Biodegradable Polyglycerol Sulfates Exhibit Promising Features for

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Biodegradable Polyglycerol Sulfates Exhibit Promising Features for Anti-inflammatory Applications Magda Ferraro, Kim Silberreis, Ehsan Mohammadifar, Falko Neumann, Jens Dernedde, and Rainer Haag Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01100 • Publication Date (Web): 09 Nov 2018 Downloaded from http://pubs.acs.org on November 20, 2018

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1

Biomacromolecules

Biodegradable

Polyglycerol

Features

for

Sulfates

Exhibit

2

Promising

Anti-inflammatory

3

Applications

4

Magda Ferraro, 1 † Kim Silberreis,1,2 † Ehsan Mohammadifar,1 Falko Neumann,1 Jens

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Dernedde,*2 Rainer Haag*1

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1Institute

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Germany

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2Charité-Universitätsmedizin

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Universität zu Berlin, and Berlin Institute of Health, Institute of Laboratory Medicine Clinical

of Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 3, 14195 Berlin,

Berlin, corporate member of Freie Universität Berlin, Humboldt-

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Chemistry and Pathobiochemistry, CVK Augustenburger Platz 1, 13353 Berlin, Germany

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ABSTRACT

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Inflammatory processes are beneficial responses to overcome injury or illness. Knowledge of the

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underlying mechanisms allows for a specific treatment. Thus, synthetic systems can be generated

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for a targeted interaction. In this context, dendritic polyglycerol sulfates (dPGS) have been

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investigated as anti-inflammatory compounds. Biodegradable systems are required to prevent

16

compound accumulation in the body.

ACS Paragon Plus Environment

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Biomacromolecules 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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Here we present biodegradable analogs of dPGS based on hyperbranched poly(glycidol-

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co-caprolactone) bearing a hydrophilic sulfate outer shell (hPG-co-PCLS). The copolymers were

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investigated regarding their physical and chemical properties. The cytocompatibility was

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confirmed using A549, Caco-2, and HaCaT cells. Internalization of hPG-co-PCLS by A549 and

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Caco-2 cells was observed as well. Moreover, we demonstrated that hPG-co-PCLS acted as a

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competitive inhibitor of the leukocytic cell adhesion receptor L-selectin. Further, a reduction of

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complement activity was observed. These new biodegradable dPGS analogs are therefore

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attractive for therapeutic applications regarding inflammatory diseases.

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KEYWORDS: sulfated polymers; biodegradable; L-selectin inhibition; complement activity

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inhibition; anti-inflammatory.

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INTRODUCTION

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The inflammatory response is an innate defense mechanism of the body when a harmful stimulus

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is recognized.1 An essential step of this process is the recruitment of leukocytes from blood

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vessels to the site of inflammation.2 This process occurs in a cascade-like fashion, during which

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the leukocytes interact with endothelial cells and can finally migrate into the inflamed tissue.3

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The steps of the leukocyte recruitment are classified by cell capturing, rolling, firm adhesion, and

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transmigration.4,5 Different cell adhesion molecules (CAMs), in particular selectins, play a

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prominent role in this process.6 Together with E- and P- selectins, the leukocytic CAM L-selectin

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mediates the initial steps of the adhesion cascade by binding glycosylated ligands on the

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endothelial surface.7,8 Although this process is necessary for the immune surveillance during an

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acute inflammation, a dysregulated ongoing extravasation of leukocytes results in tissue damage,


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which is the case in chronic diseases.9,10 Affecting the interaction between the selectins and their

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ligands is therefore a way to control the inflammatory process.11,12 Additionally, the leukocyte

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recruitment is enhanced by activation of the complement system. In this way, the supplementary

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reduction of complement activity would be beneficial.

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Several sulfated polyanions were tested for the treatment of inflammation; the most

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famous example is heparin13. Due to its favorable characteristics, heparin is mostly utilized as an

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anticoagulant.14 However, this property has a major drawback, as it can induce uncontrolled

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bleeding. Therefore, its dosage is limited. Moreover, due to its mammalian origin,

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contaminations might occur, and thus synthetic analogs are highly desired.

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The development of heparin-like structures resulted in a variety of compounds, which

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found their employment in diverse biomedical applications. For example, macromolecular

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carbohydrates such as pentosane polysulfate sodium have been investigated as potential

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candidate in the treatment of diseases like allergic rhinitis.15 Glycosylated polymers have also

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been shown to reduce liver inflammation.16 Heparin based polymers have been demonstrated to

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be suitable carriers for the delivery of DNA and small proteins.17,18 Surface functionalization has

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also been investigated, which highlights the importance of sulfonate groups and saccharides in

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polymers mimicking heparin.19

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Dendritic polymers, i.e., dendritic polyglycerol, have entered the field of medicine and

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pharmacology over the last decade.20–22 Dendritic polyglycerol sulfate (dPGS) has been

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developed as suitable heparin analog.23 In particular, it has already been demonstrated that dPGS

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possesses a more distinct anti-inflammatory potential as it competes for leukocyte adhesion and

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complement activation, but only shows a moderate antithrombotic behavior at the same time.24


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More studies have been performed in order to understand the features controlling the

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anti-inflammatory capacity of dPGS.24,25 In particular, the influence of high molecular weight

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and of the architecture has been shown. dPGS has also been compared with other anionic

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functionalities and their superiority in relation to the inhibitory potential was demonstrated.26

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Moreover, it has recently been demonstrated that dPGS-shelled micelles can enhance tumor

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targeting in vivo.27 Dendritic polyglycerol sulfate, however, tends to accumulate in organs such

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as the spleen and liver.28 In order to implement the biodegradability of dPGS, dendritic

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polyglycerol functionalized with cleavable groups in the shell has been developed. In a recent

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work by our group,29 hydroxyl terminal groups were substituted with linkers, such as amido-

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glyceryl succinate, thioglyceryl pentanoate, and thioglyceryl methyl propanoate, and then were

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further sulfated. This architecture allows for the construction of core-shell systems that are able

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to undergo acidic or enzymatic cleavage. However, the synthesis of these polysulfate-containing

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cleavable moieties still requires a multistep procedure.

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In this work, we present a simple approach based on the sulfation of hyperbranched

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polyglycerols containing oligocaprolactone chains (hPG-co-PCLS) in the branched scaffold.

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These novel polymers were tested regarding their biodegradability and cell compatibility and

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were compared to their unsulfated analogs. We also labeled some of the hPG-co-PCLS using an

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ICC NHS ester dye in order to monitor cellular uptake. Beside an efficient synthesis and

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biodegradability, hPG-co-PCLS reduces L-selectin ligand interactions and complement activity

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comparable to dPGS. Thus, hPG-co-PCLS seems to be a promising candidate for anti-

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inflammatory applications.

22 23


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Biomacromolecules

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EXPERIMENTAL SECTION

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General

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Chemicals were reagent grade, were purchased from Acros Organics and Fischer Chemical, and

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used without further purification. All solvents were purchased from Sigma-Aldrich and used

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without further purification. Dialysis was performed in benzoylated cellulose tubing purchased

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from Sigma-Aldrich (MWCO 2000 g/mol) and standard regenerated cellulose tubing purchased

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from Spectrumlab (MWCO 1000 g/mol, 2000 g/mol, 3500-5000 g/mol).

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1H

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(JEOL GmbH) or a Bruker Avance III 700 (Bruker Corporation). Chemical shifts δ were

10

reported in ppm using the deuterated solvent peak as the internal standard. Elemental analysis

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was performed with a VARIO EL III (Elementar). GPC measurements in water were performed

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using an Agilent 1100 equipped with a manual injector, isopump, and Agilent 1100 differential

13

refractometer (Agilent Technologies, Santa Clara, CA, USA). The separation of the polymer

14

samples was performed using three 30 cm columns. The flow rate of the mobile phase (water)

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was set at 1.0 mL/min; the columns were held at room temperature. For each measurement, 100

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µL samples with a concentration of 5 mg/mL was injected. The data were acquired from seven

17

scattering angles (detectors) and a differential refractometer WinGPC Unity from PSS.

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Molecular weights and molecular-weight distributions were determined by comparison with

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pullulan standards. Dynamic light scattering (DLS) and ζ-potential measurements were carried

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out with a Zetasizer Nano ZS (Malvern Instruments Ltd.) and a Zetasizer Ultra equipped with a

21

He-Ne laser (633 nm) in the backscattering mode (detector angle 173°). Particle size was

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measured in UV-transparent disposable cuvettes (Plastibrand®micro cuvette, Brand GmbH + Co

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KG) at 25 °C. Samples were dissolved in phosphate buffer (PB, 10 × 10−3 M, pH 7.4) at a

NMR spectra were recorded on a Bruker AMX 500 (Bruker Corporation), Jeol ECP 500


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concentration of 1 mg/mL. The solutions were filtered once through a 0.2 µm regenerated

2

cellulose (RC) syringe filter. The measurement was performed with 13 scans per sample. For the

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ζ -potential measurements, the samples were dissolved in phosphate buffer (PB, 10 × 10−3 M, pH

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7.4) at a concentration of 1 mg/mL. The solutions were measured by applying an electric field

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across at 25 °C in folded DTS 1060 capillary cells (Malvern Instruments Ltd.) Data evaluation

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was performed with the Malvern Zetasizer Software 6.12. The stated values and standard

7

deviations are the mean of three independent measurements with 15 scans each and are based on

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the Smoluchowski model. IR spectra were recorded on a Nicolet AVATAR 320 FT_IR 5 SXC

9

(Thermo Fischer Scientific) with a detector from 4000 to 650 cm-1.

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General procedure for the synthesis of sulfated hPG-co-PCLS

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hPG-co-PCL was prepared using a method previously described by our group.30 Briefly, glycidol

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and ε-caprolactone were copolymerized at 50 °C in a molar ratio 4:1 and in the presence of

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Sn(oct)2 catalyst. It was possible to obtain polymers with different molecular weights by tuning

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the reaction condition, the monomer to catalyst ratio, as well the stirring and the time of reaction.

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The sulfation was performed using a previously established protocol.23 The synthetic

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pathway is reported in Figure 1. The unsulfated precursor was pre-treated overnight in vacuum,

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at 60 °C. Dry DMF was added and the solution was homogenized at 60 °C for 1 h. A solution of

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sulfur trioxide pyridine complex (1.5 eq/OH groups) in dry DMF was added over a period of 3 h.

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The reaction was stirred at 60 °C for 24 h, under Ar atmosphere. The reaction was quenched with

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water and the pH was adjusted to 7 by addition of NaOH solution (1 M).


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The solvent was evaporated under reduced pressure and the product was dissolved in

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brine. Dialysis was performed with a NaCl-solution (MWCO = 1 kDa), using an ever-decreasing

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NaCl concentration, until the medium was changed with distilled water. The dialysis process was

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performed for 96 h and the medium was changed at least 12 times. The product was lyophilized

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and obtained as a yellow-brown solid in an almost quantitative yield (ranging from 77 to 96%;

6

1H

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elemental analysis. The conversion of hydroxy to sulfate groups was further confirmed by IR

8

analysis, through the appearance of a new strong band at 1200 cm-1 (Figure S1).

NMR in Figure 2). Based on the sulfur content, the degree of sulfation was determined by

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hPG-co-PCLS functionalization, dye conjugation, and sulfation

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The dye conjugate was prepared as follows: hPG-co-PCL10 (0.1 g, 0.012 mol OH, 1 eq) was

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dissolved in dry DMF (2 mL), then triethylamine (0.07 mL, 2 eq/20% OH-groups) was added

13

and the solution cooled in an ice bath. Mesyl chloride (0.02 mL, 1 eq) was added and the

14

reaction was stirred overnight at room temperature, under an Ar atmosphere. The product was

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dialyzed for 24 h against MeOH (MWCO = 3.5-5 kDa). 1H NMR enabled the quantification of

16

the degree of functionalization. (DF): 5% (Figure S2).

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The mesyl derivative was dissolved in DMF (5 mL) and sodium azide (0.08 g, 0.0012

18

mol, 5 eq) was added. The reaction was performed at room temperature for 24 h. After filtration,

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the product was dialyzed for 2 days, first in H2O, then in MeOH (MWCO = 3.5-5 kDa). The

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conversion of the azide was monitored by IR, represented by the appearance of a stretching band

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at 2100 cm-1 (Figure S3).

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The azide derivative was dissolved in water/THF mixture and PPh3 (0.2 g, 0.00076 mol,

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5 eq) was added. The solution was stirred for 48 h, and then dialyzed in MeOH (MWCO = 3.5-


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5 kDa; overall yield almost quantitative) for 24 h. The full conversion of the azide to amine was

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verified by the disappearance of the stretching band of the amine in the IR spectrum. (Figure

3

S4).

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Amine-functionalized hPG-co-PCL (8.5 mg, 0.07 mmol NH2) was dissolved in DMF

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(1 mL). The ICC NHS ester dye was dissolved in a mixture of H2O/DMF (2 mg, 2 eq) and

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simultaneously added with DIPEA (2 eq) to the reaction mixture. The solution was stirred

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overnight at room temperature (Figure S5). The product was purified by dialysis in H2O

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(MWCO = 2 kDa) for 96 h. The medium was changed at least 12 times, until no more free dye

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was detectable in the medium. The conjugation was controlled by reverse-phase thin layer

10

chromatography (RP TLC). Afterwards, the product was sulfated using the described procedure.

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The dialysis process was performed for 96 h; medium was changed at least 12 times. Yield:

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54%; DS: 88%

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Cell culture, cellular uptake, and FACS

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Viability tests were performed using the epithelial human lung cancer cell line A549 and the

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epithelial human colorectal adenocarcinoma cell line Caco-2. The A549 cells were propagated in

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Dulbecco’s modified eagle medium (DMEM, low glucose) with 10% fetal bovine serum (FBS)

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and 1% penicillin/streptomycin. Cells were seeded in medium at 1 x 105 cells/mL, cultured at

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37 °C with 5% CO2 and passaged 1:5 two times a week. The propagation of Caco-2 cells was

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done in minimum essential medium (MEM) with 1% MEM non-essential amino acid solution

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100 x, 1% sodium pyruvate, 20% FBS, and 1% penicillin/streptomycin. Cells were seeded in

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medium at 1 x 105 cells/mL, cultured at 37 °C with 5% CO2, and passaged 1:5 two times a week.

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Viability was determined using a CCK-8 kit (Sigma-Aldrich). Cells were cultured in 96-well


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plates at a density of 4 x 104 cells/mL (A549) or 1 x 105 cells/mL (Caco-2) with normal culture

2

medium or medium containing the sulfated polymer for 24 h. Absorbance measurements at a

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wavelength of 450 nm and a reference wavelength of 650 nm enabled viability quantification.

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Tests were conducted using a Tecan plate reader (Infinite pro200, TECAN-reader Tecan Group

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Ltd). Measurements were performed in pentaplicate and repeated three times. The cell viability

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was calculated by setting the negative control, corresponding to untreated cells, equal to 100%;

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the positive control was represented by cells treated with 0.1% SDS. The background signal was

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subtracted prior to calculation.

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Viability before and after degradation was tested with spontaneously transformed

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keratinocytes HaCaT cells. Degradation was observed after compound’s exposure to air moisture

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at room temperature beyond 16 months. This led to an acidic pH, which caused the cleavage of

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the ester bonds. HaCaT cells were propagated in RPMI 1640 medium with L-glutamine, 10%

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FBS, 1% penicillin/streptomycin. Cells were seeded in medium at 1 x 105 cells/mL, cultured at

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37 °C with 5% CO2 and passaged 1:5 two times a week. The viability assay was performed using

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a CCK-8 Kit (Sigma Aldrich). Cells were seeded in 96-well plates at a density of 5 x 104

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cells/mL with culture medium. Samples were added in serial dilution, including positive and

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negative controls. After 24 h incubation absorbance was measured as previously described, using

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a Tecan plate reader (Infinite pro200, TECAN-reader Tecan Group Ltd). Measurements were

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performed in triplicate and repeated three times. The cell viability was calculated as described

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above.

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Cellular uptake was verified using a confocal laser-scanning microscope (Leica SP8).

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Cells were seeded in 24-well plates at 2 x 104 cells/mL, on a transparent dish and incubated with

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culture medium or medium-containing, dye-labeled substance (final concentration 0.5 mg/mL)


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for 1, 4, and 24 h at 37 °C. The culture medium was then replaced with 500 µL staining agent

2

solution and followed by several washes with sterile Dulbecco’s Phosphate Buffered Saline

3

(DPBS) after ten minutes. 250 µL of a 4% PFA (p-formaldehyde) solution was added for

4

crosslinking, then, after 15 minutes, the solution was replaced with DAPI and incubated for 20

5

minutes. After several washes, the dishes were glued onto a microscope glass slide and used for

6

analysis. Images of different groups were acquired with the same laser and detector settings

7

using the Leica LAS X software.

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For the flow cytometry analysis, cells were seeded at a density of 5 x 104 cells/mL

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(A549) or 1 x 105 cells/mL (Caco-2) and cultivated for 24 h at 37 °C. After washing with sterile

10

DPBS, cells were detached with trypsin, recovered with DPBS, and centrifuged for 4 minutes at -

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4 °C and at 140 rpm. After the supernatant removal, the cells were suspended in PBS and

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immediately analyzed with an Accuri C6 analysis instrument (BD Bioscience).

13 14

Competitive selectin ligand binding assay

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The measurements were performed by surface plasmon resonance (SPR) by using a BIAcore

16

X100 (GE Healthcare Europe GmbH) at 25 °C and a constant flow rate of 20 µL/min. Since the

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procedure has been described in detail by S. Enders et al.,31 here it will be only briefly described.

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To mimic leukocytes, Au nanoparticles (15 nm diam., Aurion Immuno Gold Reagents &

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Accessories) were coated with L-selectin-IgG chimera (R&D Systems GmbH), while the SPR

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sensor chip’s surface imitated the vascular endothelium. Therefore, SiaLex-(20 mol%)-PAA-

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sTyr-(5 mol%) (PAA: polyacrylamide) (Lectinity Holdings, Inc.) and N-acetyllactosamine-PAA

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(Lectinity Holdings) were immobilized on the measuring and reference channels, respectively.

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The preparation of the selectin nanoparticles and the measurements were conducted with 20 mM


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HEPES-Buffer (pH 7.4) containing 150 mM NaCl and 1 mM CaCl2 (all Carl Roth GmbH & Co.

2

KG except CaCl2 from Sigma Aldrich Chemical Co.). The substances were preincubated to

3

assess the inhibitory potential of the polyglycerol derivatives against the binding of L-selectin to

4

its ligand on sensor chip surfaces. The results of L-selectin binding to its ligand without inhibitor

5

were given in response units and set to 100%, after reference subtraction. Due to the inhibitor

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concentrations (0.05 pM – 100 nM), the reduced binding signal was calculated as x% binding of

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the control. Hence, the necessary inhibitor concentration for binding reduction of 50% was set as

8

IC50 value. Measurements were performed in duplicates.

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Blood clotting assay

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The influence of different polyglycerol derivatives on blood coagulation was estimated by

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determining the partial thromboplastin time (PTT). Therefore, an Amelung coagulometer (Type

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410A4MD) was used. The mixture of 100 µL standard citrated human plasma, 100 µL Actin FS

14

solution (both from Siemens Healthcare) and optionally 4 µL of the hPGOCS were incubated

15

(3 min, 37 °C). The final concentration of the test compounds was ranging from 0.05 µg/mL to

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50 µg/mL. The anticoagulant heparin (Sigma-Aldrich) was used as a control in the same

17

concentration range. By adding 100 µL pre-warmed (37 °C) CaCl2 solution (Siemens

18

Healthcare) the blood coagulation was started. Measurements for each concentration were

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determined in triplicates. Finally, the untreated standard human plasma was used as a control and

20

its clotting time was set to 100%.

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Complement system activity


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The complement activity was tested by using the WIESLAB complement system classical

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pathway kit (Euro Diagnostica). The procedure is described in literature in detail.32 Briefly,

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human serum was diluted 1:101 with diluent CP and treated, if applicable, with different final

4

concentrations (1000, 500, 250, 100, 50, 25 nM) 1:50 of the hPGOCS. After a 5 min

5

preincubation step at room temperature, the samples were distributed into the respective

6

manufacturer’s well plate and were again incubated (1 h, 37°C). Subsequently, the wells were

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washed three times with the washing buffer and refilled with 100 µL antibody-enzyme conjugate

8

solution. Another incubation step (30 min, 20-25 °C) was followed by a second washing step as

9

described before. Finally, 100 µL of the substrate solution were added and incubated for 30 min

10

at room temperature before the well absorbance at 405 nm was measured on a microplate reader

11

(Tecan). The untreated serum was set to 100% activity and the potency of the inhibitor

12

concentrations was calculated. The concentration where the complement activity was reduced by

13

50% was set as IC50 value.

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Degradation studies

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The enzymatic degradation studies were performed in PB solution at pH = 7.4, at 37 °C. Samples

17

(C = 5 mg/mL) were incubated with 50 µL human leukocyte esterase (HLE) (5.0-6.0 U/mL)

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solution for different timeframes (from 4 to 24 h). For each time point, a sample was first shock

19

frozen in liquid nitrogen and then lyophilized. The degradation profile was analyzed by 1H NMR

20

and GPC. Moreover, HLE alone and hPG-co-PCLS together with HLE were also analyzed

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directly after the addition of the enzyme (0 h) and after an incubation time of 8 h regarding their

22

activity towards the complement activity inhibition. The experiment procedure was performed as


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described in the section “Complement system activity.” Instead of a concentration range, final

2

concentrations of 250 nM for each sample were used.

3 4 5 6

RESULTS AND DISCUSSION

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Recently our group reported a straightforward synthesis of a biodegradable hyperbranched

8

polyglycerol containing oligocaprolactone segments.30 Due to its favorable characteristics such

9

as the biocompatibility and biodegradability, the hPG-co-PCL was chosen as a candidate for

10

further sulfation, in order to develop a dPGS analog with a biodegradable polymer backbone.

11

The facile preparation of this polymer on a gram scale reduces the synthesis to only two steps. In

12

particular, in this work we wanted to create an outer layer of sulfate groups and investigate the

13

new polymeric scaffold on biological systems, in order to confirm that degradable hydrophobic

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oligocaprolactone segments have no detrimental effect. The conversion of the hydroxyl groups to

15

sulfates was performed in the presence of sulfur trioxide pyridine complex. The synthetic

16

pathway is shown in Figure 1.


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Figure 1. Two-step approach for the synthesis of the biodegradable hPG-co-PCLS.

3 4

The successful transformation of the hydroxyl groups was confirmed by 1H NMR

5

analysis. In Figure 2, the 1H NMR spectra before (in blue) and after (in red) the sulfation are

6

depicted. A shift to lower field of signals assigned to the polyglycerol backbone could be

7

observed, in the region between 3 and 4.5 ppm, which corresponded to the formation of the

8

sulfate groups. Also, the appearance of new peaks, partially covered by the solvent signal (D2O),

9

was a further confirmation of the presence of the sulfated moieties. It was also possible to

10

observe that the process did not lead to degradation of the core structure and the ratios of the two

11

monomers composing the copolymer were almost unchanged.


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Figure 2. Superimposition of the 1H NMR spectra of hPG-co-PCL (blue) and its derivative hPG-

3

co-PCLS (red).

4 5

Various studies have already been performed on dPGS that highlight how composition,

6

molecular weight, and degree of sulfation play a pivotal role on their performance as anti-

7

inflammatory compounds.24–26 This research mainly dealt with polymers ranging from several

8

kDa up to MDa (Million Dalton) polymers. In this work, two sulfated copolymers (hPG-co-

9

PCL50S0.80 and hPG-co-PCL50S0.95) were synthesized from a 50 kDa molecular weight starting

10

material and one from a 10 kDa (hPG-co-PCL10S0.90). The polymers hPG-co-PCL50S0.80 and

11

hPG-co-PCL50S0.95 consistently varied their degree of sulfation, while the low molecular weight

12

one, hPG-co-PCL10S0.90, was prepared only with a high functionalization. Elemental analysis was

13

performed to determine the sulfur content of each polymer and to define the degree of sulfation


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(DS) of each derivative. The molecular weight (MW) of the hPG-co-PCLS was calculated based

2

on the data of the precursors and together with the degree of functionalization by the elemental

3

analysis (Table 1).

4

The polymers were also investigated in relation to their physical properties. In Table 1,

5

the size and the surface charge for the synthesized compounds, measured by DLS and by ζ -

6

potential, are displayed. It clearly shows that all the three sulfated compounds possessed similar

7

sizes around 10 nm, which resulted in slightly bigger unsulfated precursors when comparing

8

each with its derivative. Moreover, all sulfated particles displayed a strong negative charge that

9

ranged from -10 to -20 mV.

10 11 12 13 14 15

Table 1. Physical properties of polymers. Compound

Mn [kDa] DS [%]

Size in PB

ζ -potential in PB

[nm]

[mV]

hPG-co-PCL high MW (50 kDa)

47a

0

17 ± 1

-11 ± 1

hPG-co-PCL50S0.80

93b

80

11 ± 1

-19 ± 2

hPG-co-PCL50S0.95

99b

95

13 ± 1

-14 ± 3

hPG-co-PCL low MW (10 kDa)

13a

0

10 ± 1

-7 ± 0.5

hPG-co-PCL10S0.90

34b

90

9±1

-10 ± 2


16

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Biomacromolecules

1 2 3 4

MW obtained by GPC. b MW calculated basing on the elemental analysis. Hydrodynamic size (mean diameter ± standard deviation (SD) as obtained from the size distribution by volume, measured in phosphate buffer (PB, C = 0.010 M), pH 7.4.

5

The biocompatibility of a polymer is a fundamental prerequisite when in vivo use is the aim.

6

Beside its hydrophilicity, size, and charge, cytotoxicity is a crucial parameter to control a long

7

bioavailability. In order to obtain in-depth information about this aspect, all the polymers were

8

tested in vitro using different cell lines. In particular, our attention was focused on the viability of

9

cells to exclude cytotoxic effects in the presence of the polymers.

10 11

a

The cytotoxicity of the sulfated copolymer was tested using two epithelial cell lines, A549 (human lung carcinoma) cells and Caco-2 (colorectal adenocarcinoma) cells.

12

Due to the resemblance of the copolymer to dPGS and based on the studies conducted on

13

the unfunctionalized copolymer, we wanted to confirm that the sulfated copolymer did not show

14

any cytotoxic effect. The viability was investigated 24 h after the treatment, using the CCK-8 test

15

(Figure S6). The response was based on the activity of cells in converting a tetrazolium salt to a

16

formazan dye. Each concentration was tested as pentaplicate and the tests were performed three

17

times. The negative control (NC) was represented by untreated cells in medium, while the

18

positive control (PC) was represented by cells exposed to the presence of sodium dodecyl sulfate

19

(SDS), which is known to lead the cells to death.

20

We observed that for all sulfated hPG-co-PCLS the cell viability was only slightly

21

reduced, which indicated a low level of toxicity by treating the A549 and Caco-2 cells.

22

Moreover, by increasing the concentration up to 500 µg/mL, there was no or less change in

23

viability, which meant that the cells could tolerate high doses of hPG-co-PCLS. These

24

experiments showed that the cell viability was neither influenced by the molecular weight nor the

25

degree of sulfation of the hPG-co-PCLS. In order to further investigate the biocompatibility of


17


Page 18 of 34

1

the polymer, HaCaT cells (human immortilized keratinocytes) were incubated with the low MW

2

compound prior and after its degradation. In both cases, only a slight reduction of viability was

3

observed. This is in accordance to the results obtained for the cancer cell lines (Figure S7).

4

Confirming the biocompatibility of all the three derivatives, we focused our attention on whether

5

cells could uptake and internalize the hPG-co-PCLS. It has already been demonstrated that the

6

polyanion dPGS can be efficently uptaken into cells. Moreover, it has been observed that size

7

plays a minor role for the uptake mechanism. In particular, the clathrin-mediated endocytosis has

8

been identified to play the major role. Processes that are not energy dependent can also

9

contribute.33 Therefore, a polysulfate-dye conjugate was also synthesized based on a low

10

molecular weight hPG-co-PCLS. A partial conversion of the hydroxyl groups to amines was

11

necessary to allow the conjugation of the ICC NHS ester dye. For that purpose, a three-step

12

procedure was applied: the formation of mesyl groups, their nucleophilic substitution with

13

sodium azide, and subsequent reduction of the latter to amine functionalities (Figure S2 – S4). A

14

degree of functionalization equal to 5%, which was confirmed by 1H NMR, was sufficient to

15

enable the conjugation of an ICC dye. After the dye conjugation, the polymer was treated with

16

sulfur trioxide pyridine complex to obtain the sulfated terminal groups. The elemental analysis of

17

the labeled polymer showed a degree of sulfation of 88% and a molecular weight of 45 kDa.

18

Both A549 and Caco-2 cells were incubated with the dye-conjugate polymer. The uptake was

19

investigated 1, 4, and 24 h after incubation. The nucleus of the cells was stained in blue with

20

4’,6-diamidino-2-phenylindole (DAPI), while the membrane was labeled with the CellMask

21

green plasma membrane stain, in order to localize the conjugated polymer, which was labeled in

22

red.


18

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Biomacromolecules

1

Merged pictures that show overlapping signals resulted in a yellow color. Images were

2

taken by using a confocal laser-scanning microscope (CLSM). Over time, the intensity increased

3

in A549 cells (Figure S8), and a remarkable cellular uptake was detected after 24 h in both cell

4

lines (Figure 3).

5 6

Figure 3. Cellular uptake images of hPG-co-PCLS -ICC with A549 cells (a-d) and Caco-2 cells

7

(e-h), after 24 h incubation. Images were taken by CLSM. The first three images report the

8

respective single channel image: (a/e) DAPI, (b/f) CellMask, (c/g) ICC dye. The last images

9

(d/h) represent the overlay.

10 11

The Caco-2 cells displayed a slightly different behavior, as shown in Figure 3. Even

12

though a small uptake could be observed after 1 and 4 h (Figure S9), a significant internalization

13

could be observed only after 24 h. These cells seemed to possess a much slower uptake activity

14

for hPG-co-PCLS-ICC. Moreover, agglomeration of cells obviously limited the uptake to the

15

periphery.


19


1

Page 20 of 34

In order to quantify the cellular uptake, flow cytometry was performed. Therefore, cells

2

were incubated for the same timeframes as in the previous experiments, then trypsinated, washed

3

with cold PBS, and centrifuged at 4 °C to slow down their metabolism. An increasing uptake

4

with time could be observed for hPG-co-PCLS-ICC when using the A549 or Caco-2 cells

5

(Figure 4).

6

7 8

Figure 4. Cellular uptake of hPG-co-PCLS-ICC into A549 cells and Caco-2 cells recorded at

9

different time points by flow cytometry.

10 11

L, P-, and E-selectins play a key role in the recruitment of leukocytes to the inflamed tissue. To

12

avoid a dysregulated activity, one may interfere with these mechanisms by blocking the binding

13

of selectins to their ligands. We used a previously established competitive SPR assay34 to

14

determine the IC50 values of all the sulfated polymers, which reflected the quantity of a substance

15

that was necessary to inhibit 50% of the binding of L-selectin to its ligands. Depending on the

16

degree of sulfation and the molecular weight of the polymer, different outcomes were expected.

17

Figure 5 shows the results of the three hPG-co-PCLS derivatives.


20

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Biomacromolecules

1 2

3 4

Figure 5. Competitive L-selectin inhibition binding assay performed with high and low

5

molecular weight hPG-co-PCLS with different degrees of sulfation.

6 7

As predicted, the molecular weight and the degree of sulfation had an influence on the

8

performance of the polymers. The high molecular weight compound that possessed the highest

9

degree of sulfation (hPG-co-PCL50S0.95) gave the best result. In particular, the IC50 value was 20

10

pM, which is one of the best ever determined in this assay. Decrease of sulfation (hPG-co-

11

PCL50S0.80) led to IC50 value of 150 pM, which was still 2,5 times better in comparison to the

12

nondegradable dPGS. The lower molecular weight compound resulted in an IC50 value of 1,5

13

nM. This behavior was also in accordance with the previous studies conducted in our group for

14

non-degradable dPGS.25 As already shown in other studies,11,25,35 unfractionated heparin (UFH,

15

mean MW ~ 15 kDa) displayed a binding affinity towards L-selectin in only the µM range.

16

Obviously, the core architecture (linear vs. dendritic) is essential and plays an important role in

17

target-binding affinity.


21


Page 22 of 34

1

In order to compare these new degradable polysulfates to heparin in more detail, we

2

investigated the influence of hPG-co-PCLS on the blood coagulation and on the complement

3

system activity. As depicted in Figure 6, all the sulfated highly branched copolymers exhibited

4

only a weak influence on blood coagulation. A moderate increase in clotting time (about 4-fold)

5

was only observed at the highest concentration of 50 µg/mL, whereby heparin prolongs the blood

6

clotting time more than 64-fold.

7 8

Figure 6. Effect of the hPG-co-PCLS on the blood coagulation in comparison to heparin. Due to

9

prolonged clotting time the experiment was discontinued after 1600 seconds and hence,

10

described as “not countable” (n.c.).

11 12

The behavior of all branched polysulfates was similar and mainly concentration-dependent. The

13

strong difference compared to heparin may refer to the more globular architectures of hPG-co-

14

PCLS. The linear heparin is known to stabilize the thrombin-anti-thrombin (TAT) complex and

15

therefore compromises blood coagulation.36 The lower influence on blood clotting is of

16

significant advantage with respect to an application as anti-inflammatory compound.

17

An effect, which usually arises during an inflammatory process, is the immediate

18

activation of the complement system, a part of our innate immune system. Compounds capable


22

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Biomacromolecules

1

to regulate the level of complement activity are highly desired to control overwhelming immune

2

reactions. Polysulfates like heparin are known to reduce complement activity. Therefore, we

3

tested the classical pathway of complement activation.

4

Figure 7 shows that all the synthetic copolymers reduce or inhibit the activity of

5

complement and displayed a better performance than heparin. In this case, the high molecular

6

weight compounds (hPG-co-PCL50S0.80 and hPG-co-PCL50S0.95) were very effective and reached

7

a similar IC50 value of 25 nM. Interestingly, the core size outcompeted the degree of sulfation,

8

because compound hPG-co-PCL10S0.90 was less effective.

9 10

Figure 7. Inhibition of complement activity by hPG-co-PCLS in comparison to heparin.

11 12

Finally, the biodegradability of these new polysulfates was investigated as a key aspect

13

concerning their intended utilization in living systems. We focused our attention on whether the

14

copolymer’s structure would be degraded in the presence of a natural occurring enzyme.

15

Previous studies30 already demonstrated that the unsulfated precursor could undergo enzymatic

16

degradation in the skin. In order to confirm whether the sulfate functionalization interferes with

17

this property, we tested the compound degradation with human leukocyte esterase (HLE), an


23


Page 24 of 34

1

enzyme which is present during various inflammatory processes37–39. The hPG-co-PCL10S0.90

2

was time-dependently incubated with HLE.

3

The ester bonds that connect the polycaprolactone to the polyglycerol represent the

4

enzymatically cleavable moieties. These bonds are recognizable in the 1H NMR spectrum as they

5

give a specific signal at 2.5 ppm. This signal is generated by the hydrogen bound to the carbon in

6

alpha position to the ester (signal highlighted in orange in Figure 8 a).

7 8

Figure 8. Time frame-dependent degradation profile of hPG-co-PCL10S0.90 evaluated by 1H

9

NMR. The red spectrum is recorded prior to incubation with the enzyme. (a) Enzyme-dependent

10

signal changes are boxed at given time frames. The disappearing ester signal is highlighted in

11

orange, while the arising signals are highlighted in blue. (b) Variation of the integral of the

12

signals highlighted in the NMR spectra versus time of incubation. The variation of the integrals

13

was plotted against the time of incubation and normalized to the signal of the hPG backbone.

14 15

The signal clearly disappeared over 24 h. Moreover, at the same time, a new signal appeared at

16

2.2 ppm (highlighted in blue in Figure 8 a), which could be related to the CH2 in alpha position

17

to the carbonyl carbon of the degradation product, formed after ester cleavage. Furthermore,

18

some new signals in the region between 0.5 and 2 ppm and between 2.8 and 3.3 ppm were


24

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Biomacromolecules

1

detected, which were due to the esterase’s presence and which changed with time. This aspect

2

may have implied the enzyme’s deactivation and the loss of its activity. The samples were also

3

analyzed by GPC to further confirm the degradation. Here, we could also observe a significant

4

reduction in molecular weight already after 4 h, which was recorded after the incubation with the

5

enzyme (Figure 9 a). In comparison to the results reported in a previous study,30 the effect of

6

HLE was not as intensive as Novozyme (a technically applied lipase), but more relevant towards

7

inflamed tissue.

8 9

Figure 9. (a) Degradation profile of hPG-co-PCL10S0.90 analyzed by GPC. The molecular weight

10

after 4, 8, and 24 h is compared to that of untreated copolymer (hPG-co-PCLS). Small

11

oscillations of 1 to 3 kDa were due to the instrumental error. b) Complement system activity

12

investigation, directly after the addition (0 h) and after incubation with the enzyme HLE (8 h).

13

An increasing percentage value corresponds to a lower inhibition of the complement system.

14

As a further confirmation of the degradation process, the samples were tested regarding

15

their bioactivity in the complement assay system. As an assumption, the degradation of the

16

multivalent hPG-co-PCL10S0.90 by HLE implies a loss of function. Complement activity was not

17

affected upon HLE addition at time point T0 (0 h incubation) and gave a value of ~23% (Figure


25


Page 26 of 34

1

9b). In contrast, after an 8 h incubation of HLE and polymer, the activity increased to ~45%,

2

which indicates the loss of the inhibitory function due to polymer degradation of more than 20%.

3

As control, we also investigated the performance of the HLE itself but could not detect any

4

significant influence on the complement activity.

5 6

CONCLUSION

7

In this work, we have shown a simple and efficient approach to synthesize degradable sulfated

8

poly(glycerol-co-caprolactone) copolymers that show a promising anti-inflammatory potential.

9

We have shown that the products are well tolerated by different cell lines and we observed

10

cellular uptake. It was further demonstrated that their performance as L-selectin inhibitor was

11

directly related to both the molecular weight and the degree of sulfation. Compared to heparin a

12

remarkable reduction in anticoagulation was detected. Further, the high molecular weight hPG-

13

co-PCLS (hPG-co-PCL50S0.80 and hPG-co-PCL50S0.95) were vastly better antagonists of the

14

classical complement pathway. In conclusion, these degradable sulfated copolymers are

15

promising candidates for a therapeutic application in inflammation.

16 17

SUPPORTING INFORMATION PARAGRAPH: Synthesis and characterization of ICC-

18

conjugated polymer, viability results, and cellular uptake after 4 h and 8 h.

19

AUTHOR INFORMATION

20

Corresponding Author: *E-mail: [email protected]

21

Authors Contributions: †These authors contributed equally.


26

Page 27 of 34 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

Biomacromolecules

1

Magda Ferraro performed the synthesis and the characterization of the copolymers, the viability

2

tests, the cellular uptake investigation, and the enzymatic degradation. Kim Silberreis performed

3

the L-selectin competitive assay, the complement activity assays and the blood clotting assay.

4

Ehsan Mohammadifar developed the synthesis of the unsulfated copolymers. Falko Neumann

5

supervised the work in the biological laboratory and took the confocal images. Jens Dernedde

6

and Rainer Haag conceived the project and supervised the work.

7 8

ACKNOWLEDGMENTS

9

Dr. Virginia Wycisk is thanked for kindly providing the ICC dye, Dr. Katharina Achazi and Elisa

10

Quaas for providing assistance in the biological laboratory. Anja Stoeshel and Cathleen

11

Schlesener are acknowledged for GPC measurements. Dr. Pamela Winchester is thanked for

12

language polishing the manuscript. Dr. Era Kapourani and Alexander Oehrl are thanked for their

13

helpful discussion. We would like to acknowledge the assistance of the Core Facility

14

BioSupraMol supported by the DFG and the Helmholtz Graduate School Macromolecular

15

Bioscience and the German Research Foundation (DFG, SFB 765) for the financial support.

16 17 18

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Table of Content Graphic


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Biodegradable Polyglycerol Sulfates Exhibit Promising Features for

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