Surface Modification of Polysaccharide-Based Nanoparticles with PEG

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Surface Modification of Polysaccharide-Based Nanoparticles with PEG and Dextran and the Effects on Immune Cell Binding and Stimulatory Characteristics Denise Bamberger, Dominika Hobernik, Matthias Konhäuser, Matthias Bros, and Peter R. Wich Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00507 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 30, 2017

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Molecular Pharmaceutics

Surface Modification of Polysaccharide-Based Nanoparticles with PEG and Dextran and the Effects on Immune Cell Binding and Stimulatory Characteristics Denise Bamberger1‡, Dominika Hobernik2‡, Matthias Konhäuser1, Matthias Bros2, Peter R. Wich1* 1

Department of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Staudingerweg 5, Mainz 55128, Germany

2

Department of Dermatology, University Medical Center, Johannes Gutenberg-University Mainz, Obere Zahlbacher Straße 63, 55131 Mainz, Germany

KEYWORDS: nanoparticles, acetalated dextran, PEGylation, DEXylation, immune cells, macrophages, dendritic cells

ACS Paragon Plus Environment

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ABSTRACT Surface modifications of nanoparticles can alter their physical and biological properties significantly. They effect particle aggregation, circulation times, and cellular uptake. This is particularly critical for the interaction with primary immune cells due to their important role in particle processing. We can show, that the introduction of a hydrophilic PEG layer on the surface of the polysaccharide-based nanoparticles prevents unwanted aggregation under physiological conditions and decreases unspecific cell uptake in different primary immune cell types. The opposite effect can be observed with a parallel-performed introduction of a layer of low molecular weight dextran (3.5 and 5 kDa) on the particle surface (DEXylation) that encourages the nanoparticle uptake by antigen-presenting cells like macrophages and dendritic cells. Binding of DEXylated particles to these immune cells results in an upregulation of surface maturation markers and elevated production of proinflammatory cytokines, reflecting cell activation. Hence, DEXylated particles can potentially be used for passive targeting of antigen presenting cells with inherent adjuvant function for future immunotherapeutic applications.


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INTRODUCTION Nanocarriers gain increasing importance in medical science for a variety of drug delivery applications.1-3 When looking at immunotherapeutic approaches, high requirements are placed on the particle systems, including biocompatibility and biodegradability, as well as lack of cytotoxicity and tailor-made immune-stimulatory effects.4-7 Potential therapeutic payloads include nucleic acid-based vaccines (e.g. DNA, mRNA, small interfering RNA) as well as protein/peptide-based

antigens,

and

various

classes

of

adjuvants.

However

today,

immunotherapy is still in need of efficient delivery systems. In this context, non-viral, cationic (bio)polymer-based nanoparticle systems of around 50–250 nm in diameter are in particular of interest because of their versatility in payload transport and the possibility of easy chemical modification.8 A highly promising carrier system for the delivery of nucleic acids as well as (bio)therapeutics represents the acetalated dextran (Ac-DEX) system, a polysaccharide-based carrier system developed by the group of Fréchet.9,

10

Benefits of Ac-DEX particles are their low-toxicity,

biocompatibility and their tunable acid degradability.11 In slightly acidic tissue, like in inflamed regions, or in the vicinity of tumor cells, as well as in specific cellular compartments like lysosomes and late endosomes the Ac-DEX hydrolyzes to water-soluble dextran and thereby releases the encapsulated cargo.9,

12

Up to now, a number of studies showed the remarkable

versatility of Ac-DEX particles to encapsulate and release e.g. siRNA13, CpG DNA oligonucleotides14, protein antigens (OVA)11 and other hydrophobic therapeutics (imiquimod).15 However, there are nearly no studies on extensive modifications of the particle surface and the


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resulting effects to alter and influence their biological characteristics, in particular stealth effects and the resulting cellular uptake by immune cells. Challenges of systemic applications in vivo are the mononuclear phagocyte system (MPS) and filtration systems like liver, spleen, and kidneys.8 Surface modifications with polyethylene glycol (PEG) or polysaccharides are described to act as stealth compounds that alter circulation times, cellular uptake and may reduce the interaction with plasma proteins.16-22 In this context, surfacebound proteins can play a significant role in the rapid elimination of the nanoparticles as well as the immunological response in vivo.23-25 In this work, we use a spermine-modified Ac-DEX (Sp-Ac-DEX) system that can encapsulate nucleic acids like DNA, mRNA, and siRNA.13 The surface of the particles was modified in parallel with linear polyethylene glycol chains (PEG) and the linear polysaccharide dextran (DEX). The resulting particles were then compared with non-modified cationic particles in respect to their stealth-like properties and cell-interacting behavior. Since immune cells play an important role for the elimination of particles from the blood and constitute the main target populations of immunotherapeutic approaches, the binding, uptake, and potential effects on cellular activation of the surface-modified Sp-Ac-DEX particles was tested on murine spleen cells and bone marrow-derived dendritic cells (BMDC) as well as macrophages (BMM).

MATERIALS AND METHODS General. All reagents were obtained from Sigma-Aldrich (München, Germany) unless otherwise noticed and were used without further purification. Dextrans 3.5 kDa and 5 kDa were purchased

from

Pharmacosmos

(Holbaek,

Denmark).

Methoxy

polyethylene

glycol

succinimidyl-active esters (NHS-PEG) 2 kDa and 5 kDa were purchased from Rapp Polymere


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(Tübingen, Germany). Fmoc-L-Cys-OH*H2O was obtained from Iris Biotech GmbH (Marktredwitz, Germany). 2-Aminoethanethiol was purchased from Acros Organics (Geel, Belgium). High purified water (dd-H2O) for buffers and particle washing steps was cleared by a Direct-Q® 5 UV Remote Water Purification System (Merck Millipore, Germany). Endotoxinfree dd-H2O (B. Braun Melsungen AG, Melsungen, Germany) was used for particle preparations. Water used during particle preparation was adjusted to pH 8 with triethylamine (TEA, Carl Roth, Germany, approx. 0.001%). All buffers were filtrated through Rotilabo®-syringe filters (sterile, CME membrane, pore size 0.22 µm, Carl Roth, Germany). Fluorescence and absorbance measurements were obtained in microplate-based assays using a TECAN Infinite® M200 Pro. rmGM-CSF (recombinant murine granulocyte macrophage colony stimulating factor) was obtained from R&D Systems (Wiesbaden, Germany). All antibodies were purchased form Biolegend (San Diego, CA) or affymetrix/eBiosciences (Santa Clara, CA). C57BL/6J mice were obtained from Harlan (Indianapolis, Indiana, USA) and were maintained in the central animal facilities of the Johannes Gutenberg-University of Mainz under specific pathogen-free conditions on a standard diet. The “Principles of Laboratory Animal Care” (NIH publication no. 85–23, revised 1985) were followed.

Synthesis of spermine-functionalized acetalated dextran. The synthesis of sperminefunctionalized acetalated dextran (Sp-Ac-DEX) was described before by Cohen et al.13 Briefly, dextran (from Leuconostoc mesenteroides, 9–11 kDa) was partially oxidized with NaIO4 followed by an acetalation of the partially oxidized dextran with 2-methoxypropene. In the last step, the oxidized and acetalated dextran was functionalized with spermine using NaBH4 for the


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reductive amination. The product Sp-Ac-DEX was obtained after purification and lyophilization as a colorless powder.

Preparation of empty particles. The preparation of dextran nanoparticles was performed similar as described previously by Bachelder et al. with minor modifications.9 Briefly, 10 mg of Sp-Ac-DEX was dissolved in 800 µL dichloromethane (DCM). A primary emulsion was prepared by adding 50 µL phosphate buffered saline (PBS) to the dextran solution and tip sonication of the sample with a Bandelin Ultrasonic Homogenisator Sonoplus UW 70 (power 75%, cycle 70%). Afterwards, 4 mL of a polyvinyl alcohol (PVA) solution (3% in PBS, 13– 23 kDa, 87–89% hydrolyzed) was added and sonicated. The secondary emulsion was stirred vigorously overnight to remove all DCM and purified by ultracentrifugation (45,000 x g, 20 min.). Finally, 100 µL 0.3% PVA was added as a cryoprotectant before lyophilization to yield a colorless, fluffy powder (approx. 42% of the initial used material).

Encapsulation of dextran-Oregon Green®. 10 mg of Sp-Ac-DEX was dissolved in 800 µL DCM and 50 µL PBS containing 5 µg dextran-Oregon Green® 488 (10,000 MW, anionic, Thermo Fisher Scientific, Darmstadt, Germany). Particles were prepared by a double emulsion technique as described above and were obtained as a bright yellow, fluffy powder (approx. 41% of the initial used material). Dextran-Oregon Green® 488 (dex-OG488) was used to directly compare the varying particle modifications by detection of the fluorescent dye. The lyophilized, dex-OG488-containing Sp-Ac-DEX nanoparticles were split in equal batches before modifying the surface to ensure comparable loading of differently functionalized nanoparticles with dexOG488.


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Synthesis of bifunctional aldehyde- and thiol-reactive linker (4-mercaptobutanehydrazide). The synthesis of 4-mercaptobutanehydrazide was described previously by von Delius et al. (2010).26

Synthesis of thiol-functionalized dextran. For the conjugation of dextran with the bifunctional linker 4-mercaptobutanehydrazide, 1.0 g dextran was dissolved in 2 mL sodium phosphate buffer (4%, pH 7.4). After total dissolution of the dextran, the crosslinker was dissolved in buffer (0.2 g·mL-1) with 5-times molar excess to the aldehyde content of the dextran. The linker was added dropwise to the dextran solution and stirred overnight. Finally, the thiolmodified dextran was purified by dialysis using a regenerated cellulose membrane (Spectra/Por® 6 dialysis membrane, regenerated cellulose, molecular weight cut off (MWCO) 1 kDa, Carl Roth, Karlsruhe, Germany) against deionized water. After lyophilization, a colorless, styrofoamlike product was obtained. The amount of thiol groups was determined by Ellman’s assay (see Supporting Information Table S1) using a protocol described by Thermo Scientific.

PEGylation of Sp-Ac-DEX nanoparticles. Particles were suspended in PBS buffer (pH 8.0, adjusted with sodium hydroxide) at a concentration of 2 mg·mL-1. PEG-NHS (10x molar excess, 50 mg·mL-1, 2 or 5 kDa) was dissolved in buffer and added dropwise to the nanoparticle suspension. For the calculation of the required amount of conjugation reagent, an amount of 150 nmol primary amines per 1 mg particles was estimated, based on average results shown by fluorescamine assays. The reaction mixture was incubated under stirring for 2 hours. Particles were purified by ultracentrifugation (45,000 x g, 20 min.) and rinsing the pellet with dd-H2O at


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pH 8 (two times, 3 mL each). Before lyophilization, 0.3% PVA was added as cryoprotectant and a colorless fluffy powder was obtained (approx. 75% of the initial weight). The degree of functionalization was determined by fluorescamine assay based on a protocol by BioTek Instruments and described in the Supporting Information (Table S2).

DEXylation of Sp-Ac-DEX nanoparticles. Nanoparticles were suspended in PBS buffer (1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4) at a concentration of 2 mg·mL-1. First, the bifunctional crosslinker 3-sulfo-N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate sodium salt (sulfo-SMCC, TCI chemicals, Eschborn, Germany) was conjugated to the primary amines on the surface of the Sp-Ac-DEX particles. The NHS ester coupling led to an introduction

of

reactive

maleimide

groups

on

the

particle

surface.

Sulfo-SMCC

(MW 436.37 g·mol-1, 10x molar excess, 2 mg·mL-1) was dissolved in buffer and added dropwise to the particle suspension. The reaction was incubated under gentle stirring for 2 hours and the unconjugated crosslinker was removed by ultracentrifugation (45,000 x g, 20 min.) and removal of the supernatant. Afterwards, the particle pellet was resuspended in buffer (2 mg·mL-1), followed by the addition of a solution of thiol-modified dextran in buffer (5x molar excess, 15 mg·mL-1). The reaction was incubated for further 4 hours and purified by ultracentrifugation (45,000 x g, 20 min.). The resulting pellet was rinsed with dd-H2O pH 8 (2-times, 3 mL). Before lyophilization, 0.3% PVA was added as cryoprotectant and a colorless fluffy powder was obtained (75–80% of the initial weight). The degree of functionalization was determined by fluorescamine assay based on a protocol by BioTek Instruments and described in the Supporting Information (Table S2).


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Particle size determination. Nanoparticle Tracking Analysis (NTA) was performed with a NanoSight LM 10 microscope (Malvern Instruments, Malvern, United Kingdom) equipped with a green laser (532 nm) and a sCMOS camera. Unless otherwise noticed, samples were prepared in PBS at concentrations of approx. 2 µg·mL-1 and sonicated (Bandelin Sonorex RK 102 H) for 20 seconds before each measurement. Movements of the particles were recorded as videos for 30 seconds at 25 °C in triplets. The size calculation was performed with NTA software version 3.1 build 3.1.54.

Zeta potential of particles. Zeta potential (particle charge) was measured with a Malvern Zetasizer Nano ZS instrument (Malvern instruments, Malvern, United Kingdom) using a clear disposable zeta cell. Three measurements with 12 individual runs were performed at 25 °C. Particle samples were prepared at concentrations of 0.1 mg·mL-1 in HEPES buffer (25 mM, pH 7.4). The refractive index (RI) of the dispersant (preset: water) was adjusted to 1.330 and the viscosity to 0.8872 cP with a dielectric constant of 78.5. The RI of the particle material dextran was set to 1.590. The resulting data was analyzed by the model of Smoluchowski with the Malvern Zetasizer software 6.20.27

Generation of bone marrow-derived dendritic cells (BMDC) and macrophages (BMM). Bone marrow-derived progenitor cells were generated as described previously28 with some modifications. Briefly, tibias and femurs of 10–12 week old C57BL/6J mice were removed and bone marrow was collected. The cells were suspended in EMEM supplemented with 2% FCS and 100 IU·mL-1 penicillin and 100 µg·mL-1 streptomycin. Erythrocytes were lysed by incubating with Geys solution (155 mM NH4Cl, 10 mM KHCO3, 100 µM EDTA, pH 7.4) for


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1 min. Cells were split and resuspended in either BMDC culture medium (IMDM supplemented with 5% (v/v) FCS, 10 ng·mL-1 GM-CSF, 50 µM β-mercaptoethanol, 2 mM L-glutamine and pen/strep (100 IU·mL-1 penicillin, 100 µg·mL-1 streptomycin) or BMM culture medium (IMDM supplemented with 5% FCS, 5% horse serum, 50 µM β-mercaptoethanol, 2 mM L-glutamine, pen/strep (100 IU·mL-1 penicillin, 100 µg·mL-1 streptomycin) and 25 ng·mL-1 recombinant murine Macrophage Colony Stimulating Factor (ImmunoTools, Frieosythe, Germany). Cells were counted and seeded in 94 mm petri dishes with a cell density of 2x106 cells/10 mL for BMDC and 3x106 cells/10 mL for BMM, respectively. Cells were cultured for 6–8 days with addition of 5 mL medium at day 3 and exchange of 5 mL medium at day 6.

Isolation of spleen cells from C57BL/6J mice. Spleens were removed from mice and mechanically disrupted by grinding through a 40 µm cell strainer (Greiner bio-one, Kremsmünster, Austria). Spleen cells were washed with EMEM supplemented with 2% FCS, 100 IU·mL-1 penicillin and 100 µg·mL-1 streptomycin and erythrocytes were lysed incubating with Geys solution. After further washing, spleen cells were seeded in IMDM supplemented with 5% (v/v) FCS, 50 µM β-mercaptoethanol, 2 mM L-glutamine and pen/strep (100 IU·mL-1 penicillin, 100 µg/mL streptomycin).

Cell Viability. Spleen cells (106) and BMDC (3x105) were seeded into wells of 24-well plates (1 mL) and were incubated with modified Sp-Ac-DEX particles (10 µg·mL-1 each) for 24 hours. Then, cells were washed and incubated with Alexa Fluor®647-labeled Annexin V (apoptosis marker) and necrosis marker 7-AAD (both from Biolegend, San Diego, CA, USA). Samples were assayed by flow cytometry.


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Binding studies of modified Sp-Ac-DEX particles. Spleen cells, BMDC and BMM were seeded into wells of 24-well plates (1 mL) with 3x105 cells and were incubated with 10 µg·mL-1 of surface-modified Sp-Ac-DEX particles. Binding of particles was monitored via flow cytometry at different time points after the start of incubation. Cells were incubated with cell type-specific antibodies specific for DC (APC-, PECy7- or BV421-conjugated anti-CD11c, clone N418), macrophages (BV421- or APC-labeled anti-F4/80, clone BM8), T cells (PerCP- or PElabeled anti-CD3e, clone 144-2C11; BV510-labeled anti-CD4, clone GK1.4; PE-labeled antiCD8a, clone 53-6.7), and B cells (PerCP-labeled anti-CD19, clone 1D3). Cell activation was assessed by co-incubation with additional antibodies (PE-labeled anti-CD86, clone GL1; APClabeled anti-CD40, clone 1C10; APC-labeled anti-CD25, clone PC61). All analyses were performed using a Canto II flow cytometer (BD, Heidelberg, Germany) and were analyzed with FlowJo software v7.6.5 (FLOWJO, Ashland, USA). To assess potential immunomodulatory effects of Sp-Ac-DEX particles on immune cells, the expression of pro-inflammatory cytokines and surface activation markers was analyzed (see below).

Confocal laser scanning microscopy. Cellular uptake of Sp-Ac-DEX particles was assayed by confocal laser scanning microscopy. BMDC (2.5x105 cells) were seeded in 48-well plates, and BMM (2.5x105 cells) in NuncTM Lab-TekTM 8-well Chamber SlidesTM (Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated with surface-modified Sp-Ac-DEX particles (each 10 µg·mL-1) for 4 hours. Cells were washed twice with PBS, and the nuclei were stained using Hoechst 33342 (1 µg·mL-1, 20 min., RT). Cells were washed twice to remove


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excess dye. Cell membranes were labeled with CellMaskTM Orange (Thermo Fisher Scientific) directly prior to measurement.

Blocking of scavenger receptors. To clarify the role of scavenger receptors in Sp-Ac-DEX particle binding, according inhibitors were applied. For this, BMDC were incubated in parallel settings with fucoidan (25 µg·mL-1), dextran sulfate and chondroitin sulfate (each 25 µg·mL-1), and polyinosinic acid and polycytidylic acid (each 12.5 µg·mL-1) for 45 min, followed by application of Sp-Ac-DEX particles (10 µg·mL-1). After another 3 hours, BMDC were incubated with anti-CD11c antibody, and assayed for Sp-Ac-DEX particle binding by flow cytometry.

Analysis of cell surface activation markers. Spleen cells (106 cells) were seeded in 24-well suspension culture plates (1 mL) and were incubated with Sp-Ac-DEX particles (each 10 µg·mL1

). After 24 hours cells were harvested, incubated with antibodies (see above), and analyzed by

flow cytometry for expression of CD11c, F4/80, CD19, CD86 (activation marker on APC, clone GL1), CD3e, CD4 (clone RM4-5), CD8 (clone 53-6.7), and CD25 (activation marker on T cells, clone PC61).

Cytokine production. Spleen cells (106) and BMDC (105) were seeded in wells (100 µL) of 96-well tissue culture plates and were incubated with 10 µg·mL-1 Sp-Ac-DEX particles. Supernatants were collected after 24 hours. TNF-α was measured using BDTM Cytometric Bead Array (BD, Heidelberg, Germany) and analyzed with FCAP ArrayTM software v.2 (BD, Heidelberg, Germany).


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RESULTS AND DISCUSSION It has been previously shown that cationic-modified Ac-DEX particles loaded with siRNA are efficiently taken up by cells and induce a gene knockdown.13 However, for advanced delivery applications, an unspecific uptake of nanoparticles has disadvantages. Hence, extended metabolic circulation times and a cell specific uptake are preferred to reduce side effects and lower the costs of the administered drugs. When particles enter the blood circulation they face several challenges. Directly after intravenous application, they are confronted by different blood components like immune cells and various proteins. These blood proteins differ in size, functionality and charge. Depending on the characteristics of the particle surface, they can adsorb on the particles and the resulting hard and soft protein corona can significantly change their properties.24,

25

This may result in

unwanted recognition by immune cells of the mononuclear phagocyte system (MPS). Binding of monocytes/macrophages as located in liver and spleen will lead to rapid elimination of the particles from the blood and degradation of the particles.29 Therefore, substantial protein adsorption and unspecific cellular uptake of the particles should be prevented. Polyethylene glycol (PEG) has been extensively described previously as a compound that modulates surface properties of (nano)materials.18,

30,

31

In comparison, the use of

polysaccharides as surface decorating material shows only limited research coverage.20,

21

Selected publications focus on the changes in protein adsorption on surface-decorated films and surfaces.32 For example, Osterberg et al. showed that a surface modification of polystyrene well plates with DEX effectively reduced adsorption of the protein fibrinogen.33 Similarly, Lemarchand et al. studied the interaction of poly(caprolactone)-dextran nanoparticles with biological media. The copolymer assembles into micelles and adsorption of specific proteins


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depending on the molecular weight of the polysaccharide.22 Karmali et al. describes crosslinked dextran as hydrogel cover of iron oxide nanoparticles. The coating increases in vivo circulating time and shows antifouling properties.34 Dextran and heparin also have been recognized as possible PEG replacement coating materials with stealth properties and abilities to inhibit opsonization and complement activation.35 Recently, first examples of polysaccharide specific interactions with selected cell types have been reported using hyaluronic acid36,

37

and

chondroitin sulfate.38 The surface-decorated NP show receptor-specific activity for a selective and efficient cellular uptake. Up to now, no cell-specific uptake using dextran as hydrophilic surface cover of nanoparticles has been reported. Therefore, we wanted to study the resulting biological effects of a dextrandecoration in combination with possible changes regarding the physical properties of the nanoparticles. For this, we developed a selective method to attach linear, low molecular weight dextran (3.5 and 5 kDa) to the surface of spermine-functionalized dextran nanoparticles (Sp-AcDEX). The method is universally applicable for nanoparticles bearing surface amines. For direct comparison, we also attached polyethylene glycol of similar molecular weight to the surface of our nanoparticles.

PEGylation and DEXylation of nanoparticles. PEGylation was performed with a commercially available NHS-PEG as described previously (Error! Reference source not found.).39


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Figure 1. Primary amines on the Sp-Ac-DEX nanoparticle surface were modified with the NHSactivated PEG chains (2 and 5 kDa) in phosphate buffer pH 8 for 2 hours. The degree of surface modification was determined by comparing the amount of surface amines on the particles before and after PEGylation using a fluorescamine assay. Before modification every particle contains approx. 90,000 primary amine groups on the surface (81.1 nmol per mg NP). After the modification with 2 kDa PEG, the amount of surface amines is reduced to approx. 77,000 per particle (69.4 nmol per mg NP) resulting in the introduction of 11.7 nmol PEG per mg particles. And after the modification with 5 kDa PEG the amount of free amines is reduced to approx. 67,000 per particle (61.2 nmol per mg NP) resulting in the introduction of 19.9 nmol PEG per mg particles. This results in an overall surface PEGylation of 14–25% based on the initial amount of accessible surface amines (see Supporting Information, Table S2). Depending on the steric hindrance of the PEG chains it might be possible that not all free amine groups can be detected after the surface modification. This will lead to a higher degree of modification determined by fluorescamine assay. Therefore, PEGylation was determined by two additional quantification methods. The methoxy group of the PEG chains on the Sp-Ac-DEX particle surface can be quantified by 1H-NMR with an internal standard, pyridine. This led to


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12.9 nmol PEG (5 kDa) per mg particles. A third method to quantify the degree of PEGylation is the fluorescence measurement of Fmoc-modified NHS-PEG (5 kDa) resulting in 12.3 nmol PEG per mg particles. Both methods, Fmoc quantification and 1H-NMR, show the same range of PEGylation but the results are minimal lower compared to the fluorescamine assay (see Supporting Information, Table S3). These results confirm the assumption that PEG chains slightly shield the primary amines from the reaction with fluorescamine.

For the DEXylation of Sp-Ac-DEX particles, dextran was activated by conjugation with a bifunctional mercaptobutane hydrazide linker resulting in thiol-modified dextran (Figure 2A). The degree of functionalization was determined by Ellman’s reagent and N-acetylcysteine as standard. The functionalization yielded in approx. 190 mmol thiols per mol dextran 3.5 kDa (19% functionalization) and 110 mmol thiols per mol dextran 5 kDa (11% functionalization). This means that every 5th dextran chain 3.5 kDa and every 10th dextran chain 5 kDa carries a thiol group at one end. For the conjugation of the dextran-thiol, Sp-Ac-DEX particles were modified with the bifunctional linker sulfo-SMCC under mild biorthogonal reaction conditions (Figure 2B). The dextran-thiol was then attached to the particle surface via the introduced maleimide groups on the particle surface (Figure 2C).


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Figure 2. Functionalization of dextran molecules (3.5 and 5 kDa) with the bifunctional thiollinker 4-mercaptobutanehydrazide (A); Reaction of Sp-Ac-DEX particles with the aminereactive linker sulfo-SMCC (B); Surface modification of SMCC-activated Sp-Ac-DEX particles with the thiol-functionalized dextran leads to DEXylated particles (C). The degree of DEXylation was determined by quantification of the amount of maleimide groups on the SMCC-activated NPs before and after the DEXylation. Therefore, we performed two orthogonal types of assays. The first method is a standard adsorption assay for the quantification of maleimides. It includes the addition of a defined excess of 2-aminoethanthiol followed by the quantification of unreacted 2-aminoethanethiol by the Ellmann`s method. The second method is a fluorescence assay for the quantification of Fmoc-cysteine that reacts with the surface maleimides of SMCC-activated particles before and after DEXylation. Both methods determine similar surface modifications. The assays show that in average around 67-69 nmol/mg maleimides were introduced with the SMCC-linker on the particle surface. (in comparison:


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initially 81.1 nmol/mg amines were available on the surface of Sp-Ac-DEX particles). After DEXylation with 3.5 kDa dextran 37–39% of maleimides were modified (25.6–26.2 nmol DEX per mg NP), whereas after DEXylation with 5 kDa dextran only 23-29% of maleimides were modified (15.7-19.8 nmol DEX per mg NP). Back-calculated to the amount of initial surface amines, this results in a surface modification of 31-32% (with 3.5 kDa dextran), and a surface modification of 19-24% (with 5 kDa dextran) (see Supporting Information, Table S4) Based on the degree of surface modification we calculated the grafting density of both types of polymers and the determined the most likely resulting polymer conformation on the particle surface. Both DEX and PEG chains are densely distributed over the particle surface and are in a brush like confirmation (see Supporting Information, Table S5).

Particle size and zeta potential. Particle size (diameter) was measured before and after surface modification using Nanoparticle Tracking Analysis (NTA) (Table 1). The average size of the entire particle sample is described by the mean value (comparable to “Z-average” in DLS), whereas the average size of the largest population of particles by numbers in the sample is described by the mode value (comparable to “number” in DLS). The aggregation behavior can be interpreted using the standard deviation (SD) value. Large values are an indicator of a polydisperse sample (comparable to PDI in DLS). Non-modified Sp-Ac-DEX particles had a mean size of approx. 150 nm in diameter right after the double emulsion preparation. However, after purification and lyophilization these particles increase their mean value to a size of 190 nm, which can be attributed to some aggregation behavior. Accordingly, SD values increase from 56 nm to 88 nm. As expected, the attachment of the additional surface layers itself does not significantly change the particle size, as can be seen in only minimal changes in the mode values, which represent the


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sizes of the main particle populations. However, when looking at the mean sizes of PEGylated particles (approx. 160 nm) and DEXylated particles (approx. 150 nm) a reduction of average size compared to the non-modified particles (approx. 190 nm) is visible. This indicates that the surface-modified particles show a reduced aggregation behavior compared to the non-modified particles due to a hydrophilic shielding of the particle material. Changes in particle properties can also be observed when looking at the net charge of the particles. The zeta potential of non-modified particles has a value of +12.8 mV due to exposed protonated spermine molecules on the particle surface. After conjugation with PEG and DEX the number of primary amines decreases. This can be seen in a lower zeta potential of PEGylated (+9.7 mV for 2 kDa; +4.6 mV for 5 kDa) and DEXylated (+3.7 mV for 3.5 kDa; +2.9 mV for 5 kDa) particles (Table 1). Particle morphologies were analyzed by scanning electron microscopy (SEM) (see Supporting Information Figure S4) and showed no obvious change in the morphology of the surfacemodified particles as well as no significant growth of the particle size.

Table 1. Overview of size and surface potential of the nanoparticles, as well as the polymer conformation of the polymers on the surface. Particle Size measured by NTA Particle Modification

Mean / d.nm

Mode / d.nm

SD / d.nm

Zeta potential / mV

Polymer Conformation on Particle Surface

Sp-Ac-DEX NP 150.1 ± 5.48 123.1 ± 6.05 56.0 ± 7.03 – – (before lyophilisation) Sp-Ac-DEX NP 190.8 ± 33.2 135.7 ± 24.1 88.4 ± 21.8 12.8 ± 1.25 – NP PEGylated 2 kDa 163.2 ± 24.8 126.3 ± 6.11 74.7 ± 20.9 9.7 ± 0.58 brush NP PEGylated 5 kDa 165.0 ± 16.1 127.6 ± 3.32 70.7 ± 19.9 4.6 ± 0.82 brush NP DEXylated 3.5 kDa 146.3 ± 39.5 106.3 ± 32.2 95.8 ± 6.36 3.7 ± 1.74 brush NP DEXylated 5 kDa 147.6 ± 22.6 88.50 ± 33.0 111.8 ± 13.6 2.9 ± 1.87 brush Mean size and SD (standard deviation) correspond to the arithmetic values calculated and based on the sizes of all particles detected in the NTA measurement. Mode values describe the average


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size of the main particle population. For details about the polymer conformation on the particle surface, see supporting information.

Particle sizes in PBS and DMEM over 3 days. As mentioned above, initial size measurements in PBS showed a tendency of aggregation for non-modified particles after lyophilization and resuspension. The stability of particles in different aqueous solutions is highly important not only regarding storage conditions but also for possible in vivo applications. For example, right after i.v. administration in the blood stream, the particles face plasma proteins, that can adsorb on the particle surface due to electrostatic interaction and can lead to particle aggregation. Therefore, we determined the particle size in PBS and DMEM (containing 10% FCS) for up to three days to investigate the particle behavior in protein-enriched media. The mean particle size of non-modified, PEGylated, and DEXylated particles in PBS or DMEM was determined by NTA measurements (Figure 3). Only minimal change in particle size was detectable after incubation in PBS up to three days. Most of the particle types stayed in a size range of 150–200 nm. However, particles in DMEM have a different size distribution over time. Whereas nonmodified and PEGylated particles only showed minimal increase in size (in range of 200– 250 nm), DEXylated particles increased their size up to 350–400 nm. This indicates a strong interaction of proteins with the particle surface of DEXylated particles followed by the aggregation of individual particles. Increased SD values of DEXylated particles in DMEM affirm these findings (see Supporting Information Figure S6).


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Figure 3. Mean particle size over 3 days, measured in PBS and DMEM (10% FCS supplement) using NTA. Lyophilized DEXylated particles show aggregation directly after resuspension. This aggregation is particularly strong in protein-enriched DMEM media (B). In comparison, low to no aggregation was observed for lyophilized and resuspended PEGylated nanoparticles over 3 days (A). In summary, all modified particles show only minimal changes in particle size when stored in PBS. Switching to protein-containing cell culture medium (DMEM supplemented with 10% FCS), non-modified and PEGylated particles show only minimal elevated sizes. In comparison, some increase of size can be observed for DEXylated particles due to aggregation.

Effect of particles on cell viability. All particles used for cellular assays contained

Surface Modification of Polysaccharide-Based Nanoparticles with PEG

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