How to tune the gene delivery and biocompatibility of poly(2-(4

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How to tune the gene delivery and biocompatibility of poly(2-(4-aminobutyl)-2-oxazoline) by self and co assembly Meike N. Leiske, Fabian H. Sobotta, Stephanie Hoeppener, Johannes C. Brendel, Anja Traeger, and Ulrich S. Schubert Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01535 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 21, 2017

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Biomacromolecules

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How to tune the gene delivery and biocompatibility

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of poly(2-(4-aminobutyl)-2-oxazoline) by self- and

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co-assembly

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Meike N. Leiske,a,b Fabian H. Sobotta,a,b Stephanie Hoeppener,a,b Johannes C. Brendel,a,b

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Anja Traeger,a,b,* Ulrich S. Schuberta,b,*

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a

Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University

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Jena, Humboldtstrasse 10, 07743 Jena, Germany. b

Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7,

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07743 Jena, Germany.

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*Correspondence to A. Traeger ([email protected]) and U. S. Schubert

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([email protected])

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Keywords: Gene-delivery, transfection, poly(2-oxazoline)s, biocompatibility, worm-like micelle

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Abstract

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Despite their promising potential in gene transfection, the toxicity and limited efficiency of

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cationic polymers as non-viral vectors are major obstacles for their broader application. The

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large amount of cationic charges, e.g. in poly(ethylene imine) (PEI) is known to be advantageous

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in terms of their transfection efficiency, but goes hand-in-hand with a high toxicity.

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Consequently, an efficient shielding of the charges is required to minimize toxic effects. In this

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study, we use a simple mixed-micelle approach to optimize the required charge density for

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efficient DNA complex formation and to minimize toxicity by using a biocompatible polymer. In

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detail, we co-assembled mixed poly(2-oxazoline) nanostructures (d ≈ 100 nm) consisting of a

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hydrophobic-cationic block copolymer (P(NonOx52-b-AmOx184)) and a hydrophobic-hydrophilic

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stealth block copolymer (P(EtOx155-b-NonOx76), in ratios of 0, 20, 40, 60, 80 and 100wt%

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P(NonOx52-b-AmOx184). All micelles with cationic polymers exhibited a very good DNA

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binding efficiency and dissociation ability, while the bio- and hemocompatibility improved with

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increasing EtOx content. Analytics via confocal laser scanning microscopy and flow cytometry

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showed an enhanced cellular uptake, transfection ability and biocompatibility of all prepared

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micelleplexes compared to AmOx homopolymers. Micelleplexes with 80 or 100wt% revealed a

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similar transfection efficiency as PEI, while the cell viability was significantly higher (80 to 90%

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compared to 60% for PEI).

15 16

Introduction

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Modern gene therapy uses two different types of gene carriers, namely viral and non-viral

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systems. Due to their high transfection efficiency and approval in clinical trials, virus based

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systems are more common in recent gene therapy approaches.1 Although viruses are

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predestinated for gene delivery, caused by their evolutionary optimization, there are some

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disadvantages, which hamper the use of viruses in gene therapy. One major drawback of viral

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vectors is the occurring immunogenicity, which may cause an activation of inflammatory cells

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leading to the degeneration of treated tissue. Moreover, toxin production and insertional


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mutagenesis were observed in some cases. Due to their size, viral vectors are limited by the

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transgenic capacity, furthermore the upscaling of these systems is challenging.2

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Thus, even if the efficiency of non-viral systems is lower compared to viral systems, there are

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significant advantages, which constitute non-viral systems as relevant alternatives in the area of

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gene delivery. In general, non-viral vectors show relatively low host immunogenicity, moreover

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they allow an almost unlimited transgene size and provide the ability of repeated application.3

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Additionally, compared to viruses, non-viral systems benefit from low cost production and their

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ability for an easy upscaling. Most commonly, cationic polymers are used for gene delivery,

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since they are capable of forming polyplexes with the negatively charged phosphate backbone of

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nucleic acids. Due to the positive charges of the polymer, the polyplex can interact with the

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negatively charged cell membrane and is consequently internalized via endocytosis.

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Subsequently, the complexes need to undergo endosomal escape into the cytoplasm, resulting in

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the release of the polyplexes.4 P. Stayton et al. demonstrated that pH-dependent amphiphilic

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nanocarriers are capable to trigger an enhanced endosomal release by interaction with the

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endosomal membrane.5 Thus, a change in the pH value and protein interactions trigger the

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dissociation of the polyplex and, thus, the genetic material can enter the nucleus to transfect the

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cells. Poly(ethylene imine) (PEI) is one of the most commonly used materials for gene delivery

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applications and for a long time it was claimed to be the gold standard for the transfection of

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genetic material.6 Its high charge density leads to the formation of physiological stable PEI-DNA

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polyplexes. In general, the high efficiency of PEI is based on the ability of the amine groups to

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buffer the pH value over a wide range, causing an efficient endosomal escape (proton sponge

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effect).7 Disadvantages of PEI-based systems are their high in vitro and in vivo toxicity and their

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resistance against biodegradation, leading to the accumulation of the polymer in the cells or


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tissue, which can elicit further toxicity effects.8 Furthermore, the cytotoxicity has been shown to

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be dependent on the molar mass of the polymers,9 but can be improved by the introduction of

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stealth units, i.e. EtOx, into the polymer chain.10-11 These drawbacks lead to a necessity to search

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for alternative polymer systems for gene delivery applications, which reveal high transfection

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efficiencies while expressing a low cytotoxicity. K. Miyata et al. introduced primary and

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secondary amines into the side chains of poly(aspartamide) to induce pH-sensitive membrane

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destabilization at an endosomal pH value of 5 a resulting enhances cytocompatibility at

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physiological pH values of 7.12 Another possibility to fulfill this aim was shown to be the

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introduction of hydrophobic units, such as cholesterol,13 stearic acid,14 to the cationic polymer

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chains. The hydrophobic units could be shown to increase the cellular uptake as well as the

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transfection efficiency, however, not the cytotoxicity.

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Aim of this study was the development of a micellar polymeric gene delivery system with low

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cytotoxicity combined with enhanced cellular uptake and transfection efficiency by adjusting the

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ratio of stealth and cationic units. Poly(2-oxazoline)s (P(Ox)s) are a class of polymers, which

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were intensively studied during the past years in the context of several potential applications.15-17

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In the field of nanomedicine, the combination of enhanced biocompatibility and structure

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variability has been shown to be an essential benefit of this polymer class.17-19 The synthesis of

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P(Ox)s by the living cationic ring-opening polymerization (CROP) provides even access to

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sophisticated polymer architectures, such as block copolymers20 and star-shaped,21-22

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hyperbranched,23 and cross-linked networks.24-25 The combination of monomer units bearing

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side-chains with different hydrophilicities leads to amphiphilic copolymers, which can self-

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assemble into different nanostructures.25 Block copolymers of two or more chemically different

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polymer chains,26 which potentially phase separate in bulk or selective solvents, provide access


4

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to several defined self-assembled structures. Furthermore, cationic P(Ox)s have already shown

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their potential in gene delivery applications.27-28 For these reasons, we synthesized two different

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amphiphilic block copolymers. The first copolymer consists of NonOx for the formation of a

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hydrophobic core and the amino-functionalized AmOx to facilitate polyplex formation with the

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genetic material. The second copolymer consisted of the same hydrophobic unit, however, EtOx

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served as the hydrophilic block, since it is known for its stealth properties.29 Finally, mixed

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nanostructures with different weight ratios of these two block copolymers were prepared and

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characterized regarding the micelle size, PDI value, pH-responsiveness, CMC, toxicity, polyplex

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formation and dissociation as well as their cellular uptake and transfection efficiency.

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Materials and methods

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Materials and Instrumentation

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Triethylamine (TEA, Sigma Aldrich), butyronitrile (VWR), 2-ethyl-2-oxazoline (EtOx, Sigma

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Aldrich), 2-nonyl-2-oxazoline (NonOx, Henkel) and methyl tosylate (MeTos, Sigma Aldrich)

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were distilled to dryness over calcium hydride (VWR) under argon atmosphere prior to usage.

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Ethyl acetate (EtOAc) and acetone, hydrochloric acid, N,N-dimethylformamide (DMF) and

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tetrahydrofuran (THF) were purchased from VWR Chemicals. Acetonitrile was obtained from a

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solvent purification system (MB-SPS-800 by MBraun) and stored under argon. All other

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solvents used were obtained from standard suppliers. Ethidium bromide solution (1%, 10 mg mL−1)

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was purchased from Carl Roth (Karlsruhe, Germany). AlamarBlue YOYO-1 iodide, Hoechst 33342

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trihydrochloride as well as all other indicated CLSM dyes were obtained from Life Technologies

22

(Thermo Fisher Scientific, Germany). If not stated otherwise, cell culture media and solutions (L-

23

glutamine, antibiotics) were obtained from Biochrom (Berlin, Germany). Plasmid eGFP (pEGFP-N1,


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4.7 kb, Clontech, USA) enhanced green fluorescent protein (eGFP) was isolated with the Giga

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Plasmid Kit provided by Qiagen (Hilden, Germany). Plasmid pCMV-GFP were obtained from

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PlasmidFactory (Bielefeld, Germany).

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The synthesis of 2-(4-((tert-butoxycarbonyl)amino)butyl)-2-oxazoline (BocOx) was described previously in our research group.24

6

Cryo transmission electron microscopy (cryoTEM) investigations were conducted with a FEI

7

Tecnai G2 20 at 200 kV acceleration voltage. Specimens were vitrified by a Vitrobot Mark V

8

system on Quantifoil grids (R2/2). The blotting time was 1 s with an amount of solution of 8.5

9

µL. Samples were plunge frozen in liquid ethane and stored under liquid nitrogen until transfer

10

to the Gatan cryo-holder and brought into the microscope. Images were acquired with an

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Olympus Mega View camera (Olympus Soft Imaging Solutions; 1376 × 1032 pixels) or an Eagle

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4 × 4 k CCD camera system.

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Proton NMR spectroscopy (1H-NMR) was performed at room temperature using a Bruker

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Avance I 300 MHz spectrometer, utilizing either CDCl3, CD3OD or D2O as solvent. The

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chemical shifts are given in ppm relative to the signal from the residual non-deuterated solvent.

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Size exclusion chromatography (SEC) of the copolymers were performed on an Agilent 1200

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series system, equipped with a PSS degasser, G1329A pump, a PSS GRAM guard/30/1000 Å

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with 10 µm particle size and a G1362 refractive index (RI) detector. DMAc containing 0.21%

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LiCl served as eluent. The column oven was set to 40 °C at a flow rate of 1 mL min-1 and

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polystyrene (PS, 400-1,000,000 g mol-1) served as the calibration.

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SEC of the Boc-protected P(EtOx3-b-BocOx157) was performed on a Shimadzu system equipped

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with a CBM-20A controller, GDU-14A degasser and a LC-10AD VP pump, a PSS SDV

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guard/linear S column with 5 µm particle size and a RID-10A RI detector. CHCl3-iso-propanol


6

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(i-PrOH)-NEt3 (94:2:4) served as eluent. The column oven was set to 40 °C using a flow rate of

2

1 mL min-1. PS (400-100,000 g mol-1) served as the calibration.

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SEC of the P(EtOx3-b-AmOx157) was conducted using a Jasco system equipped with a DG-980-

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50 degasser and a PU-980 pump, PSS SUPREMA-MAX guard/300 Å column with 10 µm

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particle size and a RI-930 RI detector. 0.3% (v/v) TFA containing 0.1 M NaCl served as aqueous

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eluent. The column oven was set to 30 °C utlizining a flow rate of 1 mL min-1. Poly(2-

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vinylpyridine) (P2VP, 1,300-81,000 g mol-1) served as the calibration.

8 9

Lyophilization of the nanostructure suspensions was conducted using an Alpha 1-2 LDplus freeze

dryer

from

Martin

Christ

Gefriertrocknungsanlagen

GmbH

(Germany).

10

Absorbance and fluorescence measurements of the bio-assays were performed at RT using a

11

TECAN Infinite M200 PRO.

12 13

Confocal laser scanning microscopy (CLSM) was performed with an LSM880 ELYRA PS.1 system (Zeiss, Oberkochen, Germany) applying a 63 × 1.4 NA plan apochromat oil objective.

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Batch dynamic light scattering (DLS) was performed on a Zetasizer Nano ZS (Malvern

15

Instruments, Herrenberg, Germany). All measurements were performed in standard

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polypropylene semi micro cuvettes, Malvern Instruments, Herrenberg, Germany). After an

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equilibration time of 180 s, 3 × 300 s runs were carried out at 25 °C (λ = 633 nm). Scattered light

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was detected at an angle of 173°. Each measurement was performed in triplicates (three

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measurements consisting of three runs each per sample). Apparent hydrodynamic radii, Rh, were

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calculated according to the Stokes–Einstein equation (1):

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R =

(1)

22 23


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Synthesis of P(Ox)s

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All polymerization solutions were prepared within a glove box under nitrogen atmosphere.

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Polymerization reactions of 2-oxazolines were performed under microwave irradiation, using

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an Initiator Sixty single-mode microwave synthesizer from Biotage, equipped with a noninvasive

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IR sensor (accuracy: 2%). Microwave vials were heated overnight at 100 °C under vacuum and

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allowed to cool to room temperature under argon before usage. Polymerizations were performed

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under temperature control. According to the polymer characteristics, size exclusion

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chromatography (SEC) of the polymers was performed on different systems and noted in the

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respective part. The synthesis of P(Ox)s was described previously.30

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P(EtOx3-b-BocOx157)

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In a microwave vial, MeTos (1.9 mg, 0.01 mmol), EtOx (3.0 mg, 0.03 mmol) and acetonitrile

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(392.6 mg) were mixed under inert conditions and heated in the microwave to 140 °C for

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63.5 min. Subsequently the vial was opened under an inert atmosphere, and BocOx (477.4 mg,

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1.97 mmol) was added. The reaction mixture was heated in the microwave at 140 °C for

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additional 18.0 min. The resulting polymer diluted with chloroform and precipitated in ice-cold

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diethyl ether. The precipitate was filtered off and redissolved in chloroform. The solvent was

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evaporated under reduced pressure to obtain the product as a white solid (391 mg, 82%).

19 20

Deprotection of P(EtOx3-b-BocOx157) yielding P(EtOx3-b-AmOx157)

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P(EtOx3-b-BocOx157) (390 mg, 10.2 mmol) was dissolved in 10 mL MeOH and 2 mL

22

concentrated hydrochloric acid were added. The reaction mixture was stirred at room

23

temperature for 24 h. Subsequently, the solvent was evaporated under reduced pressure and the


8

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crude product was redissolved in 10 mL MeOH and precipitated in ice cold diethyl ether. Then,

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the precipitate was filtered off and redissolved in 100 mL MeOH. Amberlyst A21 was added and

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the mixture was stirred slowly (100 rpm) overnight at room temperature. Then, the Amberlyst

4

A21 was filtered off and the organic solvent was evaporated under reduced pressure. The

5

polymer was redissolved in 10 mL deionized water and lyophilized to obtain the product as a

6

white powder (186 mg, 81%).

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P(EtOx155-b-NonOx76)

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In a microwave vial, MeTos (24.8 mg, 0.13 mmol), EtOx (1.98 g, 20.0 mmol), butyronitrile

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(13.0 g) were mixed under inert conditions and heated in the microwave to 140 °C for 130 min.

11

Subsequently, the vial was opened under an inert atmosphere, a sample of 100 µL was taken and

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NonOx (1.58 g, 8.0 mmol) was added. The reaction mixture was heated in the microwave at

13

140 °C for another 120 min. The resulting polymer was precipitated in ice-cold diethyl ether and

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centrifuged at 11,000 rpm for 5 min. The supernatant was discarded and the solid was

15

redissolved in CH2Cl2. The solvent was evaporated under reduced pressure to obtain the product

16

as a white solid (1.7 g, 86%).

17 18

P(NonOx52-b-BocOx184)

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In a microwave vial, MeTos (7.8 mg, 0.04 mmol), NonOx (493 mg, 2.5 mmol), butyronitrile

20

(6.1 g) were mixed under inert conditions and heated in the microwave to 140 °C for 120 min.

21

Subsequently, the vial was opened under an inert atmosphere, a sample of 100 µL was taken and

22

BocOx (1.75 g, 7.2 mmol) was added. The reaction mixture was heated in the microwave at

23

140 °C for another 90 min. The resulting polymer was precipitated in ice-cold diethyl ether and


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the solid was resuspended in deionized water and centrifuged at 11,000 rpm for 5 min. The

2

supernatant was discarded and the solid was suspended in deionized water. The solvent was

3

lyophilized under reduced pressure to obtain the product as a white powder (740 mg, 33%).

4 5

Deprotection of P(NonOx52-b-BocOx184) yielding P(NonOx52-b-AmOx184)

6

P(NonOx52-b-BocOx184) (700 mg, 12.8 mmol) was dissolved in 5 mL TFA and stirred at room

7

temperature overnight. Subsequently, 5 mL MeOH were added and the polymer was precipitated

8

in ice-cold diethyl ether. The precipitate was redissolved in MeOH, Amberlyst® A21 was added

9

and the solution was stirred slowly at room temperature for 72 h. Afterwards, Amberlyst® A21

10

was filtered off and the solvent was evaporated under reduced pressure. The polymer was

11

resuspended in deionized water and the solvent was lyophilized under reduced pressure to obtain

12

the product as a white powder (457 mg, 98%).

13 14

Self-assembly

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50 mg polymer was dissolved in 10 mL DMAc by vortexing and ultrasonification.

16

Subsequently, 10 mL of ultra-pure water were added slowly using a syringe pump (5 mL h-1)

17

under continuous stirring (1000 rpm). After that, the resulting solution was transferred to a

18

dialysis tube (cellulose, MWCO 3.5 kDa) and dialyzed against distilled water for four days by

19

daily water exchange. Subsequently, it was diluted using a 1.8wt% aqueous NaCl solution to

20

adjust the salt concentration to 0.9wt% (pH = 6). The resulting nanostructures were filtered using

21

a 0.2 µm syringe filter and characterized by DLS measurements.

22

The concentration of the polymer in the resulting solution was determined gravimetrically

23

(n = 3) after lyophilization of the samples. For this reason, also a 0.9wt% aq. NaCl solution was


10

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lyophilized (n = 3). The mean of the mass of the NaCl samples was subtracted from each

2

polymer containing sample to obtain the absolute polymer mass.

3

The average molar mass of the micelles was calculated by using equation 2.

4

=

∙% !" ∙%# $% ! &''

(2)

5 6

pH responsive behavior

7

The pH-responsiveness of the nanostructures was determined by mixing 1 mL of a

8

1.0 mg mL-1 solution with 1 mL of the following buffers (Table S1).

9

The solutions were incubated at room temperature overnight at 200 rpm (BioShake iQ,

10

Qantifoil Intruments GmbH, Jena, Germany). Afterwards the pH value was checked and the

11

z-average and PDI were determined by DLS measurements.

12 13

Determination of the critical micelle concentration (CMC)

14

The determination of the CMC via pyrene method was described previously.31 Fluorescence

15

was recorded with a Jasco FP 6500. A serial dilution of the nanostructure suspension in 0.9wt%

16

aq. NaCl was prepared.

17

A saturated pyrene solution in 0.9wt%aq. NaCl was prepared as follows. Pyrene was dissolved

18

in acetone (1 mg mL-1) and added dropwise to a solution of 0.9wt% in ultra-pure water, until a

19

slight precipitation (visible turbidity) occurred. The solution was stirred for 48 h at room

20

temperature (1000 rpm) to evaporate the acetone. Then, the solution was filtered using a pleated

21

filtered to remove any precipitate. Subsequently, the same volume of the saturated solution of

22

pyrene in 0.9wt% aq. NaCl was added to each dilution of the micelles to obtain a total volume of

23

2 mL. The mixtures were incubated overnight at room temperature at 200 rpm (BioShake iQ,


11


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Qantifoil Intruments GmbH, Jena, Germany). Excitation spectra were collected at λEm = 390 nm

2

and λEx = 300 to 380 nm. The pyrene stock solution served as the calibration sample and was

3

subtracted from all spectra prior to calculations to remove any fluorescence artifacts. For

4

calculation of the CMC, the resulting spectra were used. The fluorescence intensity at

5

λEm = 390.0 nm while exciting at λEx2 = 338.0 nm was divided by the fluorescence intensity at

6

λEm = 390.0 nm while exciting at λEx2 = 332.5 nm and plotted against the log of the polymer

7

concentration. A non-linear Boltzmann fitting and a subsequent linear fitting were conducted

8

using OriginPro 2015G. Hereby, the Boltzmann fitting was used to visualize visible areas, while

9

the linear fit was utilized to obtain the cross-point of two different linear areas, which

10

corresponds to the CMC of the nanostructures.

11 12

Cell culture

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HEK-293 cells (CRL-1573, ATCC) were cultured in DMEM medium (Merck, Darmstadt,

14

Germany) supplemented with 1 g L-1 glucose, 10% fetal calf serum (FCS, v/v), 100 µg mL-1

15

streptomycin, 100 IU mL-1 penicillin and 2 mM L-glutamine at 37 °C in a humidified 5% CO2

16

atmosphere.

17 18

Cytotoxicity

19

The cytotoxicity was tested with L929 cells, as this cell line is recommended by ISO10993-5.

20

In detail, cells were seeded at 104 cells per well in a 96-well plate and incubated for 24 h. No

21

cells were seeded in the outer wells. After exchanging the media with fresh one and 30 min

22

incubation, polymers at the indicated end concentrations were added, and the cells were

23

incubated at 37 °C for additional 24 h. Subsequently, the medium was replaced by fresh media


12

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Biomacromolecules

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and AlamarBlue (Life Technologies, Darmstadt, Germany) as recommended by the supplier.

2

After incubation for 4 h, the fluorescence was measured at λEx = 570 nm, λEm = 610 nm, with

3

untreated cells on the same well plate serving as controls. The experiments were performed

4

independently three times on three different well-plates.

5 6

Hemolysis assay and erythrocyte aggregation

7

All animal husbandry is performed in compliance with the relevant European and German laws,

8

institutional guidelines and to state the institutional animal committee. The sheep blood was

9

taken for general veterinary management of the animal health.

10

To assess the hemolytic activity of the polymer solutions, blood from sheep, collected in

11

heparinized-tubes (Institut für Versuchstierkunde und Tierschutz / Laboratory of Animal Science

12

and Animal Welfare, Friedrich Schiller University Jena), was centrifuged at 4500 × g for 5 min,

13

and the pellet was washed three times with cold 1.5 mmol L-1 phosphate buffered saline (PBS,

14

pH = 7.4). After dilution with PBS in a ratio of 1:7, aliquots of erythrocyte suspension were

15

mixed 1:1 with the polymer solution and incubated in a water bath at 37 °C for 60 min. After

16

centrifugation at 2400 × g for 5 min the hemoglobin release into the supernatant was determined

17

spectrophotometrically using a microplate reader at λEx = 544 nm wavelength. Complete

18

hemolysis (100%) was achieved using 1% Triton X-100 serving as positive control. Thereby,

19

PBS served as negative control (0%). A value less than 2% hemolysis rate was taken as non-

20

hemolytic. Experiments were run in triplicates and were performed with three different blood

21

donors. The haemolytic activity of the polycations was calculated by equation (3):

22

%Hemolysis = 100 ×

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How to tune the gene delivery and biocompatibility of poly(2-(4

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