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Serum protein adsorption enhances active leukemia stem cell targeting of mesoporous silica nanoparticles Michaela Beck, Tamoghna Mandal, Christian Buske, and Mika Lindén ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 20, 2017

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ACS Applied Materials & Interfaces

Serum Protein Adsorption Enhances Active Leukemia Stem Cell Targeting of Mesoporous Silica Nanoparticles Michaela Beck1‡, Tamoghna Mandal2‡, Christian Buske2*, Mika Lindén1* 1

University of Ulm, Institute of Inorganic Chemistry II, Albert-Einstein-Allee 11, 89081 Ulm, Germany

2

University Hospital Ulm, Institute of Experimental Cancer Research, Albert-Einstein-Allee 11, 89081 Ulm, Germany

‡ Authors contributed equally * Corresponding authors

KEYWORDS: Mesoporous silica nanoparticles, targeting, leukemia, stem cells, drug delivery

ACS Paragon Plus Environment

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ABSTRACT

The functionalization of nanoparticles with a ligand targeting receptors overexpressed by the target cells is a commonly used strategy when aiming at nanoparticle-based, cell type-specific drug delivery.1–4 However, the influence of particle surface chemistry on the targetability has received much less attention. The surface charge is known to directly or indirectly affect the nanoparticle cellular uptake kinetics by influencing serum protein adsorption.5–7 Thus, it is fair to assume that both the specificity and cellular uptake kinetics of targeted nanoparticles are influenced by the nanoparticle charge, both of which are important parameters for controlling cell-specific drug delivery efficiency. We therefore studied the influence of the surface chemistry of mesoporous silica nanoparticles (MSNs) carrying identical amounts of a specific antibody (anti-B220) on the selectivity towards B220-positive leukemia stem cells. The uptake by these cells was higher compared to the nanoparticle uptake by B220-negative leukemia stem cells, demonstrating uptake specificity. In addition, the adsorption of serum proteins onto the differently charged MSNs was studied by SDS-PAGE. Interestingly, the highest selectivity was not observed for the MSNs with the lowest level of serum protein adsorption, which suggests that proteins present in the protein corona of the MSNs may positively influence the selective uptake of targeted nanoparticles. For the particles exhibiting the highest selectivity, successful selective delivery of cargo to the B220-positive cells was demonstrated. Taken together, our results indicate that nanoparticle surface charge and adsorption of serum proteins is an important factor for enhancing selectivity in targeted delivery of drugs using nanoparticulate vectors, an observation tentatively attributed to enhanced cellular internalization kinetics in the presence of adsorbed serum proteins on the nanoparticles.


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1. Introduction Nanoparticulate drug vectors are promising carrier systems for a wide range of therapies, due to their capability of passing biological barriers, the potential for carrying high payloads, and the possibility for targeting of nanoparticles to specific target organs and cells.8–13 Active targeting of nanoparticles to target cells rely on identifying receptors that are over-expressed on these cells, and to functionalize the nanoparticles with the corresponding ligand in order to allow recognition of the target cells through specific nanoparticle-cell interactions.1–4 There are numerous reports in the literature where this approach has been shown to be successful in enhancing the specificity of nanoparticulate uptake in vitro and more recently also in vivo. Evidently, the most specific interactions can be expected for antibody-based targeting, but also peptide and small molecule targeting ligands like folic acid have also showed high promise.14–18 However, in addition to specific targeting ligand-receptor interactions, the importance of additional nanoparticle-cell interactions like those between proteins adsorbed on the nanoparticles and receptors for these on the cell surface, as well as “passive” effects of protein adsorption to nanoparticles on the cellular internalization kinetics are receiving increasing attention. On this note, pronounced protein adsorption to the nanoparticles could also lead to a lower level of specificity as the targeting ligands may be rendered inaccessible.19 The current level of understanding of these combined effects is limited, due to the fact that serum protein adsorption to the nanoparticulate drug vectors has often not been a part of the nanoparticle targetability evaluations. In addition, comparison between results obtained in different studies is difficult, as typically only one type of nanoparticle has been studied where the uptake of the nanoparticle carrying a targeting ligand is compared with the uptake of the corresponding particle void of targeting ligand. We therefore studied the influence of surface charge on the


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selective uptake of mesoporous silica nanoparticles (MSNs) covalently functionalized by a cell specific antibody by acute myeloid leukemia (AML) stem cells. Protein adsorption was also quantified for all particles. The chosen biological system is of high practical relevance, as AML is a cancer with poor prognosis and clinically aggressive course leading to death of the majority of patients still today. Fatal outcome is finally due to resistance to chemotherapy, which is believed to be mediated by AML stem cells, which are the responsible cell subpopulation initiating and maintaining the disease. Thus, there is a high unmet medical need for innovative therapeutic approaches, preferentially targeting AML stem cells, for this disease. The clinical therapy of AML is mainly based on chemotherapeutics, such as daunorubicin or cytarabine sometimes followed by stem cell transplantation.20 This therapy approach is associated with pronounced side effects and is based on a non-targeted delivery of high doses of chemotherapeutics to malignant as well as normal hematopoietic cells. A targeted delivery of chemotherapeutics to AML stem cells would promise to increase the therapeutic window and by this improve therapeutic outcome and reduce treatment related toxicity. The challenge here is the similarity between normal hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) as well as the fact that in leukemia circulating cells have to be targeted in comparison to organ specific static solid tumors, which is why the EPR (enhanced permeability and retention) effect will not contribute to enhanced nanoparticle accumulation in the target cells. In the investigated murine AML model of CALM-AF10 leukemia propagating LSCs are characterized by the expression of the surface B220 antigen.21 Using this model we hypothesized that functionalization of drug vectors, in the present study mesoporous silica nanoparticles, MSNs, with an anti-B220 antibody targeting the B220 antigen will enhance the specificity of drug delivery to LSCs. The choice of MSNs as drug vectors in this study was made based on the many


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attractive properties of MSNs for targeted drug delivery. Successful targeting in vitro and in vivo15,22–24 of MSNs functionalized with a monoclonal antibody has already been demonstrated. Their high specific surface area and pore volume allow for high drug loading levels, the flexibility in controlling MSN size and shape allow optimization towards a given route of administration, their biocompatibility and biodegradability together with the possibility of controlled surface functionalization are attractive features of this drug vector platform.9 Furthermore, MSN surface charge is a crucial factor for cellular uptake, and positively charged particles typically show a higher passive uptake in serum-free medium as compared to corresponding negatively charged particles due to electrostatic interactions with the negatively charged cell surface.25,26 In the presence of serum proteins, the opposite influence of surface charge on particle uptake by osteoblasts has been observed. This was attributed to changes in the surface charge of the MSNs upon serum protein adsorption, rendering the MSNs with almost equal effective surface charges irrespective of the surface charge of the original MSNs.5 Other studies related to the uptake of MSNs in macrophages suggest no dependency on protein corona formation on particle uptake.27 Thus, the influence of the protein corona on nanoparticle uptake remains open, but recent studies focusing on the cellular specificity of nanoparticulate uptake suggest an important role of adsorption of specific proteins to the nanoparticles.28 Zwitterionic MSNs carrying both amino- and carboxylic acid groups were evaluated, as zwitterionic surface functionalization of nanoparticles has been shown to be a means for decreasing serum protein adsorption.29 The effect on zeta potential, protein adsorption, dispersion stability and hydrolytic stability was analyzed as well as their cell specificity. Importantly, the highest cell specificity was not observed for the particles with the lowest level of serum protein adsorption. Our results highlight the importance of serum proteins adsorbed


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onto nanoparticles for enhancing the selectivity in targeted drug delivery approaches using MSNs.


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2. Materials and Methods

2.1 Materials Tetramethoxysilane

(TMOS),

3-aminopropyltrimethoxysilane

(APTMS),

cetyltrimethylammonium bromide (CTAB), N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), dimethylsulfoxide (DMSO), glycerol, sodium azide and bisacrylamide were obtained from Sigma-Aldrich Chemie GmbH (Munich, Germany). Ammonium nitrate, methanol, ethanol, 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES), succinic anhydride, potassium chloride, sodium hydroxide, bromphenol blue and Comassie® brilliant blue G250 were purchased from Merck KGaA (Darmstadt, Germany). Aluminium sulfate, phosphoric acid and sodium dodecyl sulfate (SDS) was obtained by VWR International GmbH (Radnor, USA) and 1X Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), fetal calf serum (FCS), recombinant mouse interleukin3 (RMIL310), Dulbecco's phosphate-buffered saline (PBS, without calcium and magnesium) and PageRuler Unstained Protein Ladder from Thermo Fisher Scientific Inc. (Waltham, USA). ATTO 594-NHS was purchased from ATTO-TEC GmbH (Siegen, Germany). The antibody anti-human/mouse CD45R (B220) functional grade purified (Clone: RA3-6B2), the fluorescent labelled antibodies anti-human/mouse CD45R (B220) (eFluor® 450, APC, PE) and the antibody anti-human CD9 purified and fluorescence labelled (PE) conjugated (Clone: eBioSN4 (SN4 C3-3A2)) were purchased from eBioscience Inc. (San Diego, USA). Dithiothreitol (DTT), glycine, acrylamide and tris(hydroxymethyl)aminomethane hydrochloride (TRIS.HCl) were obtained by Carl Roth GmbH & Co. KG (Karlsruhe, Germany).


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Ethanol was dried with sodium under reflux conditions and then stored under inert atmosphere over a 3 Å molecular sieve. All chemicals were used as received without further purification.

2.2 Synthesis of mesoporous silica nanoparticles (MSN_NH2) MSN_NH2 particles were synthesized according to a modified procedure described by Rosenholm et al.30. CTAB (7.9 g; 21.6 mmol) was dissolved in methanol (640.0 g; 20.0 mol) and water (962.3 g; 53.5 mol) followed by the addition of caustic soda solution (2.3 mL; 2 M; 4.6 mmol) and a mixture of TMOS (2.2 mL; 14.8 mmol) and APTMS (360.0 µL; 2.1 mmol) with continuous stirring. After 24 h of stirring at room temperature the particles were separated by centrifugation and surfactant removal was performed by extraction to obtain aminofunctionalized particles (MSN_NH2). The particles were dispersed in a mixture of ammonium nitrate in ethanol (6 g/L) and treated in an ultrasonic bath for 1 h. After centrifugation this procedure was repeated another two times. Afterwards the particles were washed twice with ethanol and dried for 24 h at 60 °C.

2.3 Particle functionalization 2.3.1 Succinic anhydride functionalization of amino functionalized MSNs (MSN_cx) Partially converting amine into carboxyl groups was performed with various amounts of succinic anhydride. To remove adsorbed water previous to the functionalization the particles were dried under vacuum for 3 h at 80 °C. Afterwards the particles (15 mg particles/mL) were dispersed in anhydrous ethanol containing different amounts of succinic anhydride (400.3, 200.1, 40.0, 10.0 and 2.0 µg/mg; 4, 2, 0.4, 0.1 and 0.02 mmol/g resulting in particles denoted MSN_c15). The reaction mixture was rotated for 24 h at room temperature before the particles were


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separated via centrifugation, washed three times with ethanol and dried for 24 h in vacuum at room temperature.

2.3.2 Fluorescent labelling For intracellular detection the particles were labelled with 0.18 wt.% ATTO 594. Therefore the particles were dispersed in HEPES buffer solution (1 mg/mL) and ATTO 594-NHS/DMSO (1 mg/mL; 1.8 µg/mg particle; 1.3 nmol/mg particle) was added. After 30 min rotation the particles were centrifuged and washed three times with ethanol before drying under vacuum for 24 h at room temperature.

2.3.3 Antibody functionalization of the MSNs (MSN_cx-B220, MSN_cx-CD9) To activate the carboxyl group on the antibody (anti-human/mouse CD45R (B220) (anti-B220) respectively anti-human CD9 (anti-CD9) respectively anti-human/mouse CD45R (B220) eFluor450® (anti-B220v450)) the antibody was suspended in HEPES buffer solution (20 µg/mL), NHS (2.3 mg/mL; 19.9 µmol/mL) and EDC (3.4 µL/mL; 19.2 µmol/mL) was added and rotated at room temperature. Meanwhile the particles were dispersed in HEPES buffer solution (2 mg/mL) and added after 30 min to the antibody solutions. The reaction mixture was rotated for another 90 min at room temperatures. Particles were centrifuged, washed twice with HEPES buffer solution and redispersed (1 mg/mL) directly in DMEM supplemented with 15 % FBS, 1 % penicillin/streptomycin and 10 ng/mL RMLI310 for cellular uptake studies. Antibody quantification was performed analogue with fluorescent labelled anti-B220v450. Supernatant and washing water were analyzed by fluorescence spectroscopy (λexc = 405 nm; λem = 444 nm) and the difference of starting fluorescence intensity minus fluorescence of supernatant


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and washing water was determined. Concentration of antibody was calculated by calibration curve of labelled antibody in HEPES buffer solution.

2.4 Particle characterization Dynamic light scattering and zeta potential measurements were performed on a Zetasizer Nano-ZS ZEN 3600 (Malvern Instruments, Germany) at particle concentrations of 0.1 mg/mL in HEPES buffer solution (pH 7.2, 25 mM). Zeta-potential titrations aiming at identifying the isoelectric point of the particles were carried out in 1 mM KCl solution using 0.25 M HCl and 0.25 M NaOH for pH adjustments at a particle concentration of 2 mg/mL. Nitrogen sorption measurements were performed at 77 K on a Quadrasorb-SI (Quantachrome Instruments, USA). For calculating the specific surface area the BET was used and for determining the pore size and pore volumes the calculation based on the non-local density functional theory kernel for silica materials with cylindrical pores was used. Particle size and morphology were analyzed via transmission electron microscopy (TEM) on a 1400 (Jeol, Germany) after embedding the particles in an epoxy resin and cutting into ultrathin slices and via scanning electron microscopy (SEM) on a S-5200 (Hitachi High Technologies America Inc., USA) after sputtering the sample with a thin layer platinum. Dynamic light scattering and zeta potential measurements were performed on a Zetasizer Nano-ZS ZEN 3600 (Malvern Instruments, Germany) at particle concentrations of 0.1 mg/mL in HEPES buffer solution (pH 7.2, 25 mM). Fluorescence properties were determined on a multiplate reader infinite M1000 (Tecan) (ATTO 594: λexc = 590 nm; λem = 600-750 nm respectively 626 nm; eFluor® 450: λexc = 405 nm; λem = 444 nm).

2.5 Particle dissolution studies and particle dispersion stability analysis


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To determine particle hydrolytic stability, particles were dispersed in PBS + 15 % FCS (0.1 mg/mL) and particles were separated after several hours (0.25, 0.5, 1, 2, 6, 24 and 48 h) by centrifugation. The supernatant was analyzed by inductively coupled - atomic emission spectrometry (ICP-AES). To determine the stability of particle dispersions, particles were dispersed in DMEM + 15 % FCS (0.1 mg/mL) and the hydrodynamic radius was determined by dynamic light scattering within 24 h at 37 °C.

2.6 Protein adsorption and detachment Protein adsorption performed by incubating anti-B220 functionalized particles in PBS + 15 % FCS (1 mg/mL) for 24 h at 37 °C with shaking. Particles were separated by centrifugation and washed twice with PBS. To detach the protein corona, particles were dispersed in SDS buffer solution (10 mg/mL), treated for 30 min in an ultrasonic bath and centrifuged before mixed with Laemmli buffer (50 µL sample + 10 µL Laemmli buffer) (1.2 mL Tris-HCl buffer (pH 6.8; 0.5 M), 1.2 g SDS, 6 mg bromphenol blue, 4.7 mg glycerol, 2.9 mL Milli-Q-water, 0.93 g DTT) and treated 10 minutes at 95 °C.

2.7 SDS-PAGE One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; separating gel: 12 %; stacking gel: 5 %) was performed on a Bio-Rad PROTEAN II XL electrophoresis chamber at constant 300 V for 3 h 10 min. The gels were washed five times with water prior to staining in Comassie staining solution (Kang et al.31) for 1 h and followed


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destaining in water for 24 h. The gel was imaged with a bioradTM geldoc system and analyzed with the image lab software.

2.8 Biological experiments 2.8.1 Cell culture and treatment CALM-AF10 positive GFP positive murine AML cells were derived from single-cell-sorted B220+Mac1- Gr1- cells and were shown to have LSC characteristics, reflected by their ability to cause AML in transplanted mice (data not shown).21 These cells are referred to as B220+-LSCs in the following. B220-negative GFP positive AML stem cells were derived from leukemic mice, which were transplanted with BM retrovirally engineered to overexpress the homeobox gene Cdx2 as previously published.32 These cells will be referred to as B220--LSCs. Cells were cultured in DMEM containing 15% FBS, 1% Penicillin-Streptomycin solution and 10 ng/mL RMIL310. Particle uptake studies in B220+-LSCs and B220--LSCs were performed for 4 and 24 h in a 37 °C incubator having relative humidity of >80 % and CO2 level of 5 % to maintain pH levels. Cells for all experiments were seeded at an initial density of 1x106 cells/mL for all biological experiments.

2.8.2 Flow Cytometry and analysis After treatment cells were washed two times with PBS before analysis. To get rid of the unnecessary antibody interaction one wash was performed with 0.01-0.1 % Tween-20 in PBS, prior to the two PBS washes. Appropriate controls for each fluorescent color were analyzed prior to recording of the samples and defining gates for further analysis. To detect live and dead cells


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sytox blue staining was performed in PBS, dead cells were positively stained by the dye. All of the flow cytometry data was generated using BD LSRFortessa (BD Biosciences. San Jose, CA). All the data and statistics were analyzed and calculated according to the controls using BD FACS Diva Software v8.0. All of the flow cytometry results were repeated in triplicates to avoid any false positives. Post analysis graphs were plotted using MS Excel 2013.


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2. Results and Discussion

MSN synthesis and functionalization Amino-functionalized, spherical MSNs, denoted MSN_NH2, with a mean size of 170 ± 20 nm and with a narrow particle size distribution were used as the starting particles. A histogram showing the particle size distribution as determined by image analysis is shown in Figure S1. Electron microscopy images of these particles are shown in Figure 1, together with a nitrogen sorption isotherm. The mesopores are clearly seen in the transmission electron microscopy image and reveals a disordered mesoporosity with a narrow pore size distribution often observed for such particles. The narrow pore size distribution is also evident from the narrow pore filling step seen centered around a relative pressure of 0.35 p/p0 in the nitrogen sorption isotherm. The particles had a BET surface area of 900 m2/g and a mean mesopore diameter of 3.5 nm as determined by non-linear density functional theory calculations (see Table 1). 800 ads. volume [cm³/g]

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600 400 200 0 0

0.2 0.4 0.6 0.8 relative pressure p/p0

1

Figure 1. Scanning electron microscopy, (SEM), image of MSN_NH2 particles (left), transmission electron microscopy, TEM, (middle) image of microtomed MSN_NH2 particles, N2 sorption isotherm measured for the MSN_NH2 particles (right).


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MSNs of varying surface charge were obtained by treating MSN_NH2 with different amounts of succinic anhydride in buffer, leading to partial conversion of –NH2 to –COOH groups, as schematically shown in Figure 2 a). MSNs treated with succinic anhydride are denoted MSN_cx, x = 1-5, where the value of x is lower for particles treated with a higher concentration of succinic anhydride, i.e. for particles that should carry a higher concentration of surface –COOH groups. The physicochemical characteristics of all particles are summarized in Table 1, where also the used succinic anhydride concentrations are given. Succinic anhydride treatment did not result in any pronounced changes in the specific surface areas, pore volumes or mesopore diameters as compared to the starting MSN_NH2 particles, highlighting that this treatment did not lead to any changes in the morphological properties of the MSNs (See Table 1). All particles were fully dispersible in HEPES buffer (pH 7.2; 25 mM), as hydrodynamic diameters in the range of 230290 nm were measured in all cases (see Table 1). The effective surface charge of the particles was evaluated by zeta potential measurements in HEPES buffer solution and by determination of the isoelectric point by zeta potential titrations in 1 mM KCl solution (see Figure 3). The higher the amount of succinic anhydride used during post-functionalization, the lower the isoelectric point (IEP) and the more negative the zeta potential value in HEPES buffer, giving strong indication for successful partial conversion of -NH2 groups to –COOH groups to an extent which is dependent on the concentration of succinic anhydride used. However, when the zeta potentials were determined in DMEM buffer supplemented with 15 % FCS, the zeta potential value for all particles was almost identical, -8 to -10 mV, (see Table 1), suggesting that the effective charge of the particles in the presence of serum protein is controlled by the adsorbed proteins and not by the zeta-potential of the starting particles, in good agreement with earlier reports.33,34


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Figure 2. (A) Schema of particle surface functionalization, (B) Schematic overview of the method used for fluorescent labeling and antibody functionalization of the particles.

8

0

isoelectric point

isoelectric point

7

-5 zeta potential (pH 7.4)

6

-10

5

-15

4

-20

3

-25

2

-30

1

-35

0

zeta potential [mV]

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-40 0

1 2 3 nsuccinic anhydride/mparticles [mmol/g]

4


16

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Figure 3. Zeta potentials measured in HEPES buffer solution (pH 7.4) and isoelectric points of particles not carrying an antibody determined by zeta potential titrations as a function of the succinic anhydride concentration used during post-functionalization of MSN_NH2.

Table 1. Characterization data of particles with different surface charges determined by nitrogen sorption measurements, dynamic light scattering and zeta potential measurements. (*) measured in HEPES buffer solution (pH 7.2; 25 mM) and (**) DMEM supplemented with 15 % FCS).

IEP ζ-Pot [mV] with w/o anti* anti B220 B220

particle

nsuccinic anhydride/ mparticle [mmol/g ]

dDLS [nm] *

MSN_NH2

0

230

-6

6.7

MSN_c5

0.02

290

-8

MSN_c4

0.1

250

MSN_c3

0.4

MSN_c2 MSN_c1

SBET [m²/g]

dNLDFT [nm]

ζ-Pot [mV]** (with antiB220)

8.0

920

3.5

-8

6.5

7.9

890

3.4

-9

-14

6.1

7.3

880

3.4

-9

260

-23

5.2

7.2

950

3.5

-10

2

250

-35

4.8

6.2

890

3.5

-10

4

230

-38

4.8

5.8

900

3.5

-9

To detect the particles in biological systems they were labelled with the fluorescent dye ATTO 594-NHS ester in HEPES buffer solution. Successful labelling was confirmed by fluorescence spectroscopy (see Figure S2).


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Antibody (anti-B220) conjugation was performed under mild conditions (pH 7.2; room temperature) through EDC/NHS coupling (see Figure 2B). Therefore the COOH-group on the antibody was used for binding to amino groups still remaining on the particles. The anti-B220 starting-concentration for coupling was fixed to 10 µg/mg particles for all experiments. The quantification of bound antibody was determined by using a fluorescent labelled anti-B220 (antiB220v450). Remaining fluorescent intensity (λexc = 405 nm; λem = 444 nm) of unbound antibody in the supernatant was used for calculation of the amount of conjugated anti-B220. There was no perceptible difference in the resulting antibody concentration on the six different particles with varying surface charges (see Figure 4). The average antibody concentration was 7.2 ± 0.3 µg anti-B220/mg particles in all cases, which corresponds to about 140 antiB220 molecules per particle. Successful anti-B220 binding shows that there were still enough –NH2 groups left on the MSNs after succinylation for an efficient antibody conjugation in all cases, and thus that all MSN_cx particles carry both –COOH and –NH2 groups. A virtually identical surface concentration of anti-B220 on all particles is important for allowing cellular uptake values to be compared without having the concentration of targeting ligand as an additional parameter.

10 manti-B220v450/mparticles [µg/mg]

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8 6 4 2 0

Figure 4. Quantification of antibody bound to the particles by fluorescence spectroscopy of the supernatant.


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The IEP of the particles increased by about 1-1.5 pH units upon anti-B220 conjugation (see Table 1) which is related to contribution of positive charges from the antibody molecules. However, the relative surface charge remained virtually unaffected, and the IEP still decreased in the expected order MSN_NH2 > MSN_c5 > … > MSN_c1. The large influence of the antibody on the zeta-potential despite the relatively low loading is ascribed to the high molecular weight of about 150 kDa of the anti-B220 antibody, and therefore the strong influence on the mean position of the shear plane, a crucial parameter for the zeta-potential reading. However, a large fraction of the surface is still “free” and should have the physicochemical properties of the “bare” particles. Finally, we determined the dissolution profiles of all studied particles and they were very similar. (See Figure S3 A) Furthermore, all MSNs were fully dispersable in 15% FCS and the dispersions were stable for at least 24 h in all cases. (Figure S3 B). Thus, the very similar physicochemical characteristics of the particles apart from their surface charge, and the virtually identical concentrations of targeting ligand, should render them ideally suited for investigating influences of particle charge on serum protein adsorption and cellular specificity.

Serum protein adsorption analysis Serum protein adsorption to the different anti-B220-functionalized particles was studied by SDS-PAGE. Here, the particles were incubated in PBS with 15 % FCS at 37 °C for 24 h, followed by thorough washing. Strongly adsorbed proteins, often referred to proteins being part of the hard corona, were then desorbed in SDS buffer for further analysis by gel electrophoresis. Protein adsorption analyses were also performed after an incubation time of 4 h, but the results were virtually identical. (See ESI Fig. S4) A typical result is shown in Figure 5. Only the results for the anti-B220-tagged particles are shown, as these are the particles that were evaluated in cell


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cultures (see below). However, the serum protein sorption to particles not tagged with antibody was very similar to that observed for the anti-B220-tagged particles, showing that the native surface of the MSNs largely controlled the protein adsorption behavior.

Figure 5. Mass-distribution of adsorbed proteins on anti-B220 functionalized particles after an incubation time of 24 h in PBS + 15 % FCS (1 mg/mL) was analyzed with SDS-PAGE and Comassie staining. PageRuler Unstained Protein ladder was used as a molecular weight standard. The overall serum protein adsorption pattern was similar for the particles MSN_NH2, MSN_c5, and MSN_c4, thus for the particles carrying the most positive charge, as judged based on the IEP values measured for the anti-B220-functionalized particles. However, the particles MSN_c3 to MSN_c1 adsorbed less protein, where the total amount of adsorbed proteins decreased with increasing negative charge of the particles. Especially the MSN_c1 particles adsorb very low amounts of low molecular weight proteins. Semi-quantitative, relative analysis of the serum protein adsorption data was performed for the most prominent bands at 69 kDa (assigned mainly to BSA)35,36 and 25 kDa (assigned mainly to Apolipoprotein 1)35,36 through integration of the


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band intensities. The correlation between the extent of protein adsorption and the isoelectric point of the MSN_cx-B220 is shown in Figure 6 for these two protein molecular weights.

120 25 kDa 69 kDa

100

relative band intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60 40 20 0 5.0

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6.0

6.5

7.0

7.5

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IEP MSN_cx-B220

Figure 6. Dependency of the relative intensities of the bands appearing at 25 and 69 kDa in the SDS-PAGE results shown in Figure 5 and the isoelectric points, IEP, of the MSNs. MSN_c3B220 has an IEP of 7.2 and thus a low level of 69 kDa protein adsorption but a high 25 kDa protein adsorption. The IEPs of the different MSN-B220 particles are given in Table 1.

As seen, there is a clear correlation between the isoelectric points and protein adsorption. A similar correlation is also obtained if the zeta-potential values measured in HEPES for the native particles would be used for comparison; again suggesting that the properties of the native particle surfaces largely controls the degree of protein binding. As discussed above, the zeta-potential values measured in 15% FCS were virtually identical for all particles. These results thus again indicate that the effective particle charge controls the amount and distribution of proteins adsorbed, which in turn determines the effective charge of the particles in serum.


21


In vitro cellular uptake studies The cell specificity of MSN uptake of the different MSNs was evaluated in vitro using a B220+-LSC, and B220--LSC as a model for specific targeting of leukemic stem cells. To mimic more relevant conditions for later in vivo applications all studies were performed in serumcontaining media. Full cellular viability was observed for all particles, as shown in Figure S6.The uptake of MSNs was measured by FACS after 4 h and 24 h of incubation, respectively. In order to have a more quantitative measure for selectivity, we also define a targeting ratio as the uptake in B220+-LSCs and uptake in B220--LSCs either based on the number of particle (ATTO594) positive cells or on the mean fluorescent intensity of the ATTO594 positive cells

80

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pos. cells (ATTO 594) [%]

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(MFI) at a given incubation time. The results are shown in Figure 7.

MFI (ATTO 594)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 7. Particle uptake of anti-B220 functionalized and ATTO594 labelled particles (particle concentration 10 µg/mL) in B220+-LSCs and B220--LSCs analyzed by FACS measurements after 4 (A and C) and 24 h (Band D) of incubation. The percentage of ATTO594 positive cells at a given incubation time is given in A and B, and the corresponding normalized mean fluorescence intensity (MFI) values are given in C and D. The MFI values were corrected for slight variations in the fluorescence intensities of the different MSNs. The ratio of particle uptake between B220+-LSCs and B220--LSCs is also indicated.

At the 4 h time point, the positively charged particles MSN_NH2-B220 to MSN_c4-B220 showed no sign of targetability when evaluated based on the number of ATTO594 positive cells, (Figure 7A) but more particles were taken up by the target B220+-LSCs as compared to the B220--LSCs as shown by the mean fluorescence intensities, MFI, shown in Figure 7C. The highest level of selectivity was observed for the MSN_c3 and MSN_c2 particles, and also here a clearly higher mean particle uptake per cell was observed for the target cells as compared to the non-target cells as judged based on the MFI values. The targeting ratio was relatively low when calculated based on the % of positive cells, but a high targeting ratio of 4.6 was observed for the MSN_c3 particles when calculated based on the MFI values. Furthermore, as the absolute level of non-specific uptake of particles is also of importance for avoiding therapy-induced sideeffects, we also note that the MFI values for MSN_c3 were clearly lower than those observed for MSN_c2. At the 24 h time-point, the number of particle positive B220+-LSCs exceeded that of B220--LSCs for all particles, as did the MFI values. While the targeting ratios calculated based on the MFI values were relatively similar for all particles after 24 h incubation, the targeting ratios calculated based on the percentage of positive cells were the highest for MSN_NH2,


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MSN_c2 and MSN_c3 particles. A lower specificity after 24 h of incubation as compared to 4h of incubation could be related to several effects, including partial detachment of the anti-B220 ligand upon partial particle dissolution, smearing of the uptake curves as the particle uptake is not as strongly under kinetic control at longer incubation times, and time-dependent differences in the composition of and amount of proteins in the corona. The composition of the protein corona will naturally change during the course of the experiments, and especially so during the first hours of incubation. However, particles with a low level of protein adsorption at 24 h can also be expected to show a lower level of protein adsorption also at short time-points. In any case, the 4 h time-point is probably of higher relevance in vivo, as the blood-circulation time of the particles could otherwise become limiting for target specificity. Despite of these inherent limitations, we further evaluate the protein adsorption to the cell specificity through correlation of the MFI-based targeting ratios measured after 4 h of incubation to the intensity of the two strongest bands observed in the SDS-PAGE analysis for the different particles under study. The corresponding plot is shown in Figure 8. As clearly seen, the cell specificity increases with increasing protein adsorption for both proteins for all particles but for MSN_c3, for which the highest selectivity was observed. For MSN_c3, the relative adsorption of proteins with a molar mass of about 69 kDa (probably mostly BSA) was low, but that of proteins with a molar mass of 25 kDa (most probably apolipoprotein A1) was still high.


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120 25 kDa

MSN_c3-B220

69 kDa

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relative band intensity

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80 60 40 20 0 0

1

2

3

4

5

MFI-based targeting ratio (4 h)

Figure 8. MFI-based targeting ratio as a function of relative contents of proteins with a molecular mass of 25 and 69 kDa at after 4 h incubation.

This data clearly suggest that serum protein adsorption can have a positive effect on targetability at this incubation time (4h), and that the composition of the protein corona also seem to play an important role for further enhancing MFI-based specificity. However, we note that the targeting ratio calculated based on the number of ATTO594 positive cells followed the same trend as the MFI-based ratios, which is why one would arrive at a similar conclusion also in this case. Our data stand in apparent contrast to some previous observations, where the targetability of transferrin functionalized silica nanoparticles was lost upon serum protein adsorption.19 The reasons for these differences could be several, but could include the absolute number and identity of proteins adsorbed in the two different cases, the size of the targeting protein/antibody used, and the different cells under study. However, one important factor may be that the B220 antigen is not internalized, and therefore serum protein adsorption can aid in increasing the endocytosis rate of the particles in our case. On the other hand, in a recent study related to the uptake of


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PEGylated polystyrene nanoparticles by RAW264.7 cells in vitro, a positive influence of the adsorption of clusterin proteins (also referred to as apolipoprotein J) on specificity was observed, which further suggests that protein adsorption could indeed have an important influence on cellular uptake kinetics, and, depending on the composition of the corona, even on the level of enhancing cell specificity.28 However, we cannot exclude that serum protein adsorption has an influence on the orientation of the anti-B220 antibody at the particle surface, which also could enhance targetability or that some specific interaction between less abundant proteins in the corona may contribute to the observed effects. Clearly more research is needed in order to shed some more light on the details of the means by which serum protein adsorption affects targetability. However, the positive influence of serum protein adsorption on cellular selectivity is still clearly demonstrated by our data.

Cellular specificity evaluations In order to evaluate if active cellular targeting involved the B220 antigen and was not only related to protein corona-mediated nanoparticle-cell interactions, the uptake of MSN_c3 particles carrying either the anti-B220 or the anti-human CD9, which is less specific to murine AML cells, at identical concentrations were investigated for both B220+-LSCs and B220--LSCs. Protein adsorption analysis of MSN_c3 with anti-B220 and anti-CD9 functionalization is shown in Figure S5. The choice of the MSN_c3-particles for this evaluation was based on the low level of unspecific uptake of MSN_c3-B220 particles in B220--LSCs, and the high targeting ratios observed after 4 h of incubation. Importantly, serum protein adsorption to the anti-B220 and antihuman CD9 antibody-tagged MSNc3 particles were identical. While an almost identical uptake of both particles was observed for the B220--LSCs, a clearly higher uptake of MSN_c3-B220 as


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compared to MSN_c3-CD9 particles was observed for the B220+-LSCs, as shown in Figure 9. There results give further support for an active role of anti-B220 antibody – B220 antigen interactions for the observed enhanced uptake of MSN_c3-B220 particles by the B220+-LSCs. The higher uptake of MSN_c3-CD9 particles in the B220+-LSCs as compared to B220--LSCs can be explained by the fact that 10 % of the B220+-LSCs are also positive for CD9, further giving support for the involvement of specific antibody-antigen interactions in enhancing cellular uptake.

100 pos. cells (ATTO 594) [%]

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80

MSN_c3-CD9 MSN_c3-B220

60 40 20 0 B220- LSCs

B220+ LSCs

Figure 9. Percentage of B220--LSCs and B220+-LSCs positive for MSN_c3-CD9 and MSN_c3B220 particles (10 µg/mL) after 24 h of incubation in 15 % FCS.


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Conclusions and outlook We have studied the influence of particle surface charge on the specificity of active targeting of antibody-tagged mesoporous silica nanoparticles, with special focus on the influence of protein adsorption on targetability. Our results suggest that serum protein adsorption can play an important role in enhancing specificity of nanoparticle uptake, which may be a combination of multivalent serum protein and targeting ligand-mediated nanoparticle-cell interactions and/or a more “passive” enhancement of cellular internalization kinetics upon the presence of adsorbed proteins on the nanoparticle surface. Here, an important reason for this observation may the fact that the B220 antigen is not an internalizing receptor, which is why serum protein adsorption could aid in enhancing the endocytosis kinetics once the particles are targeted to the outer cell membrane of the target cells. Influences on the results originating from differences in particle dispersion stabilities, targeting ligand concentration, and particle size could be excluded. MSN particles carrying a zwitterionic surface containing both –NH2 and –COOH groups in the right proportion appear to be promising candidates for antibody-based actively targeted drug delivery to AML cancer stem cells, but the results are suggested to also be of general importance for nanoparticle-based drug delivery. On the other hand, it is also known that the extent of serum protein adsorption also strongly affects blood-circulation times of nanoparticles in vivo, and an effective targeted drug delivery using nanoparticulate vectors is thus suggested to need optimization in terms of a long enough blood circulation time and the kinetics of cellular internalization of the particles by the target cells, parameters which generally show opposite dependencies on serum protein adsorption.

ASSOCIATED CONTENT


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Particle size histogram, fluorescence spectra of antibody functionalized particles, particle dissolution and dispersion stability studies, protein adsorption analysis of anti-B220 and antiCD9 functionalized MSN_c3, cytotoxicity analysis of particles in B220+-LSCs and B220—LSCs as well as quantification of anti-human/mouse B220 and anti-human CD9 epitopes on murine B220- and B220+-LSCs.

AUTHOR INFORMATION Corresponding Authors Mika Lindén *Email: [email protected]. Tel: +49 731 50 22730 Fax: +49 731 50 22733 Christian Buske *Email: [email protected]. Tel: +49 731 500 65888 Fax: +49 731 500 65822

ACKNOWLEDGMENT We like to thank Cornelia Egger for carrying out the nitrogen sorption measurements, and Felix Pascher for technical support regarding SDS-PAGE measurements. We also thank Margit Lang from the Institute for Analytical and Bioanalytical Chemistry for carrying out the ICP-AES


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measurements. We also would like to thank the FACS core facility team for allowing access to the BD LSR fortessa II.

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