Smart pH-sensitive Nanogels for Controlled Release in Acidic

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Smart pH-sensitive Nanogels for Controlled Release in Acidic Environment Peng Wei, Gauri Gangapurwala, David Pretzel, Meike N. Leiske, limin wang, Stephanie Hoeppener, Stephanie Schubert, Johannes C. Brendel, and Ulrich S. Schubert Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01228 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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Biomacromolecules

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Smart pH-sensitive Nanogels for Controlled Release in Acidic Environment

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Peng Wei, a,b Gauri Gangapurwala,a,b David Pretzel, a,b Meike N. Leiske,a,b,# Limin Wang, a,b Stephanie

3

Hoeppener,a,b Stephanie Schubert,b,c Johannes C. Brendel, a,b Ulrich S. Schubert *, a,b a Laboratory

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of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstraße 10, 07743 Jena, Germany

5 b

6

Jena Center for Soft Matter (JCSM), Friedrich-Schiller-University, Philosophenweg 7, 07743 Jena, Germany

7 8

c

Institute of Pharmacy and Biopharmacy, Department of Pharmaceutical Technology, Friedrich Schiller University Jena, Lessingstrasse 8, 07743 Jena, Germany

9 10 11 12

# Current

address: ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash

Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia *Correspondence

to: U. S. Schubert (E-mail: [email protected])

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ABSTRACT

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The encapsulation of therapeutic compounds into nanosized delivery vectors has become an important

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strategy to improve efficiency and reduce side-effects in drug delivery applications. Here, we report the

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synthesis of pH-sensitive nanogels, which are based on the monomer N-[(2,2-dimethyl-1,3-

17

dioxolane)methyl]acrylamide (DMDOMA) bearing an acid cleavable acetal group. Degradation studies

18

revealed that these nanogels hydrolyze under acidic conditions and degrade completely, depending on the

19

crosslinker, but are stable in physiological environment. The best performing system was further studied

20

regarding its release kinetics using the anticancer drug doxorubicin. In vitro studies revealed a good

21

compatibility of the unloaded nanogel and the capability of the doxorubicin loaded nanogel to mediate 1

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Page 2 of 34

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cytotoxic effects in a concentration and time dependant manner with an even higher efficiency than the

2

free drug. Based on the investigated features, the presented nanogels represent a promising and

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conveniently prepared alternative to existing carrier systems for drug delivery.

4

KEYWORDS:

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precipitation polymerization.

Nanogel; pH-sensitive; drug delivery; selective release; stimuli responsive;

6 7

Introduction

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The encapsulation of pharmaceutically active ingredients into nanosized carrier systems, which are most

9

commonly based on polymers, has become a major strategy in research to improve the efficacy of these

10

drugs by a more localized delivery and a controlled release. In particular, great efforts have been paid to

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the development of carrier systems for potent chemotherapy agents.1-3 Among them, smart nanoscale drug

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delivery system, such as liposomes, polymeric micelles, metallic nanoparticles and nanogels have been

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intensively investigated due to their advantage of enhancing the delivery efficiency of anticancer drugs by

14

enhanced permeability and retention (EPR), while reducing side effects at a certain level.4-7 In particular,

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nanogels with three-dimensional polymer networks have attracted more and more attention because of

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their excellent properties, including high loading capacity,8 high cellular uptake,9 large surface area

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allowing multivalent bioconjugation,10 and smart responsiveness to environmental stimuli such as pH

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value,11 redox potential,12, 13 temperature,14 and enzymatic activity.15

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The responsive character is useful to control the release of the encapsulated active ingredients. For instance,

20

the variation of the pH value from the extracellular compartments (pH = 7.4) to the intracellular lysosomes

21

(pH = 4.0 to 4.5) and endosomes (pH = 5.0 to 5.5)16 can be used to trigger such an intracellular drug release

22

by incorporating acid-cleavable groups or cross-linkers into the nanogels. Moreover, the increased

23

intracellular glutathione (GSH) concentration results in a reducing environment within the cytosol of the 2


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Biomacromolecules

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cell,17 which facilitates the cleavage of disulfide bonds and, thus, a degradation of an appropriately

2

modified nanogel.

3

To develop such smart nanogels an increasing number of different methods have been studied in the past

4

decade, such as emulsion polymerization,18,

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polymerization.21 Among them, the latter turned out to be one of the most popular methods due to the ease

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of use, the high conversion of monomers, and the wide range of options to incorporate responsive groups.

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In particular, the use of water as solvent and the application of a straightforward free radical polymerization

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technique render this method attractive for biomedical applications, as potentially harmful solvents can be

9

excluded, while still well-defined nanostructures are obtained. Furthermore, the nanogels can conveniently

19

inverse emulsion polymerization,20 and precipitation

10

be purified by centrifugation or dialysis in water.

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N-Isopropylacrylamide (NIPAm),22-24 poly(ethylene glycol) (PEG)8, 25 and N-vinylcaprolactam (VCL)14,

12

26, 27

13

nanogels by precipitation polymerization. In addition, acrylic acid (AA) is commonly incorporated to

14

stabilize the nanogels because of the negative charge.28 Various crosslinkers are also applied, including

15

crosslinkers based on ketal or disulfide bonds, which can be cleaved by the decrease of the pH value or a

16

reductive environment, respectively.8, 11 This degradation of crosslinking points in the nanogel results in

17

the dissolution of the material, which is so far the most commonly used trigger to release any encapsulated

18

compounds. However, the polymer chains themselves retain their partially hydrophobic character, which

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might impede the accessibility of the labile links or the complete release of the active ingredients. Nanogels,

20

which do not only rely on the cleavage of the crosslinking points, but facilitate a change, for example, of

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the polarity of the whole polymer chains upon a trigger, have so far not been considered most probably as

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a result of the limited number of suitable monomers reported up to now.

are the most common monomers that have been used to construct a large number of responsive

3



Page 4 of 34

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N-[(2,2-Dimethyl-1,3-dioxolane)methyl]acrylamide (DMDOMA) is a recently introduced monomer,

2

which has a similar structure like NIPAm and also features a comparable thermoresponsive behavior.29-34

3

However, in contrast to NIPAm, the acetal group of the monomer endows the resulting polymer with a

4

degradable side chain, which at low pH values will result in a cleavage of the acetal and finally renders

5

the polymer very hydrophilic due to the formation of a dihydroxy moiety in the side chain. Despite the

6

similarity between NIPAm and DMDOMA, the preparation of nanogels using this pH-sensitive monomer

7

has so far not been reported.

8

In this work, we prepared a series of a new type of nanogels based on DMDOMA applying three different

9

crosslinkers: N,N’-Methylene bisacrylamide (BIS), N,N’-bis(acryloyl)cystamine (BAC) and (propane-2,2-

10

diylbis(oxy))bis(ethane-2,1-diyl)diacrylate (KTDA) (Scheme 1). Acrylic acid is additionally integrated in

11

order to stabilize the resulting nanogel and circumvent the use of additional surfactants which would be

12

difficult to remove. At low pH values, the acetal groups of the used DMDOMA will be hydrolyzed

13

resulting in a change of hydrophilicity and consequently can trigger the release of an encapsulated drug

14

(Scheme 1). Applying a degradable crosslinker, the proceeding degeneration of these nanogels would lead

15

to the formation of linear water-soluble polymers, which should be sufficiently small enough for further

16

elimination from the body, for example via the kidney.

4


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Biomacromolecules

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Scheme 1: Illustration of the nanogel-preparation, drug loading, and acid-triggered drug release from the

3

nanogels.

4 5

Experimental

6

Materials

7

The monomer N-[(2,2-dimethyl-1,3-dioxolane)methyl]acrylamide (DMDOMA) and the crosslinker

8

(propane-2,2-diylbis(oxy))bis(ethane-2,1-diyl)diacrylate (KTDA) were synthesized according to a

9

procedure reported elsewhere.35,

36

Acrylic acid (AA), potassium persulfate (KPS), N,N’-methylene

10

bisacrylamide (BIS), Nile red (NR) and doxorubicin hydrochloride (DOX) were purchased from Sigma-

11

Aldrich. N,N’-Bis(acryloyl)cystamine (BAC) was purchased from ABCR. 5



Page 6 of 34

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Instrumentation

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Proton NMR (1H NMR) spectra were recorded at room temperature in CDCl3 or D2O on a Bruker Avance

3

300 MHz. The chemical shifts are given in ppm.

4

Dynamic light scattering (DLS) was performed on a Zetasizer Nano ZS (Malvern Instruments, Herrenberg,

5

Germany). The 1 mg mL-1 nanogel suspensions were measured at 25 °C or 37 °C (λ = 633 nm) at an angle

6

of 173 ° after an equilibration time of 120 s. The size distribution of the nanospheres was calculated

7

applying the nonlinear least-squares fitting mode.

8

A M200 Pro fluorescence microplate reader (Tecan) and a spectrometer FP-8300 (JASCO) were used for

9

fluorescence measurements.

10

Scanning electron microscopy (SEM) imaging was conducted with a Zeiss LEO 1530 Gemini utilizing the

11

lnlense detector. The morphology of the nanogels was observed by SEM. Briefly, 0.7 mg mL-1 nanogel

12

suspension was dropped on a silicon wafer and dried at room temperature.

13

Size-exclusion chromatography (SEC) measurements were performed on an Jasco equipped with a PU-

14

980 pump, a RI-930 refractive index detector and a PSS NOVEMA-MAX column with H2O + 0.1% TFA

15

+ 0.1 M NaCl as eluent. The column oven was set to 30 °C, and a poly(2-vinylpyridine) (P2VP) standard

16

was used for calibration.

17

A TECAN Infinite M200 PRO plate reader (TECAN, Crailsheim, Germany) was used to determine the

18

cell viability using AlamarBlue assay with excitation/emission wavelength at λEx = 570 nm and λEm = 610

19

nm in fluorescence bottom reading mode and at ambient conditions. Each well was measured with 25

20

flashes per scan.

21

Evaluation of uptake of DOX loaded nanogels and free DOX was performed by flow cytometry measured

22

on a Beckmann Coulter Cytomics FC-500 (Beckman Coulter, Krefeld, Germany) equipped with an

23

Uniphase Argon ion laser, λ = 488 nm, 20 mW output and fluorescence signal emission collected in FL-3

24

with λ = 620 nm filter. Analysis was performed using Cytomics CXP software. 6


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Biomacromolecules

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For confocal laser scanning microscopy (CLSM) uptake analysis of DOX loaded nanogels and free DOX

2

as well as co-localization with cell organelles, cell membrane, nuclei and the late endosomes/lysosomes

3

were stained with WGA Alexa 633, Hoechst 33342 and LysoTracker Green, respectively. CLSM images

4

were acquired using a Zeiss LSM 880 (Carl Zeiss, Oberkochen, Germany) with the following settings:

5

WGA Alexa 633: λEx = 633 nm, λEm = 638 to 747 nm; LysoTracker Green: λEx = 488 nm, λEm = 490 to

6

535 nm, DOX: λEx = 488 nm, λEm = 535 to 677 nm, Hoechst 33342: λEx = 405 nm, λEm = 410 to 499 nm

7

and transmission signal with λ = 405 nm laser and PMT detector. Images were captured with a Plan-

8

Apochromat 63× objective in multitrack mode, enabling single excitation and emission of fluorescence

9

dyes. Colocalization was visualized in overlay images of the multiple channels.

10 11

Synthesis

12

Degradation study of monomer (DMDOMA)

13

To evaluate the stability of the DMDOMA, 5 mg of the monomers were dissolved into pure D2O and D2O

14

at pH 10 in NMR tubes, respectively. Then, the tubes were incubated at 70 °C and measured at room

15

temperature at each time point.

16 17

Preparation of nanogels

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The procedure for synthesis of PDA-KTDA via precipitation polymerization is exemplarily described here:

19

33 µL (9.8 mol% of total monomers) acrylic acid was dissolved in 15 mL H2O in a three-neck flask

20

equipped with a reflux condenser, NaOH (1 M) solution was used to adjust the pH value around 10. 800

21

mg (88.2 mol% of total monomers) DMDOMA and 26 mg (2 mol% of total monomers) KTDA were added

22

and NaOH solution was used to keep the pH value at 10 and fill the total volume of the reaction system to

23

30 mL. After degassing for 45 min, the flask was placed in a pre-heated oil bath at 70 °C, subsequently 7

24

mg KPS in 3 mL degassed H2O were added to initiate the polymerization. The reaction was cooled to room 7



Page 8 of 34

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temperature after 16 h and dialyzed at pH 8 (phosphate buffer, 10 mM) for 3 days (molar mass cut off

2

12000 to 14000 g mol-1) to get a PDA-KTDA nanogels’ suspension in the yield of 93%. For the synthesis

3

of other nanogels, PDA-BIS (88%) and PDA-BAC (96%) were used in the same molar ratio of monomers,

4

crosslinkers, and KPS.

5 6

pH-Dependent hydrolysis of nanogels

7

For the 1H-NMR investigations, the three nanogels (each 1 mg) were suspended in acetate buffer (10 mM,

8

pH = 5.1) and D2O containing 5 mg of trioxane as internal standard and incubated at 37 °C. At each time

9

point, samples were measured by 1H-NMR and the integral of trioxane was set as 100 as a standard. The

10

acetone peak, which is the hydrolysate, at 2.1 ppm was tracked for the hydrolysis study.

11

For the DLS investigation, 1 mg mL-1 nanogel suspension was dispersed in phosphate buffer (10 mM, pH

12

= 7.4) or acetate buffer (10 mM, pH = 5.1) and incubated at 37 °C. At each time point, the size and mean

13

count rate of the nanogels were measured at 37 °C with the fixed position and attenuator.

14 15

Degradation of nanogels by DL-Dithiothreitol (DTT)

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10 mM DTT was added to 1 mg mL-1 PDA-BAC nanogel in phosphate buffer (10 mM, Ph = 7.4) and

17

incubated at 37 °C. At each time point, the size and mean count rate of the nanogels were measured by

18

DLS at 37 °C with the fixed position and attenuator.

19 20

Loading of Nile red (NR) and doxorubicin (DOX) into nanogels

21

Nile red (NR) loading experiment was performed according to procedures reported in literature.21 2 mg

22

NR were dissolved in 2 mL THF and mixed with 2 mL nanogel suspension (1 mg mL-1). After stirring at

23

room temperature for 24 h, the dispersion was dialyzed (molar mass cut off of the membrane: 12,000 to

24

14,000 g mL-1) to remove the THF. Afterwards, the suspension was filtered to remove any remaining free 8


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Biomacromolecules

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NR. In addition, free DOX in the same buffer solution was also dialyzed under the same conditions to

2

evaluate the speed of passing the dialysis membrane.

3

In case of DOX loading, 1.5 mg DOX was first dissolved in 10 mL phosphate buffer (pH 7.4) containing

4

25 mg of PAD-KTDA nanogel. After stirring for 24 h at room temperature, the dispersion was dialyzed

5

for 30 h (molar mass cut off of the membrane: 12,000 to 14,000 g mL-1) to remove any remaining free

6

DOX. This extensive washing procedure was further used to ensure that no weakly bound DOX remains

7

on the surface or inside of the nanogels, which may result in an uncontrolled burst release in the following

8

degradation experiments, and only sufficiently well-bound drug is tested. To determine the final loading

9

capacity and encapsulation efficiency, 300 µL of the dispersion was completely degraded by addition of

10

50 µL HCl. Then, 40 µL of the solution was diluted with 2 mL dimethyl sulfoxide. The DOX content of

11

this solution was determined by fluorescence measurements according to a previously recorded calibration

12

(SI, Figure S18). Drug loading capacity (DLC) and encapsulation efficiency (EE) of the nanogels were

13

defined by the following equations:

14

DLC (%) =

weight of DOX in nanogels weight of DOX loaded nanogels

× 100%

15 16

EE (%) =

weight of DOX in nanogels initial weight of DOX

× 100%

17

Fluorescence quenching assay for pH dependent degradation of the nanogels

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The nanogel suspension loaded with NR was first incubated at 37 °C for 3 h, then, phosphate buffer (pH

19

= 7.4) or acetate buffer (pH = 5.1) was added and incubated for another 15 minutes. The intensity of the

20

NR fluorescence at λEm = 640 nm was then measured as time point zero. Subsequently, at regular time

21

intervals 100 µL solvent was taken out and the fluorescence intensity was immediately measured.

22 9



Page 10 of 34

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Release study of DOX

2

The release experiment of DOX from nanogels was carried out by a dialysis method.12 Briefly, 1 mL DOX

3

loaded nanogels solution was injected into 2 mL of phosphate buffer (10 mM, pH = 7.4) or acetate buffer

4

(10 mM, pH = 5.1), respectively. For dialysis (molar mass cut off of the membrane: 12000 to 14000 g

5

mL-1) the samples were immersed into 20 mL of the respective buffer at 37 °C. At regular intervals, 2 mL

6

of the outer medium was taken for fluorescence measurements (calibration SI, Figures S19 and S20) and

7

refilled with the same amount of fresh buffer.

8 9

Cytocompatibility of nanogels and cytotoxicity of DOX loaded nanogels and free DOX

10

Effects on the cellular metabolism upon incubation with unloaded and DOX loaded nanogels as well as

11

free DOX were evaluated using the established L929 mouse fibroblast cell line.37 The in vitro cytotoxicity

12

experiments were performed using commercial AlamarBlue assay following supplier’s instructions. The

13

cells were routinely cultured as follows: Dulbecco's modied eagle's medium (DMEM) supplemented with

14

10% FCS, 100 U mL-1 penicillin and 100 µg mL-1 streptomycin (all components from Biochrom, Berlin,

15

Germany) at 37 °C in a humidified atmosphere with 5% (v/v) CO2. L929 cells were seeded in 96-well

16

plates at a density of 1 × 104 cells/well and were grown as monolayer cultures for 24 h. Cells were then

17

incubated with different concentrations of unloaded nanogels (10, 100 and 1000 µg polymer mL-1), DOX

18

loaded nanogels (10, 100 and 1000 µg polymer mL-1containing 0.25, 2.5 and 25 µg DOX mL-1) and free

19

DOX (0.25, 2.5 and 25 µg mL-1) for 24, 48 and 72 h at 37 °C (n = 6 for each experimental group). Control

20

cells were incubated with fresh culture medium. After incubation, culture medium was aspirated and

21

100 µL fresh media containing 10 µL AlamarBlue reagent prepared according to the manufacturer’s

22

instructions were added to each well. After 4 h at 37 °C, 90 µL of each solution were transferred to a new

23

microtiter plate and the fluorescence was measured at λEx = 560 nm and λEm = 590 nm. The negative

24

control was standardized as 0% of metabolism inhibition and referred as 100% viability. 10


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Biomacromolecules

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Flow cytometric (FC) investigations on time dependent uptake

3

FC measurements were performed to gather quantitative information about the time dependent uptake of

4

the DOX loaded nanogels (1000 µg polymer and 25 µg DOX mL-1) in comparison to the free DOX (25

5

µg DOX mL-1). For this purpose, cells were incubated for 1, 4, 24 and 48 h (n = 3 for each experimental

6

group) and then the cells were washed with PBS to remove remaining material. Subsequently, the cells

7

were detached by trypsin treatment and directly subjected to FC analysis, where 5000 individual

8

cells/sample were measured for their fluorescence intensity derived from the cell associated DOX loaded

9

nanogel and the free DOX, respectively. Obtained mean fluorescence intensities of three individual

10

measurements per sample were then plotted against time.

11 12

Internalization and co-localization with cellular compartments

13

The cellular internalization and intracellular localization of DOX loaded nanogels and the free DOX in

14

L929 cells was further elucidated by confocal laser scanning microscopy (CLSM) investigations. For this

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purpose, the cells were incubated for 4 or 24 h with DOX loaded nanogels (500 µg polymer and 12.5 µg

16

DOX mL-1) and the free DOX (12.5 µg DOX mL-1) at 37 °C. In order to assign the localization of the

17

fluorescent DOX to cellular structures, the living adherent cells were stained with specific dyes for the cell

18

plasma membrane (wheat germ agglutinin Alexa 633, 5 µg mL-1 for 30 min), the nuclei (Hoechst 33342,

19

1 µg mL-1 for 30 min) and the acidic late endosomes/lysosomes (LysoTracker Green, 1 µM for 30 min),

20

respectively.

21 22

Results and Discussion

23

Synthesis and characterization of nanogels 11



Page 12 of 34

1

Scheme 1 shows all monomers and crosslinkers which were used in this work. The monomer DMDOMA

2

and the crosslinker KTDA were synthesized according to procedures reported in literature.30,

3

nanogels were prepared in aqueous medium by precipitation polymerization without the use of surfactants

4

(Scheme 1). NaOH was used to maintain the basic pH value, which is required to preserve the pH-sensitive

5

monomer DMDOMA and to deprotonate the acrylic acid (AA). The anionic form of AA stabilizes the

6

nanogels and inhibits their aggregation due to electrostatic repulsion.28

7

In order to evaluate the stability of the monomers, DMDOMA was incubated in pure water and buffer (pH

8

10) at 70 °C to mimic the conditions during the nanogel synthesis. As shown in Figure S1 (SI), DMDOMA

9

degraded fast within 1 hour in pure water. However, it was very stable at pH 10 for at least 24 hours. This

31

All

10

proves the potential polymerization of DMDOMA at pH 10 and 70 °C.

11

Three different crosslinkers were incorporated into the nanogels, which allow a long-term storage at

12

neutral conditions. The average diameter of the nanogels was measured by DLS in pure water (SI, Table

13

S1) and phosphate buffer (Table 1) as well as by SEM in the dry state (Figure 1, SI Figures S2, S3). A

14

significant decrease of size is observed in DLS when changing the solvent from pure water to the buffer

15

solution, which is in accordance with previous reports on similar systems.38 We assume that the addition

16

of the buffer results in a more collapsed state of the gel, as the repulsive forces of the negatively charged

17

acrylic acid moieties are shielded. The three different nanogels differ significantly in their size and PDI

18

value. This phenomenon was also observed in previous studies.10, 24 The nanogels swell more strongly

19

during the polymerization process with increasing water solubility of the crosslinker, leading to larger

20

sizes and higher PDI values. At the same time, the longer the crosslinking chains are the larger become

21

the nanogels. SEM measurements revealed sizes of 160 nm to 360 nm, however, it has to be taken into

22

account that these samples are in the dried state, which might cause shrinkage or a flattening of the gel.

23

12


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1

2

Biomacromolecules

Table 1: Characterization of nanogels by DLS measurements.

a

Sample

Z-Avea (nm)

PDIa

PDA-BIS

150

0.217

PDA-BAC

97

0.093

PDA-KTDA

202

0.159

Size was measured in phosphate buffer (10 mM, pH 7.4) at 25 °C.

3

4 5

Figure 1 SEM image of PDA-KTDA.

6 7

Hydrolysis of DMDOMA in the nanogels

8

The presented hydrophobic nanogels were designed to facilitate a progressing hydrolysis of the acetal

9

groups under mildly acidic conditions, which turns the polymer in these hydrogels from partially

10

hydrophobic into very hydrophilic.

13



Page 14 of 34

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1H NMR was used to monitor the hydrolysis of the acetal groups at a pH value of 5.1. Therefore, an internal

2

standard (trioxane, 5.1 ppm) was set as 100 and used to compare the integrals of the signal of the released

3

acetone. All nanogels displayed similar hydrolyses rates over a time of up to 168 h (Figure 2A, SI, Figures

4

S4-S6).

5

In addition, hydrolysis of the nanogels was investigated by DLS. Nanogels were incubated at 37 °C under

6

acidic (pH = 5.1) and also neutral conditions (pH = 7.4) for up to 168 h, and the change of the relative

7

count rate was assessed. Assuming a degradation of the acetal groups only at acidic conditions and thus a

8

release of the more hydrophilic dihydroxy side chains, an increased swelling of the nanogels with time can

9

be expected, while the count rate should decrease as a consequence of the lower density of the nanogel.

10

As seen in Figure 2 B, the relative count rates of all three nanogels (all the three curves are overlapping)

11

kept constant at physiological conditions (pH = 7.4), while a considerable decrease is observed at acidic

12

conditions (pH = 5.1). Among the differently crosslinked nanogels, the relative count rate of PDA-BAC

13

decreased less prominent, which can be explained by the fact that during the polymerization of the nanogel

14

the disulfide bonds in the crosslinker might be attacked by radicals resulting in thiol ether bonds which, in

15

turn, would result in higher crosslinking density (SI Scheme S1).39, 40 At the same time, the relative count

16

rate of PDA-KTDA showed the strongest decrease due to the pH-sensitive ketal crosslinker, which could

17

additionally be hydrolysed besides the acetal groups.

18

The zeta potential of the nanogels was also measured before and after hydrolysis (SI, Figures S8-S10). As

19

expected, all nanogels possess a negative surface potential due to the acrylate, which may improve the

20

circulation time in blood by reducing the interaction with negatively charged proteins.24

21 22

14


Page 15 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 2

Figure 2: Measurement of the hydrolysis of nanogels at 37 °C at various time points by 1H-NMR (A) and

3

by DLS (B). All the samples were measured in triplicate.

4 5

Degradation of crosslinkers

6

Three different crosslinkers were applied in this study, which comprise either non-degradable amide bonds

7

(PDA-BIS), cleavable disulfide bonds (PDA-BAC) or acid labile ketal bonds (PDA-KTDA). The

8

degradability of these three nanogels was tested by time-dependent DLS measurements in acidic

9

conditions or in the case of PDA-BAC additionally under reductive environment (Scheme 2). PDA-BIS is

10

considered to be a control sample, which was incubated at 37 °C under acidic (pH = 5.1) as well in neutral

11

conditions (pH = 7.4) for ten days. After ten days, the size did not change at neutral conditions but

12

increased to 170 nm at a pH value of 5.1(Figure 3), which is related to the hydrolysis of the acetal groups

13

within the polymer main chain leading to a swelling of the particle as a result of the increased

14

hydrophilicity of the formed dihydroxy moiety (Figure 2). 15



1

2 3

Scheme 2: Illustration of the degradation pathways for the different nanogels.

4

5 6

Figure 3: Degradation study of PDA-BIS at different pH using DLS at 37 °C.

7 16


Page 16 of 34

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1

PDA-BAC was also hydrolyzed at different pH condition for 10 days. The diameter increases to 116 nm

2

under acidic condition (pH = 5.1) and is stable at neutral condition (pH = 7.4) (Figure 4 A). However, no

3

degradation under these conditions is expected for this system.

4

The presence of disulfide bonds in the BAC crosslinker and the resulting PDA-BAC nanogel should be

5

sensitive to cleavage by reductive agents. DTT (10 mM), a commonly used reducing agent, was added to

6

the nanogel suspension at neutral conditions (pH 7.4), incubated at 37 °C, and monitored by DLS for 10

7

days. Figure 4 B shows that both the diameter and the mean count rate remain constant within the whole

8

process. As already mentioned, the disulfide bonds may undergo a side reaction during the polymerization

9

leading to stable thiol ether bonds (SI Scheme S1).39 ,40 In order to ensure that this effect is not only related

10

to the concentration and strengths of the reducing agent, an even higher concentration of DTT (100 mM)

11

and stronger reducing agents (TCEP·HCl) were used (SI, Figures S11, S12). In addition, the nanogel was

12

first completely hydrolyzed by HCl to guarantee the access of the reducing agent to the crosslinking points,

13

then 100 mM DTT or TCEP·HCl were added to the solutions. After two days incubation at 37 °C, DLS

14

measurements revealed no change in size, only the count rate decreased slightly, which revealed that non-

15

cleavable crosslinking points were formed during the synthesis and only the remaining disulfide bonds can

16

be cleaved.

17



Page 18 of 34

1 2

Figure 4: Degradation study of PDA-BAC using DLS at 37 °C at different pH value (A) with DTT (10

3

mM) (B).

4

The third nanogel, PDA-KTDA, only contains pH-sensitive groups. When incubating the nanogel under

5

physiological (pH = 7.4) and acidic conditions (pH = 5.1) at 37 °C for 10 days, the size of the remaining

6

structures decreased to 14 nm and the mean count rate decreases from 991 to 60 in acidic medium, while

7

it remained constant at physiological pH value (Figure 5 A, B). These results prove that the ketal linkage

8

is sensitive towards acidic conditions and thus facilitates the degradation into smaller polymer chains, but

9

remains quite stable at neutral conditions. In order to confirm the presence of single polymer chains, SEC

10

was also used to characterize the hydrolyzed product (SI, Figure S13,). In a further experiment, the

11

preparation of the nanogels was performed under the same conditions but without the crosslinker. The

12

resulting polymer has a similar distribution like the degradation product of PDA-KTDA as shown by DLS

13

(SI, Figure S14). Self-crosslinking processes might have occurred as already reported in literature.41

18


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Biomacromolecules

1

However, it is an enormous analytical challenge to qualify and quantify these undefined cross-linking

2

processes, which was also not the scope of this work.

3 4

Figure 5: Degradation study of PDA-KTDA at different pH using DLS at 37 °C, diameter change (A)

5

mean count rate at pH 5.1 (B).

6 7

A closer look on the pH response of the nanogels immediately after an induced pH change (time point 0)

8

shows further that the diameters of PDA-BIS and PDA-BAC decrease below a pH of 5, but remain

9

constant above that point (Figure S15). In contrast, the diameter of PDA-KTDA already decreases below

10

a pH of 6.2. This supports the assumption that the different crosslinkers can influence the pKa of the

11

acrylate, which is around 4.8.21

12 13

NR encapsulation and fluorescence measurement

19



Page 20 of 34

1

As reported previously, similar nanogels are able to encapsulate hydrophobic compounds because of

2

hydrophobic microdomains within the gel network.10 Nile red (NR), a commonly used hydrophobic model

3

compound, displays a strong fluorescence if dispersed in a hydrophobic environment, but is almost not

4

fluorescent in aqueous dispersions (SI, Figure S16).25 Due to the hydrophobic domains formed by the

5

acetal groups, NR could be loaded into the presented nanogels resulting in a strong fluorescence intensity

6

of the dye (λmax = 650 nm). Monitoring the fluorescence intensity over time in different media allows to

7

follow the degradation of the nanogels, as they become increasingly hydrophilic and, thus, the fluorescence

8

of NR is quenched. Figure 6 shows the time-dependent relative fluorescence intensity of the different

9

nanogels loaded with NR when incubated in neutral or acidic media, respectively. As expected, the

10

fluorescence signal is stable under physiological conditions, which proves a stable loading of the nanogels

11

with NR at a neutral pH value. The pH-sensitive properties of the nanogels became obvious at a decreased

12

pH level of 5.1, where fluorescence intensities decreased clearly over time in all nanogels in a cumulative

13

manner indicating a constant hydrolysis of the acetal groups and also the degradation of pH-sensitive

14

crosslinkers in PDA-KTDA. Among all, PDA-BAC decreased slowest, which is again related to the

15

increased crosslinking density resulting in a stronger NR association.10 In contrast, the intensity in PDA-

16

KTDA decreased fastest due to the additional hydrolysis of the ketal groups in the crosslinker.

17

20


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Biomacromolecules

1

Figure 6: Change of relative fluorescence intensity over time of different nanogels loaded with NR and

2

incubated in neutral (pH = 7.4) or acidic buffer (pH = 5.1) at 37 °C.

3 4

Taking together results from hydrolysis and degradation evaluation by NMR, DLS, and the NR experiment,

5

it can be concluded that all three presented nanogels are capable of a pH-dependent change from partially

6

hydrophobic to very hydrophilic. However, the PDA-KTDA nanogel displayed the fastest response and it

7

was the only sample which could be fully degraded due to the labile linkers.

8

Therefore, we focused in the further experiments exclusively on this PDA-KTDA nanogel, which included

9

loading and release studies with Doxorubicin (DOX), as well as in vitro experiments on cytotoxicity,

10

cellular uptake and distribution.

11 12

DOX loading and release

13

DOX, a prominent anti-cancer drug also known as adriamycin or doxorubicin, was investigated regarding

14

its ability to be encapsulated into and released from the nanogel PDA-KTDA. Because of the positive

15

charge of DOX (pKa = 8.2) at physiological conditions and the hydrophobic domains, we expected strong

16

hydrophobic and electrostatic interactions with the acetal based chains and anionic acrylic acid,

17

respectively. To estimate a loading capacity (LC) and encapsulation efficiency (EE), DOX dispersed in an

18

aqueous buffer was added to the nanogel dispersion and the combined mixture was dialyzed against pure

19

buffer solution for 30 h to remove any free DOX, which was not entrapped in the nanogel. Dialysis

20

effectively removes unloaded DOX from the DOX-loaded nanogels, which was proven by tests on free

21

DOX, which easily and fast passes the membrane within 4 hours. This unhindered passage was monitored

22

by the measurement of the fluorescence intensity within the dialysis bag over time (SI, Figure S17). The

23

final LC and EE was determined to be 2.9% and 52%, respectively, which demonstrated that the PDA-

24

KTDA nanogel represents a promising candidate for drug delivery. This loading is already well suited for 21



Page 22 of 34

1

further biological investigations,10, 24 but an improvement of the loading capacity is still considered in our

2

ongoing work.

3

As the nanogels were designed not only to encapsulate a drug such as DOX, but selectively release it

4

within the acidic environment of cellular endosomal/lysosomal compartments (pH = 5.0 to 6.0) we further

5

analyzed the release behavior of DOX from nanogels under both physiological condition (pH = 7.4) and

6

acidic condition (pH = 5.1). Incubating the loaded nanogels at 37°C in neutral or acidic buffer, respectively,

7

revealed that around 50% DOX was released from the PDA-KTDA nanogel at pH 5.1 within 4 days, while

8

only 20% DOX was released within the same time at a pH value of 7.4 (Figure 7). The increased release

9

at acidic pH is most probably caused by a partly protonation of the acrylic acid but also a hydrolysis of the

10

nanogel network. These results indicate that a release of DOX as model drug will preferably occur after

11

cellular uptake at the acidic conditions of the lysosomal compartments. Hence, the PDA-KTDA nanogel

12

features a controlled release of the drug, which is essential for an efficient transport if applied in vivo.

13 14

15 16

Figure 7: Release profile of DOX from DOX-loaded nanogel (PDA-KTDA) at different pH values. The

17

samples were incubated at 37 °C within a dialysis bag and samples of the outer medium were taken to

22


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Biomacromolecules

1

evaluate the amount of released DOX. Three samples for each pH conditions were measured at the same

2

time.

3 4

Cytocompatibility and cytotoxicity tests

5

The use of the presented nanogels as versatile carriers for e.g. pharmaceutical substances implies a

6

cytocompatible character of the unloaded nanogels to prevent unwanted side effects and to enable the

7

analysis of drug mediated effects. After loading with cytostatic DOX, the nanogels should ideally result in

8

a cytotoxic response.

9 10

11 12

Figure 8: Cell viability of L929 mouse fibroblasts after incubation with unloaded nanogels (100 µg mL-

13

1),

14

24, 48 and 72 h, respectively. Data represent mean values ± SD of 6-fold measured samples.

15

* p ≤ 0.05 Mann-Whitney test compared to free DOX.

DOX loaded nanogels (100 µg polymer and 2.5 µg DOX mL-1) and free DOX (2.5 µg DOX mL-1) for

16 17

At all incubation time points, the metabolic activity of cells treated with unloaded nanogels was found to

18

be at the level of the untreated controls or even higher. The absence of harmful effects on cell integrity

19

demonstrates the general cytocompatibility of the polymeric material or the nanogel, respectively (Figure 23



Page 24 of 34

1

8, for higher and lower DOX dosages see SI Figure S21, S22). In contrast, DOX loaded nanogels as well

2

as free DOX induced a clear dose and time dependent decrease in cell viability. Interestingly, DOX

3

entrapped in the nanogels was more potent than the respective free DOX dosage in terms of mediating a

4

more drastic cytotoxic effect. Exemplarily, the mean viability of cells treated for 24 h with DOX loaded

5

nanogels (100 µg polymer and 2.5 µg DOX mL-1) was found to be only 68%, whereas cells exposed to the

6

corresponding dose of free DOX (2.5 µg mL-1) still showed a viability of 82% – this trend even remained

7

visible over longer incubation times with viability rates of 10% vs. 36% after 72 h for the above mentioned

8

example. While the significant reduction of cell viability by the established cytostatic drug DOX was

9

expected, an enhancement of this effect by the application of DOX containing nanogels was somewhat

10

unexpected, but complies with other reports on nanocarriers in literature.42, 43 Release studies, which are

11

described above, indicated that at acidic pH value the release of the entrapped drug from the nanogel takes

12

about 100 h for a release of 50% of the loaded drug. Thus, it was rather expected that also in vitro

13

cytotoxicity would occur at later time points than the observed time frame between 24 h and 72 h. Most

14

likely, the studied nanogels release their load in the cell much faster than measured by ex vitro experiments

15

and additional release stimuli, such as enzymatic degradation, may contribute to the fast liberation of the

16

load.44 Also a slightly lower pH value in the lysosome (pH = 4 to 4.5) compared to early and late

17

endosomes can accelerate the release. In order to verify this hypothesis, the PDA-KTDA was incubated

18

under pH 4.5 at 37 °C and tracked by DLS (SI, Figure S23). The result shows that PDA-KTDA hydrolyzes

19

considerably faster at pH 4.5 than at pH 5.1.

20

The enhanced cytotoxic effect of DOX carrying nanogels is possibly mediated by an increased cellular

21

uptake efficiency of the nanoformulated carrier bearing the DOX load in a more concentrated form

22

compared to the free drug, which was dissolved in the culture media.

23 24

Time dependent uptake of DOX loaded nanogels and free DOX 24


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Biomacromolecules

1

As mentioned previously, an increased uptake of DOX mediated by the nanogel might be a reason for the

2

enhanced cytotoxicity of the delivery system. For this purpose, the uptake kinetics of DOX loaded

3

nanogels in comparison to free drug were evaluated by flow cytometry. The obtained mean fluorescence

4

intensities of the cell populations clearly display a time dependent increase for both the DOX loaded

5

nanogel and the free DOX (Figure 9). Whereas the majority of the DOX loaded nanogels was internalized

6

by the cells already within the first 4 h, free DOX uptake remained relatively constant over the whole

7

experiment without reaching a plateau. Interestingly, the DOX entrapped in nanogels is taken up much

8

more efficiently than the free drug. Taking the mean fluorescence intensity value as read out, the

9

internalization rate of the nanogels exceeds the uptake of free DOX by the factor of 2.5 to 3 over the whole

10

examined period of time. As reported for other nanostructured carriers, this effect can be attributed to an

11

increased uptake rate of the nanogels into the cells via various endocytotic pathways.

12 13

Figure 9: Flow cytometry investigation on the time dependent uptake of DOX loaded nanogels (1000 µg

14

polymer and 25 µg DOX mL-1) and the free DOX (25 µg DOX mL-1) by L929 mouse fibroblasts at 37 °C

15

following incubation times of 1, 4, 24 and 48 h. Cells incubated only with culture medium served as

16

control. Line plot depicts mean fluorescence intensities obtained from flow cytometry of the analyzed cell

17

populations. The data are expressed as mean ± SD of triplicate samples.

18 25



Page 26 of 34

1

Internalization of DOX loaded nanogels in comparison to free DOX

2

CLSM investigations with precise optical z-sectioning through the cell body of treated cells proved that

3

free DOX as well as the DOX loaded nanogels entered the cells showing both lysosomal and cytoplasmatic

4

localization, and in selected cell samples even an entry into the nucleus could be observed (Figure 10 and

5

SI, Figures S24-S26). Quantitative image analysis of cell associated fluorescence intensity values of

6

internalized fluorescent doxorubicin confirmed the results from flow cytometry in terms of an increased

7

uptake of nanogel loaded drug vs. free drug (Figure S27).

8 9 10 B

A

10 µm

D

C

E

10 µm

10 µm

10 µm

F

10 µm

10 µm

11

Figure 10: CLSM images of adherent L929 cells after 4 h incubation at 37 °C with DOX loaded nanogels

12

at a concentration of 500 µg polymer and 12.5 µg DOX/mL. Transmitted light (A), Cell membranes (B),

13

cell nuclei (C) and late endosomes/lysosomes (D) were specifically stained and correlated with the 26


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Biomacromolecules

1

fluorescence signal of DOX loaded nanogels (E). Overlay of all channels proves (F) an intracellular

2

localization of the DOX loaded nanogels in the cytoplasm (F, orange color) but also a colocalization with

3

lysosomal structures (F, yellow color) and nuclear regions (F, pink color).

4

Results for free DOX show similar intracellular distributions of the DOX (SI Figure S24).

5 6

A colocalization of nanogel associated DOX with lysosomal compartments was expected due to the

7

proposed uptake mechanism via endocytotic pathways – the concurrent cytoplasmic presence of the DOX

8

signal most probably results from drug released from lysosomes by endosomal escape events.45 50 Identical

9

results for cellular distribution were obtained for the free DOX treated cells, even though due to an uptake

10

via passive diffusion a prominent cytoplasmatic localization would be expected – the additional presence

11

in lysosomal compartments might be explained by a further diffusion into lysosomal compartments where

12

it accumulates due to cation trapping.46, 47, 49 The relatively low intracellular concentration of the free DOX

13

as observed by flow cytometry and CLSM image analysis can be caused by a lower uptake rate of passive

14

diffusion vs. active endocytosis. Additionally, export processes via MDR transporters very likely more affect

15

the free drug located in cytoplasmic regions than nanogel associated DOX present in the lysosomal

16

compartment.48, 49

17

In context with the outcome of the cytotoxicity assay, results support the interpretation that the presented

18

nanogels are feasible carriers for transporting and protecting their specific load in combination with promoting

19

uptake, accumulation and intracellular release of the drug. The higher uptake rate could in turn be beneficial

20

to e.g. lower the required dose for in vivo application of anticancer therapeutics. In combination with the

21

shielding effect of the nanogel, the occurrence of unwanted side effects in healthy tissues and systemic

22

toxicity could be reduced.

23 24

Conclusion 27



Page 28 of 34

1

In summary, a new type of pH-sensitive nanogels was successfully synthesized by surfactant-free

2

precipitation polymerization methods applying on the monomer DMDOMA in combination with AA for

3

the first time. Due to the acetal groups, the nanogel can be hydrolyzed at acidic condition, which is present

4

for example in intracellular lysosomal compartments, while it remains stable at neutral physiological

5

environment. In addition, an implemented ketal containing crosslinker (PDA-KDTA) facilitates the full

6

degradation of the nanogel into smaller polymers chains under acidic condition. On the contrary, no

7

degradation was observed for the nanogel applying a disulfide crosslinker (PDA-BAC) even in the

8

presence of strong reducing agents. All nanogels are able to encapsulate hydrophobic compounds within

9

the hydrophobic microdomains in the three-dimensional network, as it was tested with NR as a model

10

compound. Furthermore, in the case of the degradable nanogel PDA-KTDA, DOX was effectively

11

encapsulated leading to loading capacities of 2.6%. While at neutral conditions only a slow release was

12

observed, the PDA-KTDA nanogel released 50% of the initial DOX amount within 4 days at acidic pH

13

values of 5.1.

14

Cell biological experiments proved that the PDA-KTDA nanogel itself is cytocompatible and capable of

15

delivering its DOX load in a highly effective manner. The enhanced uptake of the DOX loaded PDA-

16

KTDA nanogel resulted in significantly enhanced cytotoxic response in comparison to the free drug–

17

rendering the PDA-KTDA nanogel as a versatile and promising drug carrier for biomedical applications.

18 19

Notes

20

The authors declare no competing financial interest

21 22

Associated Content

23

Supporting information 28


Page 29 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

The Supporting Information is available free of charge on the ACS Publications website at DOI:

2

SEM, DLS, 1H NMR, DOX calibration, SEC, the mean fluorescence intensities of DOX for all the tested

3

groups in CLSM images, the viability of L929 mouse fibroblasts incubated with free DOX or DOX-loaded

4

nanogels by different concentration.

5 6

Acknowledgements

7

We acknowledge funding from the collaborative research center PolyTarget (SFB 1278, projects A01, A04

8

to A06, C01, C04) by the German Science Foundation (DFG). P. W. was financially supported by the

9

China Scholarship Council. J. C. B. acknowledges the DFG for generous funding within the Emmy-

10

Noether-Programme (BR 4905/3-1). M. N. L. acknowledges the German Federal Ministry of Education

11

and Research (BMBF, #13N13416 smart-dye-livery) for funding. We acknowledge Dr. Grit Festag for the

12

SEC measurement.

13 14

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