Smart pH-Sensitive Nanogels for Controlled Release in an Acidic

26 Oct 2018 - The encapsulation of therapeutic compounds into nanosized delivery vectors has become an important strategy to improve efficiency and ...
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Smart pH-Sensitive Nanogels for Controlled Release in an 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 Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 12/02/18. For personal use only.

Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstraße 10, 07743 Jena, Germany ‡ Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany ∥ Institute of Pharmacy and Biopharmacy, Department of Pharmaceutical Technology, Friedrich Schiller University Jena, Lessingstrasse 8, 07743 Jena, Germany S Supporting Information *

ABSTRACT: The encapsulation of therapeutic compounds into nanosized delivery vectors has become an important strategy to improve efficiency and reduce side effects in drug delivery applications. Here, we report the synthesis of pHsensitive nanogels, which are based on the monomer N-[(2,2dimethyl-1,3-dioxolane)methyl]acrylamide (DMDOMA) bearing an acid cleavable acetal group. Degradation studies revealed that these nanogels hydrolyze under acidic conditions and degrade completely, depending on the cross-linker, but are stable in physiological environment. The best performing system was further studied regarding its release kinetics using the anticancer drug doxorubicin. In vitro studies revealed a good compatibility of the unloaded nanogel and the capability of the doxorubicin loaded nanogel to mediate cytotoxic effects in a concentration and time-dependent manner with an even higher efficiency than the free drug. Based on the investigated features, the presented nanogels represent a promising and conveniently prepared alternative to existing carrier systems for drug delivery.

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INTRODUCTION The encapsulation of pharmaceutically active ingredients into nanosized carrier systems, which are most commonly based on polymers, has become a major strategy in research to improve the efficacy of these drugs by a more localized delivery and a controlled release. In particular, great efforts have been paid to the development of carrier systems for potent chemotherapy agents.1−3 Among them, a smart nanoscale drug delivery system, such as liposomes, polymeric micelles, metallic nanoparticles, and nanogels, have been intensively investigated due to their advantage of enhancing the delivery efficiency of anticancer drugs by enhanced permeability and retention (EPR), while reducing side effects at a certain level.4−7 In particular, nanogels with three-dimensional polymer networks have attracted more and more attention because of their excellent properties, including high loading capacity,8 high cellular uptake,9 large surface area allowing multivalent bioconjugation,10 and smart responsiveness to environmental stimuli such as pH value,11 redox potential,12,13 temperature,14 and enzymatic activity.15 The responsive character is useful to control the release of the encapsulated active ingredients. For instance, the variation of the pH value from the extracellular compartments (pH = 7.4) to the intracellular lysosomes (pH = 4.0−4.5) and endosomes (pH = 5.0−5.5)16 can be used to trigger such an intracellular drug release by incorporating acid-cleavable © XXXX American Chemical Society

groups or cross-linkers into the nanogels. Moreover, the increased intracellular glutathione (GSH) concentration results in a reducing environment within the cytosol of the cell,17 which facilitates the cleavage of disulfide bonds and, thus, a degradation of an appropriately modified nanogel. To develop such smart nanogels an increasing number of different methods have been studied in the past decade, such as emulsion polymerization,18,19 inverse emulsion polymerization,20 and precipitation polymerization.21 Among them, the latter turned out to be one of the most popular methods due to the ease of use, the high conversion of monomers, and the wide range of options to incorporate responsive groups. In particular, the use of water as solvent and the application of a straightforward free radical polymerization technique render this method attractive for biomedical applications, as potentially harmful solvents can be excluded, while still welldefined nanostructures are obtained. Furthermore, the nanogels can conveniently be purified by centrifugation or dialysis in water. N-Isopropylacrylamide (NIPAm),22−24 poly(ethylene glycol) (PEG),8,25 and N-vinylcaprolactam (VCL)14,26,27 are the Special Issue: Biomacromolecules BPC Received: August 13, 2018 Revised: October 25, 2018 Published: October 26, 2018 A

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Scheme 1. Illustration of the Nanogel-Preparation, Drug Loading, and Acid-Triggered Drug Release from the Nanogels

most common monomers that have been used to construct a large number of responsive nanogels by precipitation polymerization. In addition, acrylic acid (AA) is commonly incorporated to stabilize the nanogels because of the negative charge.28 Various cross-linkers are also applied, including crosslinkers based on ketal or disulfide bonds, which can be cleaved by the decrease of the pH value or a reductive environment, respectively.8,11 This degradation of cross-linking points in the nanogel results in the dissolution of the material, which is so far the most commonly used trigger to release any encapsulated compounds. However, the polymer chains themselves retain their partially hydrophobic character, which might impede the accessibility of the labile links or the complete release of the active ingredients. Nanogels, which do not only rely on the cleavage of the cross-linking points, but facilitate a change, for example, of the polarity of the whole polymer chains upon a trigger, have so far not been considered most probably as a result of the limited number of suitable monomers reported up to now. N-[(2,2-Dimethyl-1,3-dioxolane)methyl]acrylamide (DMDOMA) is a recently introduced monomer, which has a similar structure like NIPAm and also features a comparable thermoresponsive behavior.29−34 However, in contrast to NIPAm, the acetal group of the monomer endows the resulting polymer with a degradable side chain, which at low pH values will result in a cleavage of the acetal and finally renders the polymer very hydrophilic due to the formation of a dihydroxy moiety in the side chain. Despite the similarity between NIPAm and DMDOMA, the preparation of nanogels using this pH-sensitive monomer has so far not been reported. In this work, we prepared a series of a new type of nanogels based on DMDOMA applying three different cross-linkers: N,N′-methylene bis(acrylamide) (BIS), N,N′-bis(acryloyl)cystamine (BAC), and (propane-2,2-diylbis(oxy))bis(ethane2,1-diyl)diacrylate (KTDA; Scheme 1). Acrylic acid is

additionally integrated in order to stabilize the resulting nanogel and circumvent the use of additional surfactants, which would be difficult to remove. At low pH values, the acetal groups of the used DMDOMA will be hydrolyzed, resulting in a change of hydrophilicity and consequently can trigger the release of an encapsulated drug (Scheme 1). Applying a degradable cross-linker, the proceeding degeneration of these nanogels would lead to the formation of linear water-soluble polymers, which should be sufficiently small enough for further elimination from the body, for example, via the kidney.

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EXPERIMENTAL SECTION

Materials. The monomer N-[(2,2-dimethyl-1,3-dioxolane)methyl]acrylamide (DMDOMA) and the cross-linker (propane-2,2diylbis(oxy))bis(ethane-2,1-diyl)diacrylate (KTDA) were synthesized according to a procedure reported elsewhere.35,36 Acrylic acid (AA), potassium persulfate (KPS), N,N′-methylene bisacrylamide (BIS), Nile red (NR), and doxorubicin hydrochloride (DOX) were purchased from Sigma-Aldrich. N,N′-Bis(acryloyl)cystamine (BAC) was purchased from ABCR. Instrumentation. 1H NMR spectra were recorded at room temperature in CDCl3 or D2O on a Bruker Avance 300 MHz. The chemical shifts are given in ppm. Dynamic light scattering (DLS) was performed on a Zetasizer Nano ZS (Malvern Instruments, Herrenberg, Germany). The 1 mg mL−1 nanogel suspensions were measured at 25 or 37 °C (λ = 633 nm) at an angle of 173° after an equilibration time of 120 s. The size distribution of the nanospheres was calculated applying the nonlinear least-squares fitting mode. A M200 Pro fluorescence microplate reader (Tecan) and a spectrometer FP-8300 (JASCO) were used for fluorescence measurements. Scanning electron microscopy (SEM) imaging was conducted with a Zeiss Sigma VP utilizing the lnlense detector. The morphology of the nanogels was observed by SEM. Briefly, 0.7 mg mL−1 nanogel B

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the size and mean count rate of the nanogels were measured by DLS at 37 °C with a fixed position and attenuator. Loading of Nile Red (NR) and Doxorubicin (DOX) into Nanogels. Nile red (NR) loading experiment was performed according to procedures reported in literature.21 A total of 2 mg NR was dissolved in 2 mL of THF and mixed with 2 mL of nanogel suspension (1 mg mL−1). After stirring at room temperature for 24 h, the dispersion was dialyzed (molar mass cut off of the membrane: 12000−14000 g mL−1) to remove the THF. Afterward, the suspension was filtered to remove any remaining free NR. In the case of DOX loading, 1.5 mg DOX was first dissolved in 10 mL of phosphate buffer (pH 7.4) containing 25 mg of PAD-KTDA nanogel. After stirring for 24 h at room temperature, the dispersion was dialyzed for 30 h (molar mass cut off of the membrane: 12000− 14000 g mL−1) to remove any remaining free DOX. This extensive washing procedure was further used to ensure that no weakly bound DOX remains on the surface or inside of the nanogels, which may result in an uncontrolled burst release in the following degradation experiments, and only sufficiently well-bound drug is tested. In addition, free DOX in the same buffer solution was also dialyzed under the same conditions to evaluate the speed of passing the dialysis membrane. To determine the final loading capacity and encapsulation efficiency, 300 μL of the dispersion was completely degraded by addition of 50 μL of HCl. Then, 40 μL of the solution was diluted with 2 mL of dimethyl sulfoxide. The DOX content of this solution was determined by fluorescence measurements according to a previously recorded calibration (SI, Figure S18). Drug loading capacity (DLC) and encapsulation efficiency (EE) of the nanogels were defined by the following equations:

suspension was dropped on a silicon wafer and dried at room temperature. Size-exclusion chromatography (SEC) measurements were performed on an Jasco equipped with a PU-980 pump, a RI-930 refractive index detector, and a PSS NOVEMA-MAX column with H2O + 0.1% TFA + 0.1 M NaCl as eluent. The column oven was set to 30 °C, and a poly(2-vinylpyridine) (P2VP) standard was used for calibration. A TECAN Infinite M200 PRO plate reader (TECAN, Crailsheim, Germany) was used to determine the cell viability using AlamarBlue assay with excitation/emission wavelength at λEx = 570 nm and λEm = 610 nm in fluorescence bottom reading mode and at ambient conditions. Each well was measured with 25 flashes per scan. Evaluation of uptake of DOX-loaded nanogels and free DOX was performed by flow cytometry measured on a Beckmann Coulter Cytomics FC-500 (Beckman Coulter, Krefeld, Germany) equipped with an Uniphase argon ion laser, λ = 488 nm, 20 mW output, and fluorescence signal emission collected in FL-3 with λ = 620 nm filter. Analysis was performed using Cytomics CXP software. For confocal laser scanning microscopy (CLSM) uptake analysis of DOX-loaded nanogels and free DOX as well as colocalization with cell organelles, cell membrane, nuclei, and the late endosomes/lysosomes were stained with WGA Alexa 633, Hoechst 33342 and LysoTracker Green, respectively. CLSM images were acquired using a Zeiss LSM 880 (Carl Zeiss, Oberkochen, Germany) with the following settings: WGA Alexa 633: λEx = 633 nm, λEm = 638−747 nm; LysoTracker Green: λEx = 488 nm, λEm = 490−535 nm, DOX: λEx = 488 nm, λEm = 535−677 nm, Hoechst 33342: λEx = 405 nm, λEm = 410−499 nm and transmission signal with λ = 405 nm laser and PMT detector. Images were captured with a Plan-Apochromat 63× objective in multitrack mode, enabling single excitation and emission of fluorescence dyes. Colocalization was visualized in overlay images of the multiple channels. Synthesis. Degradation Study of Monomer (DMDOMA). To evaluate the stability of the DMDOMA, 5 mg of the monomers were dissolved into pure D2O and D2O at pH 10 in NMR tubes, respectively. Then, the tubes were incubated at 70 °C and measured at room temperature at each time point. Preparation of Nanogels. The procedure for synthesis of PDAKTDA via precipitation polymerization is exemplarily described here: 33 μL (9.8 mol % of total monomers) acrylic acid was dissolved in 15 mL of H2O in a three-neck flask equipped with a reflux condenser, NaOH (1 M) solution was used to adjust the pH value around 10. A total of 800 mg (88.2 mol % of total monomers) DMDOMA and 26 mg (2 mol % of total monomers) KTDA were added, and the NaOH solution was used to keep the pH value at 10 and fill the total volume of the reaction system to 30 mL. After degassing for 45 min, the flask was placed in a preheated oil bath at 70 °C; subsequently, 7 mg KPS in 3 mL of degassed H2O was added to initiate the polymerization. The reaction was cooled to room temperature after 16 h and dialyzed at pH 8 (phosphate buffer, 10 mM) for 3 days (molar mass cut off 12000−14000 g mol−1) to get a PDA-KTDA nanogels’ suspension in the yield of 93%. For the synthesis of other nanogels, PDA-BIS (88%) and PDA-BAC (96%) were used in the same molar ratio of monomers, cross-linkers, and KPS. pH-Dependent Hydrolysis of Nanogels. For the 1H NMR investigations, the three nanogels (each 1 mg) were suspended in acetate buffer (10 mM, pH = 5.1) and D2O containing 5 mg of trioxane as internal standard and incubated at 37 °C. At each time point, samples were measured by 1H NMR and the integral of trioxane was set as 100 as a standard. The acetone peak, which is the hydrolysate, at 2.1 ppm was tracked for the hydrolysis study. For the DLS investigation, 1 mg mL−1 nanogel suspension was dispersed in phosphate buffer (10 mM, pH = 7.4) or acetate buffer (10 mM, pH = 5.1) and incubated at 37 °C. At each time point, the size and mean count rate of the nanogels were measured at 37 °C with the fixed position and attenuator. Degradation of Nanogels by DL-Dithiothreitol (DTT). 10 mM DTT was added to 1 mg mL−1 PDA-BAC nanogel in phosphate buffer (10 mM, pH = 7.4) and incubated at 37 °C. At each time point,

DLC(%) =

EE(%) =

wt of DOX in nanogels × 100% wt of DOX‐loaded nanogels

wt of DOX in nanogels × 100% initial wt of DOX

Fluorescence Quenching Assay for pH-Dependent Degradation of the Nanogels. The nanogel suspension loaded with NR was first incubated at 37 °C for 3 h, then, phosphate buffer (pH = 7.4) or acetate buffer (pH = 5.1) was added and incubated for another 15 min. The intensity of the NR fluorescence at λEm = 640 nm was then measured as time point zero. Subsequently, at regular time intervals 100 μL solvent was taken out and the fluorescence intensity was immediately measured. Release Study of DOX. The release experiment of DOX from nanogels was carried out by a dialysis method.12 Briefly, 1 mL of DOX-loaded nanogels solution was injected into 2 mL of phosphate buffer (10 mM, pH = 7.4) or acetate buffer (10 mM, pH = 5.1), respectively. For dialysis (molar mass cut off of the membrane: 12000−14000 g mL−1) the samples were immersed into 20 mL of the respective buffer at 37 °C. At regular intervals, 2 mL of the outer medium was taken for fluorescence measurements (calibration SI, Figures S19 and S20) and refilled with the same amount of fresh buffer. Cytocompatibility of Nanogels and Cytotoxicity of DOX-Loaded Nanogels and Free DOX. Effects on the cellular metabolism upon incubation with unloaded and DOX loaded nanogels as well as free DOX were evaluated using the established L929 mouse fibroblast cell line.37 The in vitro cytotoxicity experiments were performed using commercial AlamarBlue assay following supplier’s instructions. The cells were routinely cultured as follows: Dulbecco’s modied eagle’s medium (DMEM) supplemented with 10% FCS, 100 U mL−1 penicillin and 100 μg mL−1 streptomycin (all components from Biochrom, Berlin, Germany) at 37 °C in a humidified atmosphere with 5% (v/v) CO2. L929 cells were seeded in 96-well plates at a density of 1 × 104 cells/well and were grown as monolayer cultures for 24 h. Cells were then incubated with different concentrations of unloaded nanogels (10, 100, and 1000 μg polymer mL−1), DOXloaded nanogels (10, 100, and 1000 μg polymer mL−1 containing 0.25, 2.5, and 25 μg DOX mL−1), and free DOX (0.25, 2.5, and 25 μg mL−1) for 24, 48, and 72 h at 37 °C (n = 6 for each experimental C

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Biomacromolecules group). Control cells were incubated with fresh culture medium. After incubation, culture medium was aspirated and 100 μL of fresh media containing 10 μL of AlamarBlue reagent prepared according to the manufacturer’s instructions were added to each well. After 4 h at 37 °C, a 90 μL aliquot of each solution was transferred to a new microtiter plate and the fluorescence was measured at λEx = 560 nm and λEm = 590 nm. The negative control was standardized as 0% of metabolism inhibition and referred as 100% viability. Flow Cytometric (FC) Investigations on Time-Dependent Uptake. FC measurements were performed to gather quantitative information about the time-dependent uptake of the DOX-loaded nanogels (1000 μg polymer and 25 μg DOX mL−1) in comparison to the free DOX (25 μg DOX mL−1). For this purpose, cells were incubated for 1, 4, 24, and 48 h (n = 3 for each experimental group) and then the cells were washed with PBS to remove the remaining material. Subsequently, the cells were detached by trypsin treatment and directly subjected to FC analysis, where 5000 individual cells/ sample were measured for their fluorescence intensity, derived from the cell-associated, DOX-loaded nanogel and the free DOX, respectively. Obtained mean fluorescence intensities of three individual measurements per sample were then plotted against time. Internalization and Colocalization with Cellular Compartments. The cellular internalization and intracellular localization of DOXloaded nanogels and the free DOX in L929 cells were further elucidated by confocal laser scanning microscopy (CLSM) investigations. For this purpose, the cells were incubated for 4 or 24 h with DOX-loaded nanogels (500 μg polymer and 12.5 μg DOX mL−1) and the free DOX (12.5 μg DOX mL−1) at 37 °C. In order to assign the localization of the fluorescent DOX to cellular structures, the living adherent cells were stained with specific dyes for the cell plasma membrane (wheat germ agglutinin Alexa 633, 5 μg mL−1 for 30 min), the nuclei (Hoechst 33342, 1 μg mL−1 for 30 min), and the acidic late endosomes/lysosomes (LysoTracker Green, 1 μM for 30 min), respectively.

Table 1. Characterization of Nanogels by DLS Measurements

a

sample

Z-avga (nm)

PDIa

PDA-BIS PDA-BAC PDA-KTDA

150 97 202

0.217 0.093 0.159

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

Figure 1. SEM image of PDA-KTDA.

their size and PDI value. This phenomenon was also observed in previous studies.10,24 The nanogels swell more strongly during the polymerization process with increasing water solubility of the cross-linker, leading to larger sizes and higher PDI values. At the same time, the longer the cross-linking chains are, the larger the nanogels become. SEM measurements revealed sizes of 160−360 nm, however, it has to be taken into account that these samples are in the dried state, which might cause shrinkage or a flattening of the gel. Hydrolysis of DMDOMA in the Nanogels. The presented hydrophobic nanogels were designed to facilitate a progressing hydrolysis of the acetal groups under mildly acidic conditions, which turns the polymer in these hydrogels from partially hydrophobic into very hydrophilic. 1 H NMR was used to monitor the hydrolysis of the acetal groups at a pH value of 5.1. Therefore, an internal standard (trioxane, 5.1 ppm) was set as 100 and used to compare the integrals of the signal of the released acetone. All nanogels displayed similar hydrolyses rates over a time of up to 168 h (Figure 2A, SI, Figures S4−S6). In addition, hydrolysis of the nanogels was investigated by DLS. Nanogels were incubated at 37 °C under acidic (pH = 5.1) and also neutral conditions (pH = 7.4) for up to 168 h, and the change of the relative count rate was assessed. Assuming a degradation of the acetal groups only at acidic conditions and thus a release of the more hydrophilic dihydroxy side chains, an increased swelling of the nanogels with time can be expected, while the count rate should decrease as a consequence of the lower density of the nanogel. As seen in Figure 2 B, the relative count rates of all three nanogels (all the three curves are overlapping) kept constant at physiological conditions (pH = 7.4), while a considerable decrease is observed at acidic conditions (pH = 5.1). Among the differently cross-linked nanogels, the relative count rate of PDA-BAC decreased less prominent, which can be explained by the fact that during the polymerization of the nanogel the

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RESULTS AND DISCUSSION Synthesis and Characterization of Nanogels. Scheme 1 shows all monomers and cross-linkers that were used in this work. The monomer DMDOMA and the cross-linker KTDA were synthesized according to procedures reported in the literature.30,31 All nanogels were prepared in aqueous medium by precipitation polymerization without the use of surfactants (Scheme 1). NaOH was used to maintain the basic pH value, which is required to preserve the pH-sensitive monomer DMDOMA and to deprotonate the acrylic acid (AA). The anionic form of AA stabilizes the nanogels and inhibits their aggregation due to electrostatic repulsion.28 In order to evaluate the stability of the monomers, DMDOMA was incubated in pure water and buffer (pH 10) at 70 °C to mimic the conditions during the nanogel synthesis. As shown in Figure S1 (SI), DMDOMA degraded fast within 1 h in pure water. However, it was very stable at pH 10 for at least 24 h. This proves the potential polymerization of DMDOMA at pH 10 and 70 °C. Three different cross-linkers were incorporated into the nanogels, which allow a long-term storage at neutral conditions. The average diameter of the nanogels was measured by DLS in pure water (SI, Table S1) and phosphate buffer (Table 1), as well as by SEM in the dry state (Figure 1 and SI, Figures S2, S3). A significant decrease of size is observed in DLS when changing the solvent from pure water to the buffer solution, which is in accordance with previous reports on similar systems.38 We assume that the addition of the buffer results in a more collapsed state of the gel, as the repulsive forces of the negatively charged acrylic acid moieties are shielded. The three different nanogels differ significantly in D

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Figure 2. Measurement of the hydrolysis of nanogels at 37 °C at various time points by 1H NMR (A) and by DLS (B). All the samples were measured in triplicate.

disulfide bonds in the cross-linker might be attacked by radicals resulting in thiol ether bonds which, in turn, would result in higher cross-linking density (SI Scheme S1).39,40 At the same time, the relative count rate of PDA-KTDA showed the strongest decrease due to the pH-sensitive ketal cross-linker, which could additionally be hydrolyzed besides the acetal groups. The zeta potential of the nanogels was also measured before and after hydrolysis (SI, Figures S8−S10). As expected, all nanogels possess a negative surface potential due to the acrylate, which may improve the circulation time in blood by reducing the interaction with negatively charged proteins.24 Degradation of Cross-Linkers. Three different crosslinkers were applied in this study, which comprise either nondegradable amide bonds (PDA-BIS), cleavable disulfide bonds (PDA-BAC), or acid-labile ketal bonds (PDA-KTDA). The degradability of these three nanogels was tested by timedependent DLS measurements in acidic conditions or in the case of PDA-BAC additionally under a reductive environment (Scheme 2). PDA-BIS is considered to be a control sample, which was incubated at 37 °C under acidic (pH = 5.1) as well in neutral conditions (pH = 7.4) for 10 days. After 10 days, the size did not change at neutral conditions, but increased to 170 nm at a pH value of 5.1 (Figure 3), which is related to the hydrolysis of the acetal groups within the polymer main chain, leading to a swelling of the particle as a result of the increased hydrophilicity of the formed dihydroxy moiety (Figure 2). PDA-BAC was also hydrolyzed at different pH conditions for 10 days. The diameter increases to 116 nm under acidic conditions (pH = 5.1) and is stable at neutral conditions (pH = 7.4; Figure 4 A). However, no degradation under these conditions is expected for this system. The presence of disulfide bonds in the BAC cross-linker and the resulting PDA-BAC nanogel should be sensitive to cleavage by reductive agents. DTT (10 mM), a commonly used

Scheme 2. Illustration of the Degradation Pathways for the Different Nanogels

reducing agent, was added to the nanogel suspension at neutral conditions (pH 7.4), incubated at 37 °C, and monitored by DLS for 10 days. Figure 4B shows that both the diameter and the mean count rate remain constant within the whole process. As already mentioned, the disulfide bonds may undergo a side reaction during the polymerization, leading to stable thiol ether bonds (SI, Scheme S1).39,40 In order to ensure that this effect is not only related to the concentration and strengths of the reducing agent, an even higher concentration of DTT (100 mM) and stronger reducing agents (TCEP·HCl) were used (SI, Figures S11 and S12). In addition, the nanogel was first completely hydrolyzed by HCl to guarantee the access of the reducing agent to the cross-linking points, then 100 mM DTT or TCEP·HCl were added to the solutions. After 2 days incubation at 37 °C, DLS measurements revealed no change in size, only the count rate decreased slightly, which revealed that E

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the preparation of the nanogels was performed under the same conditions but without the cross-linker. The resulting polymer has a similar distribution like the degradation product of PDAKTDA as shown by DLS (SI, Figure S14). Self-cross-linking processes might have occurred, as already reported in literature.41 However, it is an enormous analytical challenge to qualify and quantify these undefined cross-linking processes, which was also not the scope of this work. A closer look on the pH response of the nanogels immediately after an induced pH change (time point 0) shows further that the diameters of PDA-BIS and PDA-BAC decrease below a pH of 5, but remain constant above that point (Figure S15). In contrast, the diameter of PDA-KTDA already decreases below a pH of 6.2. This supports the assumption that the different cross-linkers can influence the pKa of the acrylate, which is around 4.8.21 NR Encapsulation and Fluorescence Measurement. As reported previously, similar nanogels are able to encapsulate hydrophobic compounds because of hydrophobic microdomains within the gel network.10 Nile red (NR), a commonly used hydrophobic model compound, displays a strong fluorescence if dispersed in a hydrophobic environment but is almost not fluorescent in aqueous dispersions (SI, Figure S16).25 Due to the hydrophobic domains formed by the acetal groups, NR could be loaded into the presented nanogels, resulting in a strong fluorescence intensity of the dye (λmax = 650 nm). Monitoring the fluorescence intensity over time in different media allows to follow the degradation of the nanogels, as they become increasingly hydrophilic and, thus, the fluorescence of NR is quenched. Figure 6 shows the timedependent relative fluorescence intensity of the different nanogels loaded with NR when incubated in neutral or acidic media, respectively. As expected, the fluorescence signal is stable under physiological conditions, which proves a stable loading of the nanogels with NR at a neutral pH value. The pH-sensitive properties of the nanogels became obvious at a decreased pH level of 5.1, where fluorescence intensities decreased clearly over time in all nanogels in a cumulative

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

noncleavable cross-linking points were formed during the synthesis, and only the remaining disulfide bonds can be cleaved. The third nanogel, PDA-KTDA, only contains pH-sensitive groups. When incubating the nanogel under physiological (pH = 7.4) and acidic conditions (pH = 5.1) at 37 °C for 10 days, the size of the remaining structures decreased to 14 nm, and the mean count rate decreases from 991 to 60 in acidic medium, while it remained constant at physiological pH values (Figure 5A,B). These results prove that the ketal linkage is sensitive toward acidic conditions and, thus, facilitates the degradation into smaller polymer chains, but remains quite stable at neutral conditions. In order to confirm the presence of single polymer chains, SEC was also used to characterize the hydrolyzed product (SI, Figure S13). In a further experiment,

Figure 4. Degradation study of PDA-BAC using DLS at 37 °C at different pH values (A) and with 10 mM DTT (B). F

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Figure 5. Degradation study of PDA-KTDA at different pH values using DLS at 37 °C: diameter change (A) and mean count rate at pH 5.1 (B).

DOX Loading and Release. DOX, a prominent anticancer drug also known as adriamycin or doxorubicin, was investigated regarding its ability to be encapsulated into and released from the nanogel PDA-KTDA. Because of the positive charge of DOX (pKa = 8.2) at physiological conditions and the hydrophobic domains, we expected strong hydrophobic and electrostatic interactions with the acetal-based chains and anionic acrylic acid, respectively. To estimate a loading capacity (LC) and encapsulation efficiency (EE), DOX dispersed in an aqueous buffer was added to the nanogel dispersion, and the combined mixture was dialyzed against pure buffer solution for 30 h to remove any free DOX, which was not entrapped in the nanogel. Dialysis effectively removes unloaded DOX from the DOX-loaded nanogels, which was proven by tests on free DOX, which easily and fast passes the membrane within 4 h. This unhindered passage was monitored by the measurement of the fluorescence intensity within the dialysis bag over time (SI, Figure S17). The final LC and EE were determined to be 2.9% and 52%, respectively, which demonstrated that the PDA-KTDA nanogel represents a promising candidate for drug delivery. This loading is already well suited for further biological investigations,10,24 but an improvement of the loading capacity is still considered in our ongoing work. As the nanogels were designed not only to encapsulate a drug, such as DOX, but selectively release it within the acidic environment of cellular endosomal/lysosomal compartments (pH = 5.0−6.0), we further analyzed the release behavior of DOX from nanogels under both physiological condition (pH = 7.4) and acidic condition (pH = 5.1). Incubating the loaded nanogels at 37 °C in neutral or acidic buffer, respectively, revealed that around 50% DOX was released from the PDAKTDA nanogel at pH 5.1 within 4 days, while only 20% DOX was released within the same time at a pH value of 7.4 (Figure 7). The increased release at acidic pH is most probably caused by a partly protonation of the acrylic acid but also a hydrolysis of the nanogel network. These results indicate that a release of DOX as model drug will preferably occur after cellular uptake

Figure 6. Change of relative fluorescence intensity over time of different nanogels loaded with NR and incubated in neutral (pH = 7.4) or acidic buffer (pH = 5.1) at 37 °C.

manner, indicating a constant hydrolysis of the acetal groups and also the degradation of pH-sensitive cross-linkers in PDAKTDA. Among all, PDA-BAC decreased slowest, which is again related to the increased cross-linking density resulting in a stronger NR association.10 In contrast, the intensity in PDAKTDA decreased fastest due to the additional hydrolysis of the ketal groups in the cross-linker. Taking together results from hydrolysis and degradation evaluation by NMR, DLS, and the NR experiment, it can be concluded that all three presented nanogels are capable of a pH-dependent change from partially hydrophobic to very hydrophilic. However, the PDA-KTDA nanogel displayed the fastest response and it was the only sample that could be fully degraded due to the labile linkers. Therefore, we focused in the further experiments exclusively on this PDA-KTDA nanogel, which included loading and release studies with doxorubicin (DOX), as well as in vitro experiments on cytotoxicity, cellular uptake, and distribution. G

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mediating a more drastic cytotoxic effect. Exemplarily, the mean viability of cells treated for 24 h with DOX loaded nanogels (100 μg polymer and 2.5 μg DOX mL−1) was found to be only 68%, whereas cells exposed to the corresponding dose of free DOX (2.5 μg mL−1) still showed a viability of 82%; this trend even remained visible over longer incubation times with viability rates of 10% versus 36% after 72 h for the above-mentioned example. While the significant reduction of cell viability by the established cytostatic drug DOX was expected, an enhancement of this effect by the application of DOX containing nanogels was somewhat unexpected, but complies with other reports on nanocarriers in literature.42,43 Release studies, which are described above, indicated that at acidic pH value the release of the entrapped drug from the nanogel takes about 100 h for a release of 50% of the loaded drug. Thus, it was rather expected that also in vitro cytotoxicity would occur at later time points than the observed time frame between 24 and 72 h. Most likely, the studied nanogels release their load in the cell much faster than measured by ex vitro experiments and additional release stimuli, such as enzymatic degradation, may contribute to the fast liberation of the load.44 Also, a slightly lower pH value in the lysosome (pH = 4−4.5) compared to early and late endosomes can accelerate the release. In order to verify this hypothesis, the PDA-KTDA was incubated under pH 4.5 at 37 °C and tracked by DLS (SI, Figure S23). The result shows that PDA-KTDA hydrolyzes considerably faster at pH 4.5 than at pH 5.1. The enhanced cytotoxic effect of DOX carrying nanogels is possibly mediated by an increased cellular uptake efficiency of the nanoformulated carrier bearing the DOX load in a more concentrated form compared to the free drug, which was dissolved in the culture media. Time-Dependent Uptake of DOX-Loaded Nanogels and Free DOX. As mentioned previously, an increased uptake of DOX mediated by the nanogel might be a reason for the enhanced cytotoxicity of the delivery system. For this purpose, the uptake kinetics of DOX-loaded nanogels in comparison to free drug were evaluated by flow cytometry. The obtained mean fluorescence intensities of the cell populations clearly display a time-dependent increase for both the DOX-loaded nanogel and the free DOX (Figure 9). Whereas the majority of the DOX-loaded nanogels was internalized by the cells already within the first 4 h, free DOX uptake remained relatively constant over the whole experiment without reaching a plateau. Interestingly, the DOX entrapped in nanogels is taken up much more efficiently than the free drug. Taking the mean fluorescence intensity value as read out, the internalization rate of the nanogels exceeds the uptake of free DOX by a factor of 2.5−3 over the whole examined period of time. As reported for other nanostructured carriers, this effect can be attributed to an increased uptake rate of the nanogels into the cells via various endocytotic pathways. Internalization of DOX-Loaded Nanogels in Comparison to Free DOX. CLSM investigations with precise optical z-sectioning through the cell body of treated cells proved that free DOX as well as the DOX loaded nanogels entered the cells showing both lysosomal and cytoplasmatic localization, and in selected cell samples even an entry into the nucleus could be observed (Figure 10 and SI, Figures S24−S26). Quantitative image analysis of cell associated fluorescence intensity values of internalized fluorescent doxorubicin confirmed the results from flow cytometry in terms of an increased uptake of nanogel loaded drug versus free drug (Figure S27).

Figure 7. Release profile of DOX from DOX-loaded nanogel (PDAKTDA) at different pH values. The samples were incubated at 37 °C within a dialysis bag and samples of the outer medium were taken to evaluate the amount of released DOX. Three samples for each pH conditions were measured at the same time.

at the acidic conditions of the lysosomal compartments. Hence, the PDA-KTDA nanogel features a controlled release of the drug, which is essential for an efficient transport if applied in vivo. Cytocompatibility and Cytotoxicity Tests. The use of the presented nanogels as versatile carriers for e.g. pharmaceutical substances implies a cytocompatible character of the unloaded nanogels to prevent unwanted side effects and to enable the analysis of drug mediated effects. After loading with cytostatic DOX, the nanogels should ideally result in a cytotoxic response. At all incubation time points, the metabolic activity of cells treated with unloaded nanogels was found to be at the level of the untreated controls or even higher. The absence of harmful effects on cell integrity demonstrates the general cytocompatibility of the polymeric material or the nanogel, respectively (Figure 8, for higher and lower DOX dosages see SI, Figures S21 and S22). In contrast, DOX-loaded nanogels as well as free DOX induced a clear dose and time-dependent decrease in cell viability. Interestingly, DOX entrapped in the nanogels was more potent than the respective free DOX dosage in terms of

Figure 8. Cell viability of L929 mouse fibroblasts after incubation with unloaded nanogels (100 μg mL−1), DOX-loaded nanogels (100 μg polymer and 2.5 μg DOX mL−1), and free DOX (2.5 μg DOX mL−1) for 24, 48, and 72 h, respectively. Data represent mean values ± SD of six-fold measured samples. *p ≤ 0.05 Mann−Whitney test compared to free DOX. H

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be expected; the additional presence in lysosomal compartments might be explained by a further diffusion into lysosomal compartments where it accumulates due to cation trapping.46,47,49 The relatively low intracellular concentration of the free DOX as observed by flow cytometry and CLSM image analysis can be caused by a lower uptake rate of passive diffusion versus active endocytosis. Additionally, export processes via MDR transporters very likely more affect the free drug located in cytoplasmic regions than nanogel associated DOX present in the lysosomal compartment.48,49 In context with the outcome of the cytotoxicity assay, results support the interpretation that the presented nanogels are feasible carriers for transporting and protecting their specific load in combination with promoting uptake, accumulation and intracellular release of the drug. The higher uptake rate could in turn be beneficial to, for example, lower the required dose for in vivo application of anticancer therapeutics. In combination with the shielding effect of the nanogel, the occurrence of unwanted side effects in healthy tissues and systemic toxicity could be reduced.

Figure 9. Flow cytometry investigation on the time dependent uptake of DOX loaded nanogels (1000 μg polymer and 25 μg DOX mL−1) and the free DOX (25 μg DOX mL−1) by L929 mouse fibroblasts at 37 °C following incubation times of 1, 4, 24, and 48 h. Cells incubated only with culture medium served as control. Line plot depicts mean fluorescence intensities obtained from flow cytometry of the analyzed cell populations. The data are expressed as mean ± SD of triplicate samples.

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CONCLUSION In summary, a new type of pH-sensitive nanogel was successfully synthesized by surfactant-free precipitation polymerization methods applying on the monomer DMDOMA in combination with AA for the first time. Due to the acetal groups, the nanogel can be hydrolyzed at acidic conditions, which is present, for example, in intracellular lysosomal compartments, while it remains stable at neutral physiological environment. In addition, an implemented ketal-containing cross-linker (PDA-KDTA) facilitates the full degradation of the nanogel into smaller polymer chains under acidic conditions. On the contrary, no degradation was observed for the nanogel applying a disulfide cross-linker (PDA-BAC) even in the

Results for free DOX show similar intracellular distributions of the DOX (SI, Figure S24). A colocalization of nanogel associated DOX with lysosomal compartments was expected due to the proposed uptake mechanism via endocytotic pathways; the concurrent cytoplasmic presence of the DOX signal most probably results from drug released from lysosomes by endosomal escape events.45,50 Identical results for cellular distribution were obtained for the free DOX treated cells, even though due to an uptake via passive diffusion a prominent cytoplasmatic localization would

Figure 10. CLSM images of adherent L929 cells after 4 h incubation at 37 °C with DOX-loaded nanogels at a concentration of 500 μg polymer and 12.5 μg DOX/mL. Transmitted light (A), cell membranes (B), cell nuclei (C), and late endosomes/lysosomes (D) were specifically stained and correlated with the fluorescence signal of DOX-loaded nanogels (E). Overlay of all channels proves (F) an intracellular localization of the DOXloaded nanogels in the cytoplasm (F, orange color), but also a colocalization with lysosomal structures (F, yellow color) and nuclear regions (F, pink color). I

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presence of strong reducing agents. All nanogels are able to encapsulate hydrophobic compounds within the hydrophobic microdomains in the three-dimensional network, as it was tested with NR as a model compound. Furthermore, in the case of the degradable nanogel PDA-KTDA, DOX was effectively encapsulated leading to loading capacities of 2.6%. While at neutral conditions only a slow release was observed, the PDA-KTDA nanogel released 50% of the initial DOX amount within 4 days at acidic pH values of 5.1. Cell biological experiments proved that the PDA-KTDA nanogel itself is cytocompatible and capable of delivering its DOX load in a highly effective manner. The enhanced uptake of the DOX-loaded PDA-KTDA nanogel resulted in significantly enhanced cytotoxic response in comparison to the free drug, rendering the PDA-KTDA nanogel as a versatile and promising drug carrier for biomedical applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.8b01228. SEM, DLS, 1H NMR, DOX calibration, SEC, the mean fluorescence intensities of DOX for all the tested groups in CLSM images, and the viability of L929 mouse fibroblasts incubated with free DOX or DOX-loaded nanogels by different concentration (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Johannes C. Brendel: 0000-0002-1206-1375 Ulrich S. Schubert: 0000-0003-4978-4670 Present Address §

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge funding from the collaborative research center PolyTarget (SFB 1278, Projects A01, A04 to A06, C01, C04) by the German Science Foundation (DFG) and funding from the ProExcellence II initiative “NanoPolar” by the State of Thuringia, Germany. P.W. was financially supported by the China Scholarship Council. J.C.B. acknowledges the DFG for generous funding within the Emmy-Noether-Programme (BR 4905/3-1). M.N.L. acknowledges the German Federal Ministry of Education and Research (BMBF, #13N13416 smart-dyelivery) for funding. We acknowledge Dr. Grit Festag for the SEC measurement and the DFG/EFRE for funding of the SEM (EM unit JCSM).

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REFERENCES

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