, we speculated that the other MSNAPTMS-linkers which could be taken up by cells, induced intracellular ROS generation through the routes of cell surface receptors and/or cell signaling transduction in the cytosol. At the same time, because the maleimide groups on MSN-APTMS-linkers can react with thio-containing biomolecules, the increased ROS could result from the decline of intracellular antioxidant GSH over the reaction. From the results of Figures 2d and 3, after delivering MSN-APTMS-linkers, the p-p38 expression profile was similar to NF-κB p65 activation due to p-p38 upregulating NF-κB p65 activation in cells. In addition, the MSN-APTMS-MBS induced stronger p-p38 expression as well as NF-κB p65 activation, compared to the other MSN-APTMS-linkers. Although p21 protein expression was observed with delivery of all of the MSN-APTMS-linkers (Figure 2d), MSN-APTMSlinkers-TAT (Figure 4b), and MSN-APTMS-linkers-BSA (Figure 4d), MSN and MSNAPTMS also activated p21 the same as did the MSN-APTMS-linkers, suggesting that these crosslinkers conjugated on MSNs probably did not activate p21 expression or just had a slight effect, which was not easy to distinguish by Western blotting. We therefore propose that p21 activation was the major form of the MSNs themselves. Among all the MSN-APTMS-linkers except for MSN-APTMS-PEG, there was obvious activation at the protein expression level of p-p38 compared to control MSNs


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and MSN-APTMS (Figure 2d). Especially interesting is that MSN-APTMS-PEG contains the lowest amount of crosslinkers compare to that in other cases of MSN-APTMSlinkers. The results demonstrated that crosslinkers conjugated on NPs were available to cause cell responses. To understand in detail if crosslinker-induced cell responses were from (1) the unconjugated MAL ends of the crosslinkers, (2) functional groups of the crosslinkers themselves, or (3) enriched crosslinkers on the surfaces of each NP, TAT or BSA was conjugated on the MSN surface to shelter MAL ends of the crosslinkers. Comparison of results of p-p38 expression (Figure 2d, 4b and 4d) showed no significant difference when the Mal ends of crosslinkers were sheltered at different levels depending on the size of TAT or BSA. It is, however, worth noting that all BSA-containing nanoparticles, including MSN-APTMS-PEG-BSA, activated p-p38 expression. Hence, we speculated that the reasons for p-p38 activation were the functional groups of the crosslinkers themselves or the enriched crosslinkers or BSA on the surfaces of each NP. Importantly, crosslinker-caused cell responses should be considered when modifying the surfaces of NPs. According to a previous study, Faure et al. reported four PEG derivatives (PEG250COOH, PEG2000-COOH, PEG2000-NH2, and PEG2000-OCH3), containing different chain lengths and end groups conjugated on NPs, exerted a significant influence on their biodistributions in vivo.58 In our study, three PEG derivatives had the same PEG lengths with different end moieties, including MAL, TAT, and BSA. Our results revealed that the different biodistributions of the three PEG derivatives depended on the end moieties of the PEG chain, which was similar to results of their study (Figure 5a, b). Coating the surface of NPs with PEG, a polymer with electrical neutrality and a hydrophilic nature, is



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a frequent strategy and the most efficient way to avoid nonspecific accumulation, provide steric hindrance to improve NP dispersion, prolong blood circulation, and prevent reticuloendothelial system (RES) uptake by suppressing protein adsorption in serum (protein corona).58-61 The protein corona has been the focus of a great deal of recent study due to its inevitable formation and its impacts on the biological consequences of nanomaterials. A few studies focused on how the surface properties influence protein corona formation and affect uptake by macrophages, as well as the in vivo biodistribution that plays a more-important role than size in NP-cell internalization.62 As shown in Figure 5a and 5b, no obvious or strong signals were observed in the liver, spleen, or kidneys, suggesting that PEG successfully helped the three PEG derivatives prevent uptake by the RES and clearance by kidney excretion. Additionally, Rasa et al. demonstrated that covalent and non-covalent binding of BSA to ZnO NPs could decrease ZnO NP-induced cytotoxicity and ROS generation through protection against formation of the protein corona.63 Hence, we demonstrated that BSA-mediated protein corona protection would lead to MSN-APTMS-PEG-BSA having the ability to prevent RES uptake better than the other PEG derivatives, resulting in no accumulation in the liver. Therefore, from the results of blood assays (Figure 5c), MSN-APTMS-PEG-BSA did not affect the liver and kidney function. In contrast, MSN-APTMS-PEG-TAT significantly caused white blood cell (WBC)-related cell and platelet abnormalities, as well as liver and kidney dysfunction (Figure 5c, Table S2). Also, among the three PEG derivatives, a significant difference in accumulation in the lungs was observed (MSN-APTMS-PEGBSA > MSN-APTMS-PEG-TAT > MSN-APTMS-PEG). In brief, we speculated these above fates of PEG derivatives probably were attributable to differences in surface


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properties, which were from the effect of the protein corona, as well as the end moieties of the PEG chain.

Conclusions In summary, we investigated the cytotoxicity of NPs, focusing on the in vitro and in vivo influences of crosslinkers conjugated onto NPs. The fate of these nanosized materials mainly depended on their structure and surface physicochemical properties. Using an in vitro cell viability assay to address NP-induced cytotoxicity is not an impersonal evaluation. Because sublethal cellular damage usually does not cause significant cell death, but might alter or influence certain cellular functions by regulating cellular signaling. This study demonstrated that crosslinkers should be considered as a parameter when designing multifunctional NPs.

Supporting Information The N2 adsorption-desorption isotherms, small-angle powder XRD, cell viability assay. , serological and hematological assays, and amounts of crosslinkers conjugated onto MSNs.

Author Information Corresponding Authors *E-mail: [email protected] Notes #

Co-first authors



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Acknowledgements This research was supported in part by grants from Taipei Medical University (TMU 104-1801-013-400 and TMU 104-1801-015-400). We thank Dr. Chung-Yuan Mou for his advice and support.


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Scheme 1. Schematic illustration of the conjugation of mesoporous silica nanoparticles (MSNs) with crosslinkers, followed by conjugation with TAT peptides or BSA.

Figure 1. TEM images of various nanoparticles (NPs).


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(a)

(b)

(c)

(d)

Figure 2. Cell responses of MSN-APTMS-linkers. (a) After treatment of RAW264.7 cells with different concentrations of nanoparticles (NPs; 50~500 µg/mL) for 4 h, cell viability was measured with a WST-1 assay. (b) Flow cytometry measured the cellular



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uptake efficiency under trypan blue conditions in RAW264.7 cells treated with 100 µg/mL of NPs for 2 and 4 h. RAW264.7 cells were treated with 100 µg/mL of NPs or LPS (1 µg/mL) for 4 h. (c) Intracellular ROS were stained by DHE and detected using flow cytometry. (d) Expression levels of p-p38 and p21 were analyzed using Western blotting. α-Tubulin was used as a loading control to ensure the equal loading of lysates.


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Figure 3. Confocal images of NF-κB p65 activation. RAW264.7 cells were treated with MSN-APTMS-linkers (100 µg/mL) for 4 h. Then, NF-κB p65 (Alexa-568-labeled antip65 antibody, red) and the cell nucleus (DAPI-labeled, blue) were stained and imaged using confocal microscopy.



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(a)

(b)

(c)

(d)

Figure 4. Cell responses of MSN-APTMS-linkers-TAT and MSN-APTMS-linkers-BSA. (a, c) Cellular uptake was measured by flow cytometry under trypan blue conditions after


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treatment of RAW264.7 cells with 100 µg/mL of (a) MSN-APTMS-linkers-TAT or (c) MSN-APTMS-linkers-BSA for 2 and 4 h. (b, d) Western blotting analysis of expression levels of p-p38 and p21 in RAW264.7 cells treated with 100 µg/mL of (b) MSNAPTMS-linkers-TAT or (d) MSN-APTMS-linkers-BSA or LPS (1 µg/mL) for 4 h. αTubulin was used as a loading control to ensure the equal loading of lysates.



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(a)

(b)

(c)


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Figure 5. Biodistribution and blood assays (biochemistry and complete blood count) of PEG derivatives. Mice were administered 50 mg/kg of various nanoparticles (NPs) through a tail vein IV injection. (a) Four and (b) 24 h later, the organs were imaged using an IVIS imaging system for the biodistribution assay. (c) Serological assays. Serum was collected from sacrificed mice treated with various NPs for 24 h. These parameters included glutamic-oxaloacetic transaminase (GOT), total bilirubin (TBIL), alkaline phosphatase (ALP), creatinine (CRE) and blood urea nitrogen (BUN). (* p < 0.05 and ** p < 0.01, compared to the control).



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Table 1. Zeta potentials and hydrodynamic diameter distributions of various nanoparticles (NPs).

Table 2. Zeta potentials and hydrodynamic diameter distributions of MSN-APTMSlinkers-TAT.


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TOC


Impacts of Cross-Linkers on Biological Effects of Mesoporous Silica

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