pH-Responsive Dendritic Core–Multishell Nanocarriers

COREL-07129; No of Pages 10 Journal of Controlled Release xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

pH-responsive dendritic core–multishell nanocarriers

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Emanuel Fleige Katharina Achazi, Karolina Schaletzki, Therese Triemer, Rainer Haag ⁎

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Freie Universität Berlin, Institut für Chemie und Biochemie, Takustraße 3, 14195 Berlin, Germany

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a r t i c l e

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Article history: Received 29 November 2013 Accepted 12 April 2014 Available online xxxx

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Keywords: Drug delivery Nile red Doxorubicin Transport capacity Dynamic light scattering Real time cell analysis

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In this paper we describe novel pH-responsive core–multishell (CMS) nanocarrier (pH-CMS), obtained by introducing an aromatic imine linker between the shell and the core. At a pH of 5 and lower the used imine linker was rapidly cleaved as demonstrated by NMR studies. The CMS nanocarriers were loaded with the dye Nile red (NR) and the anticancer drug doxorubicin (DOX), respectively. The transport capacities were determined using UV/Vis spectroscopy and the sizes of the loaded and unloaded CMS nanocarriers were investigated using dynamic light scattering (DLS). We could show that CMS nanocarriers efficiently transported NR in supramolecular aggregates, while DOX was transported in a unimolecular fashion. After cellular uptake the DOX-loaded pH-responsive nanocarriers showed higher toxicities than the stable CMS nanocarriers. This is due to a more efficient DOX release caused by the cleavage of the pH-labile imine bond at lower pH within the intracellular compartments. © 2014 Published by Elsevier B.V.

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Most clinically used drugs encounter the problem of short half-life times in blood and a high overall clearance rate. Due to their small size they rapidly diffuse into tissue and are therefore distributed throughout the whole body, which is the main reason for undesired side effects. New drug-delivery concepts rely on the use of polymeric drug delivery systems (DDSs) [1–3]. Among these one can find polymer conjugates [4,5], macromolecular prodrugs [6] and drug-delivery systems based on polymeric core– shell architectures [7]. The most famous examples of such core–shell architectures are polymeric micelles [8,9] and liposomes [10,11]. Nowadays, unimolecular core–shell particles have also become increasingly interesting because they do not fall apart upon dilution. For example, our group recently developed an efficient unimolecular core–shell architecture which consists of a dendritic hydrophobic poly(ethylene) core and a grafted, dendritic, hydrophilic polyglycerol (dPG) shell [12]. Furthermore, we developed a new type of unimolecular liposome-like

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1. Introduction

Abbreviations: DDS, drug delivery system; dPG, dendritic polyglycerol; mPEG, poly(ethylene glycol) methyl ether; PAMAM, poly(amido amine); HPMA, N-(2hydroxypropyl)methacrylamide; CMS, core–multishell; EPR, enhanced permeation and retention; PEI, poly(ethylene imine); pH-CMS, pH-responsive CMS nanoparticles, C18diCOOH, 1,18-octadecanedioic acid; mPEG1000, mPEG with an average number averaged molecular weight of 1000 g/mol; GPC, gel permeation chromatography; DLS, dynamic light scattering; DOX · HCl, doxorubicin hydrochloride, DOX, doxorubicin, free base; DMF, dimethylformamide; NR, Nile red; THF, tetrahydrofurane; SEC, size exclusion chromatography; DMEM, Dulbecco's Modified Eagle Medium; PBS, phosphate buffered saline; rt, room temperature; THBA, trihydroxybenzaldehyde; MWCO, molecular weight cut-off; RTCA, real time cell analysis. ⁎ Corresponding author. Tel.: +49 30 83852633; fax: +49 30 83853357. E-mail address: [email protected] (R. Haag). URL: http://www.polytree.de (R. Haag).

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multishell system which is based on a hydrophilic dPG core, a hydrophobic inner alkyl shell, and a hydrophilic outer poly(ethylene glycol) methyl ether (mPEG) shell. This system is characterized by its high solubility in a wide range of solvents and its ability to encapsulate hydrophobic as well as hydrophilic guest molecules [13]. The transport of guest molecules did not occur via unimolecular core–multishell (CMS) nanocarriers but via the formation of CMS aggregates [14]. Compared to polymer-drug conjugates, like for example PAMAM-based (poly(amidoamine)) star HPMA (N-(2-hydroxypropyl)methacrylamide) [15,16] or dPG-PEG polymer-drug conjugates [17], the loading of these architectures with active agents does not require a synthetic step. On the other hand the loading capacities of DDS using simple entrapment as loading technique are usually lower than the ones for drug conjugates. The CMS nanocarriers have already been used in biomedical applications, e.g., for the modulation of the copper level in eukaryotic cells and the in vivo targeting of a F9 teratocarcinoma tumor [18,19]. Additionally, we were able to show that the nanocarriers can passively target tumors based on the enhanced permeability and retention (EPR) effect [20,21] which is one of the major benefits of macromolecular DDS. The CMS nanocarriers specifically accumulated in tumor tissue and therefore delivered their guest molecules more selectively to the desired site of action [18]. Furthermore, the skin penetration of the hydrophilic dye rhodamin B and the hydrophobic dye Nile red could be greatly improved in comparison to other pharmaceutical formulations [22,23]. The CMS nanocarriers were also able to enhance the delivery of an electron paramagnetic resonance spin label into the upper layers of the stratum corneum [24]. One of the drawbacks of some DDS is their inability to release the encapsulated guest after reaching the desired site of action. For this reason, many DDSs have been developed which are able to release their cargo upon action of an external stimulus like light, ultrasound, magnetic

http://dx.doi.org/10.1016/j.jconrel.2014.04.019 0168-3659/© 2014 Published by Elsevier B.V.

Please cite this article as: E.F.K. Achazi, et al., pH-responsive dendritic core–multishell nanocarriers, J. Control. Release (2014), http://dx.doi.org/ 10.1016/j.jconrel.2014.04.019

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2. Experimental section

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2.1. General

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Reactions requiring dry conditions were carried out in dried Schlenk glassware under argon. Analytical grade solvents and chemicals were purchased from Acros or Sigma Aldrich and used as received. The 1,18-octadecanedioic acid (C18diCOOH) was a kind gift of Cognis. Sephadex LH-20 was purchased from GE Healthcare. Dry solvents were obtained from a MBraun SPS-800 solvent purification system. dPG amine (Mn 10,000 g/mol) was prepared with a degree of amination of 70% analogous to the published method [40]. Dendritic CMS nanocarriers with mPEG1000 (mPEG with an average number averaged

2.2. Preparation of DOX

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Doxorubicin hydrochloride (DOX · HCl) was transferred into the free base doxorubicin (DOX) similar to the published method [42]. DOX · HCl was dissolved in dimethylformamide (DMF) and stirred with 2 eq. of triethylamine overnight.

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molecular weight of 1000 g/mol) as the outer shell were synthesized as described in the literature [13,23]. NMR spectra were recorded on a Jeol ECX 400 or a Jeol Eclipse 500 MHz spectrometer. Proton and carbon NMR were recorded in ppm and were referenced to the indicated solvents [41]. NMR data were reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), integration, and coupling constants (s) in Hertz (Hz). Multiplets (m) were reported over the range (ppm) at which they appear at the indicated field strength. Mass spectrometry was performed on an Agilent 6210 ESI-TOF spectrometer. Gel permeation chromatography (GPC) data was obtained by measurements using an Agilent 1100 solvent delivery system with pump, manual injector, and an Agilent differential refractometer. Three 30 cm Suprema columns (PPS: Polymer Standards Service GmbH, Germany; Suprema 100 Å, 1000 Å, 3000 Å with 5 and 10 μm particle size) were used to separate aqueous polymer samples using water with 0.05% NaN3 as the mobile phase at a flow rate of 1 mL·min− 1. The columns were operated at ambient temperature with the RI detector at 50 °C. The calibration was performed by using linear pullulan calibration standard (PPS GmbH, Germany). Measurements were carried out under highly diluted conditions (10 mg/mL, injected volume 20 μL). WinGPC Unity software from PSS was used for data acquirement and interpretation. UV/Vis spectra were recorded on a Scinco S-3100 UV/Vis spectrometer. The dynamic light scattering (DLS) measurements for the size determination were performed on a Malvern Zetasizer Nano equipped with a He–Ne laser (633 nm) using backscattering mode (detector angle 173°). The samples were filtered through 0.2 μm regenerated cellulose syringe filters prior to the DLS measurement and left for 24 h to equilibrate. 100 μL of the solution to be analyzed was added to a disposable microcuvette (Plastibrand) with a round aperture. The autocorrelation functions of the backscattered light fluctuation were analyzed using Zetasizer DTS software from Malvern to determine the size distribution by intensity and volume. The measurements were performed at 25 °C, equilibrating the system on this temperature for 120 s.

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field, or a change in temperature, redox potential, or pH of the environment [25]. The use of pH-sensitive DDS is of special interest since pH gradients are found in many biological systems. In some tumor tissues the pH easily drops from the physiological pH of 7.4 to values of 6 and lower [26]. During cellular uptake the pH-value can even drop to values of around 4–5 in the late lysosomes [27]. By taking this into consideration, different pH-cleavable core–shell type architectures have been developed within the last years, e.g., pH-responsive polymeric micelles [28,29] and polymersomes [30,31]. Our group reported a number of unimolecular pH-responsive core–shell DDS based on dendritic poly(ethylene imine) (PEI) and dPG using different functional groups to attach pH-cleavable shells. These dendritic architectures released various guest molecules at pH values between 5 and 7 depending on the functional group used for the attachment of the shell [32–36]. Only few examples of pH-sensitive DDS based on unimolecular core–multishell architecture have been reported so far. For instance, Shen et al. described a pH-sensitive core– double shell system based on PAMAM dendrimers [37] and Tian et al. developed a triple-shell DDS which is responsive to pH and temperature [38]. In order to combine the benefits of our unimolecular CMS nanocarriers with the ability to release the cargo due to a drop in pH, we introduced an aromatic imine bond for attachment of the double shell (pH-CMS, Fig. 1). The aromatic imine bond can be hydrolyzed at the required pH-value to achieve the cleavage of the carrier after cellular uptake. Additionally, it allows for further fine-tuning of the release due to the possibility of introducing substituents at the aromatic ring to adjust the pH to the required value for the cleavage of the shell [39].

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E.F.K. Achazi et al. / Journal of Controlled Release xxx (2014) xxx–xxx

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Fig. 1. Schematic structure of a core–multishell nanoparticle based on a dendritic polyglycerol core (dPG, gray), an inner hydrophobic alkyl shell (green), and an outer hydrophilic shell made out of poly(ethylene glycol) methyl ether with an average number averaged molecular weight of 1000 g/mol (mPEG1000, blue). The pH-sensitive CMS nanoparticle (pH-CMS) is obtained by introduction of an aromatic imine functionality which is derived from a trihydroxybenzaldehyde derivative. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: E.F.K. Achazi, et al., pH-responsive dendritic core–multishell nanocarriers, J. Control. Release (2014), http://dx.doi.org/ 10.1016/j.jconrel.2014.04.019

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2.4. Transport capacity determination

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1 mL of the aqueous solution of the different nanocarriers with solubilized NR was freeze-dried and redissolved in 2 mL methanol. The concentrations of NR in methanol (MeOH) were estimated using the molar extinction coefficient (ε) of 45,000 M− 1 cm− 1 at 552 nm [43]. The concentrations of DOX in PBS were estimated using the molar extinction coefficient (ε) of 10,645 M−1 cm−1 at 495 nm [44].

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2.5. Cleavage experiment

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The cleavage of the pH-CMS was evaluated with a two phase system. 10 mg pH-CMS with mPEG350 was dissolved in 1 mL CDCl3. The chloroform phase was overlaid with different buffer solutions (pH = 4, 5, 6, 7.4; acetate buffer or PBS) and stirred at 1100 rpm. Over five hours every hour the chloroform phase was investigated via 1H NMR. The cleavage was monitored by the diminishing imine signal (8.2–7.6 ppm) and the growing aldehyde signal (9.8 ppm).

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2.6. Stability and release experiments

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The stability of DOX-loaded pH-CMS nanocarriers as well as the release of DOX was evaluated via SEC combined with UV/Vis measurements. DOX-loaded pH-CMS nanocarrier solutions were kept at pH 7.4, pH 5, and pH 4 at 37 °C for different time intervals. Directly after preparation as well as after certain time intervals 200 μL of the sample was applied to a small SEC column (Sephadex LH-20 equilibrated in PBS pH 7.4). Additional PBS pH 7.4 was added to elute the sample and achieve separation of encapsulated DOX from released DOX. The DOX loaded pH-CMS nanocarrier fractions were collected and the absorption of DOX at 495 nm was measured. The amount of released DOX was calculated with the help of the remaining absorption in comparison to the absorption at the beginning of the measurements.

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2.7. Confocal laser scanning microscopy

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Cellular uptake of DOX-loaded CMS nanocarriers and intracellular release of DOX was monitored by confocal microscopy. For the uptake study 50,000 A549 cells were seeded on 9 mm glass coverslips in each well of a 24-well plate and cultured for 24 h before adding the DOXloaded CMS nanocarriers in a final concentration of 0.1 mg/mL and 0.01 mg/mL, respectively. PBS and DOX (20 μM and 2 μM) treated cells served as controls. After 1, 4 or 24 h, respectively, cells were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min. Afterwards, cells were permeabilized with 0.1% TritonX for 5 min and washed 2 times with PBS. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). For observation, imaging and

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The cell experiments were performed with an xCELLigence RTCA SP (Roche) using A549 lung cancer cells. The xCELLigence RTCA SP was used to dynamically monitor cell proliferation and viability in real time, based on impedance. For background measurement 50 μL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin, streptomycin and glutamine was added to each well of a 96-well E-plate (Roche). Afterwards, a total of 1 × 104 A549 cells in 50 μL supplemented DMEM were seeded into each well and the measurement started. On the next day, 20 μL of either the unloaded or DOX-loaded CMS nanocarriers in PBS in different concentrations plus 80 μL fresh medium were added to the wells in duplicates. All samples were calibrated on the DOX concentration using UV/Vis spectroscopy. DOX and PBS treated cells served as controls. The E-plate was incubated during the whole measurement with 5% CO2 at 37 °C and monitored on the RTCA SP system with time intervals of at least 15 min for 72 h after treatment. Analysis was performed using the RTCA 1.2.1 software and GraphPad Prism 5. IC50 values were as well calculated with GraphPad Prism (equation: log(inhibitor) vs. response − variable slope (four parameters)) using end points generated by the RTCA software.

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2.8.1. Octadecane-1,18-dimethylester (C18diCOOMe) In a 1000 mL flask C18diCOOH (20.5 g, 63.7 mmol, 1.0 eq.) was added to 600 mL of MeOH. After the addition of sulfuric acid (10 drops) the mixture was stirred for 16 h under reflux. The C18diCOOH dissolved upon heating. The reaction mixture was allowed to reach room temperature (rt). The product crystallized as a colorless solid and was filtered and washed with cold MeOH. The colorless solid was purified by flash column chromatography (hexane:ethyl acetate = 9:1) to receive 21.7 g of C18diCOOMe (99%) as colorless powder. 1 H NMR (400 MHz, CDCl3, TMS): δ (ppm) = 3.65 (s, 6H,\O\CH3), 2.29 (t, 4H, J = 7.56 Hz,\CO\CH 2 \CH 2 \), 1.60 (m, 4H,\CH 2 \ CH2\CH2\), 1.37–1.20 (m, 24H,\CH2\(CH2)12\CH2\). 13 C NMR (100 MHz, CDCl3, TMS): δ (ppm) = 174.3 (\CH2\CO\ O\CH3), 51.4 (\O\CH3), 34.1 (\CH2\CO\), 29.6–29.2 (\CH2\ (CH2)12\CH2\), 29.1 (\CH2\(CH2)2\CO\), 24.9 (\CH2\CH2\CO\). IR (cm−1): 3451, 2916, 2847, 1738, 1472, 1463, 1435, 1412, 1382, 1370, 1350, 1316, 1273, 1233, 1191, 1163, 1117, 1091, 1033, 991, 968, 882, 824, 769, 732, 719, 700. MS (EI): m/z (%) calculated for C20H38O4Na: 365.2668; found: 365.2674.

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2.8.2. Octadecane-1,18-diol (C18diol) In a 1000 mL flask C18diCOOMe (14.8 g, 43.1 mmol, 1 eq.) was dissolved in 700 mL dry THF and cooled to 0 °C. Then LiAlH4 (4.1 g, 107.8 mmol, 2.5 eq.) was added slowly. The resulting mixture was stirred for 1 h at rt before refluxing it for 6 h. The mixture was cooled to 0 °C and slowly quenched with a cold H2SO4/water mixture (4 eq.: 12 eq. in relation to LiAlH4) in THF. The precipitate was filtered off and washed with THF (3 × 400 mL). The resulting solution was concentrated under reduced pressure yielding the product as a white solid (12.3 g, 98%). 1 H NMR (400 MHz, THF-d8, TMS): δ (ppm) = 3.45 (dd, J = 11.35, 6.15 Hz, 4H, HO\CH2\CH2\), 2.49 (s, 2H, HO\CH2\), 1.46 (m, 4H, HO\CH2\CH2\), 1.39–1.25 (m, 24H,\CH2\(CH2)12\CH2\). 13 C NMR (100 MHz, THF-d8, TMS): δ (ppm) = 62.7 (HO\CH2\), 34.3 (HO\CH2\CH2\), 30.9–30.7 (\CH2\(CH2)12\CH2\), 27.1 (HO\(CH2)2\CH2\). IR (cm−1): 3414, 3355, 2917, 2846, 1468, 1352, 1334, 1059, 1046, 1015, 979, 723. MS (EI): m/z (%) calculated for C18H38O2Na: 309.2770; found: 309.2770.

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Stock solutions of the stable CMS nanocarriers with concentrations of 1 and 5 mg/mL in dist. water were prepared. The solution of the pH-CMS nanocarriers with the same concentrations was always freshly prepared before usage. A 5 mg/mL guest (Nile red (NR) or DOX) stock solution in tetrahydrofuran (THF) or DMF in case of DOX was prepared. 200 μL of the guest stock solution was transferred into small sample vials and the solvent was evaporated. Afterwards, 3 mL of the different CMS nanocarrier stock solutions or dist. water as control was added. The samples were stirred at 1200 rpm for 6 h. Subsequently, the NR samples were filtered through 0.45 μm regenerated cellulose syringe filters to remove unsolubilized NR. The DOX-loaded CMS nanocarriers were freeze-dried, taken up with 0.5 mL dist. water, and purified by size exclusion chromatography (SEC) using Sephadex LH-20 in dist. water. After SEC the samples were freeze-dried and dissolved in phosphate buffered saline (PBS) to obtain concentrations of 1 or 5 mg/mL CMS nanocarriers.

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image processing a Leica (DMI6000CSB stand) confocal laser scan 212 microscope and the Leica LAS AF software were used. 213

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2.3. Loading of CMS nanocarriers

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Please cite this article as: E.F.K. Achazi, et al., pH-responsive dendritic core–multishell nanocarriers, J. Control. Release (2014), http://dx.doi.org/ 10.1016/j.jconrel.2014.04.019

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2.8.3. Octadecane-1,18-dibromide (C18diBr) In a 1000 mL Schlenk-flask under argon atmosphere C18diol (6.7 g, 23.4 mmol, 1 eq.) was dissolved in 700 mL dry THF. Then CBr 4 (19.4 g, 58.5 mmol, 2.5 eq.) was added and the reaction mixture was heated to 60 °C resulting in a yellow solution. Finally PPh 3 (18.4 g, 70.2 mmol, 3 eq.) was added in small portions. After complete addition the reaction was heated to reflux for 4 h, cooled to rt and the formed precipitate was filtered off. The solvent was evaporated and the remaining crude product was dissolved in a small amount of CHCl3 and loaded onto silica gel. The final product was obtained as white crystals after column chromatography using hexane as the eluent (9.1 g, 94%). 1 H NMR (400 MHz, CDCl3, TMS): δ (ppm) = 3.41 (t, J = 6.88 Hz, 4H,\CH2\Br), 1.88–1.78 (m, 4H,\CH2\CH2\Br), 1.46–1.37 (m, 4H,\CH2\(CH2)2\Br), 1.34–1.20 (m, 24H,\CH2\(CH2)12\CH2\). 13 C NMR (100 MHz, CDCl3, TMS): δ (ppm) = 34.0 (\CH2\Br), 32.8 (\CH2\CH2\Br), 29.7–29.4 (\CH 2\(CH2) 12\CH2\), 28.8 (\CH2\(CH2)3\Br), 28.2 (\CH2\(CH2)2\Br). MS (ESI): m/z (%) calculated for C18H36Br2: 410.1184; found: 410.1177. IR (cm−1): 2914, 2849, 1470, 1338, 1313, 1278, 1241, 1206, 716.

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Fig. 2. Synthesis of the pH-cleavable core–multishell architectures (pH-CMS).

2.8.4. Poly(ethylene glycol) methyl ether octadecane-18-bromides (mPEGxC18Br) In a 250 mL Schlenk-flask under argon atmosphere mPEGx (6.8 mmol, 1.0 eq.) was dissolved in 150 mL of dry THF and a catalytic amount (10 μL) of 15-crown-5 ether was added. NaH (1.94 mmol, 1.5 eq.) was added slowly and the mixture was stirred for 3 h. The resulting solution was transferred with a transfer cannula into a 250 mL dropping funnel attached to a 1000 mL Schlenk-flask filled with C18diBr (20.4 mmol, 3.0 eq.) in 500 mL dry THF. The solution in the dropping funnel was added dropwise. The resulting mixture was stirred for 16 h before evaporating the solvent. Purification was accomplished using dry column chromatography (CHCl3 to CHCl3 + 10% MeOH), followed by a filtration

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Table 1 GPC data for the CMS nanocarriers used for the in vitro experiments. Nanocarrier

Mn [g/mol]

Mw [g/mol]

# of arms attached

Dispersity

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94,100 95,800

26–65 17–22

2.15 1.25

t1:4 t1:5

Please cite this article as: E.F.K. Achazi, et al., pH-responsive dendritic core–multishell nanocarriers, J. Control. Release (2014), http://dx.doi.org/ 10.1016/j.jconrel.2014.04.019

E.F.K. Achazi et al. / Journal of Controlled Release xxx (2014) xxx–xxx t2:1 t2:2 t2:3

Table 2 Hydrodynamic radii of unloaded CMS and pH-CMS nanocarriers in methanol and PBS at different nanocarrier concentrationsa. Nanocarrier

Carrier conc. [mg/mL]

Size in MeOH [nm]

Size in PBS pH 7.4 [nm]

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73.5 5.1 74.7 6.0 9.9 96.2 9.1 93.6

5 pH-CMS

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± ± ± ± ± ± ± ±

3.4 (65%) 0.1 (35%) 2.7 (57%) 0.2 (43%) 0.4 (58%) 6.2 (42%) 0.2 (67%) 3.2 (33%)

rH vol

rH int

4.4 ± 0.1

89.9 6.9 98.7 6.7 8.9 115.1 9.8 109.8

4.0 ± 0.1 6.9 ± 0.2 5.9 ± 0.1

rH vol ± ± ± ± ± ± ± ±

3.1 (69%) 0.2 (31%) 2.1 (67%) 0.4 (33%) 0.2 (55%) 5.7 (45%) 0.2 (63%) 4.2 (37%)

5.6 ± 0.2 5.2 ± 0.2 7.4 ± 0.2 7.7 ± 0.1

a r Hint and r Hvol are the average hydrodynamic radii calculated by intensity and volume given with the mean standard deviation. In the case of two visible peaks in the size distribution the abundance is given in brackets.

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through a pad of RP silica gel (Chromabond® C18ec, MeOH + 60% water to MeOH) to afford the desired product as colorless wax.

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mPEG350C18Br: yield = 1.8 g, 80% 1 H NMR (500 MHz, CDCl3, TMS): δ (ppm) = 3.64–3.56 (m, 24H, \(O\CH2\CH2)n\), 3.54–3.48 (m, 4H, \(O\CH2\CH2\OCH3)), 3.39 (t, J = 6.8 Hz, 2H, \O\CH2\(CH2)17\), 3.35 (t, J = 6.9 Hz, 2H, \CH2\CH2\Br), 3.32 (s, 3H, \O\CH3), 1.83–1.74 (m, 2H, \CH2\CH2\Br), 1.55–1.48 (m, 2H, \O\CH2\CH2\(CH2)n\), 1.41–1.33 (m, 2H, \CH2\(CH2)2\Br), 1.30–1.11 (m, 26H, \CH2\(CH2)13\CH2\). 13 C NMR (100 MHz, CDCl3, TMS): δ (ppm) = 71.8 (\CH2\OCH3), 71.4 (\O\CH2\(CH2)n\), 70.4–70.3 (\(O\CH2\CH2)n\), 69.9 (\CH2\O\(CH2)n\), 58.9 (\O\CH3), 33.8 (\CH2\Br), 32.7 (\CH2\CH2\Br), 29.5–29.3 (\CH2\(CH2)12\CH2\, \OCH2\ CH2\(CH2)n\), 28.6 (\CH2\(CH2)3\Br), 28.0 (\CH2\(CH2)2\Br), 25.9 (\O\(CH2)2\CH2\). IR (cm−1): 2924, 2854, 1466, 1359, 1342, 1280, 1240, 1146, 1112, 1060, 958, 842, 715. mPEG1000C18Br: yield = 3.5 g, 39% 1 H NMR (500 MHz, CDCl3, TMS): δ (ppm) = 3.74–3.48 (m, 84H, \(O\CH2\CH2)n\), 3.42 (t, J = 6.8 Hz, 2H, \O\CH2\(CH2)17\), 3.38 (t, J = 6.9 Hz, 2H, \CH2\CH2\Br), 3.35 (s, 3H, \O\CH3), 1.87–1.77 (m, 2H, \CH 2 \CH 2 \Br), 1.59–1.49 (m, 2H, \O\ CH 2 \CH 2 \(CH 2 ) n \), 1.44–1.34 (m, 2H, \CH 2 \(CH 2 ) 2 \Br), 1.33–1.10 (m, 26H, \CH2\(CH2)13\CH2\). 13 C NMR (125 MHz, CDCl3, TMS): δ (ppm) = 71.9 (\CH2\OCH3), 71.5 (\O\CH2\(CH2)n\), 70.7–70.4 (\(O\CH2\CH2)n\), 70.0 (\CH2\O\(CH2)n\), 59.0 (\O\CH3), 34.0 (\CH2\Br), 32.8 (\CH2\CH2\Br), 29.7–29.3 (\CH2\(CH2)12\CH2\, \OCH2\ CH2\(CH2)n\), 28.7 (\CH2\(CH2)3\Br), 28.1 (\CH2\(CH2)2\Br), 26.0 (\O\(CH2)2\CH2\).

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Table 3 Hydrodynamic radii of loaded CMS and pH-CMS nanocarriers in PBS at different nanocarrier concentrationsa. b

Carrier conc. [mg/mL]

Size of NR-loaded CMS nanocarrier [nm]

Size of DOX-loaded CMS nanocarrier [nm]

r H int

rH vol

rH int

1

134.1 ± 4.7

141.0 ± 7.1

5

131.6 ± 1.6

137.2 ± 2.9

1

114.0 ± 1.9

111.2 ± 2.4

5

126.9 ± 5.3 (73%) 10.4 ± 1.0 (27%)

94.2 9.7 88.6 10.5 10.7 104.9 8.7 101.9

t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12

THBA-C18-mPEG350: yield = 0.6 g, 80% 1 H NMR (500 MHz, CDCl3, TMS): δ (ppm) = 9.78 (s, 1H, (\CHO)), 7.03 (s, 2H, Ar\H), 4.02 (t, J = 6.4 Hz, 2H, Ar\O\CH2\), 3.98 (t, J = 5.9 Hz, 4H, 2 × Ar\O\CH2 \), 3.66–3.56 (m, ~ 72H, \(O\CH2\CH2)n\), 3.39 (t, J = 6.8 Hz, 6H, \O\CH2\(CH2)17\), 3.33 (s, 9H, \O\CH3), 1.82–1.74 (m, 4H, \CH2\CH2\O\Ar), 1.74–1.66 (m, 2H, \CH2\CH2\O\Ar), 1.56–1.48 (m, 12H, 3 × \O\CH2\CH2\(CH2)n\, 3× Ar\O\CH2\CH2\(CH2)n\), 1.47– 1.38 (m, 6H, 3 × Ar\O\(CH2 ) 2\CH2\), 1.34–1.12 (m, 78H, \CH2\(CH2)13\CH2-). 13 C NMR (100 MHz, CDCl3, TMS): δ (ppm) = 191.1 (Ar\COH), 153.4 (Ar\O\), 143.7 (Ar\O\)131.3 (Ar\COH), 107.7 (Ar\H), 73.5 (\O\CH2\(CH2) n \), 71.8 (\CH2\OCH3 ), 71.4 (\O\CH2 \(CH2) n \), 70.4–70.3 (\(O\CH2 \CH2 ) n \), 69.9 (\CH2 \O\(CH2) n \), 69.1 (Ar\O\CH2 \), 58.8 (\O\CH3 ),

U

337

t3:1 t3:2

O

T

321

R O

313

P

312

340 341

D

311

2.8.5. 3,4,5-Trioctadecane poly(ethylene glycol) methyl ether benzaldehyde (THBA-C18-mPEGx) In a 250 mL Schlenk-flask 3,4,5-trihydroxybenzaldehyde monohydrate (THBA, 0.58 mmol, 1 eq.), mPEGxC18Br (2.03 mmol, 3.5 eq.), potassium carbonate (0.32 g, 2,32 mmol, 4 eq.) and potassium iodine (0.05 g, catalytic) were mixed in 160 mL DMF. The reaction mixture was degassed by three consecutive cycles of applying vacuum and bubbling argon through the DMF. The reaction was heated to 80 °C and stirred for three days. After reaching rt, DMF was evaporated and the residue taken up with CHCl3. The mixture was filtered through a pad of silica gel (CHCl3 + 5% MeOH). The solvent was removed and the residue dissolved in water and dialyzed (water, MWCO 2000 Da). The product solution was freeze-dried yielding the product as slightly yellow foam.

E

310

IR (cm−1): 2915, 2883, 1466, 1359, 1342, 1280, 1240, 1146, 1104, 338 1060, 958, 842, 715. 339

CMS

pH-CMS

6.6 ± 0.4

rH vol ± ± ± ± ± ± ± ±

2.4 (82%) 0.7 (18%) 1.5 (64%) 0.7 (36%) 0.2 (52%) 6.6 (48%) 0.1 (52%) 1.9 (48%)

6.8 ± 0.6 6.4 ± 0.2 8.1 ± 0.3 7.7 ± 0.3

a r Hint and r Hvol are the average hydrodynamic radii calculated by intensity and volume given with the mean standard deviation. In the case of two visible peaks in the size distribution the abundance is given in brackets.

Please cite this article as: E.F.K. Achazi, et al., pH-responsive dendritic core–multishell nanocarriers, J. Control. Release (2014), http://dx.doi.org/ 10.1016/j.jconrel.2014.04.019

342 343 344 345 346 347 348 349 350 351 352 353

355

356 357 358 359 360 361 362 363 364

365 366 367 368

384

385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404

405 406 407 408 409 410

411 412 413 414 415 416 417

418 419 420 421 422 423

424 425 426 427

2.8.6. pH-responsive core–multishell nanocarrier (pH-CMS) The functionalized benzaldehyde THBA-C18-mPEGx (1.0 eq.) dissolved in MeOH (1 mL per 0.1 mmol) was added dropwise to a mixture of dPG amine (70% amine functionalization, 9.45 mmol (\NH2) per g) in MeOH (1 mL per 0.1 mmol (\NH2)) and anhydrous sodium sulfate (1 g per 1 mL MeOH). The mixture was refluxed for 24 h. After reaching rt, the solution was filtered through Celite and concentrated under vacuum. The crude product was purified by ultrafiltration (MWCO 10 kDa) in methanol with 0.1% triethylamine to give the final product with a degree of functionalization of 31% (based on the amount of OH groups of the dPG).

3. Results and discussion 3.1. Synthesis

41.7 12.6 51.2 19.4

t4:8 t4:9 t4:10 t4:11

F

383

t4:7

3.0 1.1 2.6 2.2

428

O

382

pH-CMS

t4:6

DOX

429

R O

381

1 5 1 5

pH-CMS with mPEG350: 1 H NMR (500 MHz, CDCl3, TMS): δ (ppm) = 8.20–7.60 (s broad, 1H, \CH_N\), 7.00–6.50 (s broad, 2H, Ar\H), 4.20–3.40 (m, ~ 72H,\(O\CH 2 \CH 2 ) n \), 3.33 (s, 9H,\O\CH 3 ), 1.90–1.60 (m, 6H, 3 × \O\CH2\CH2\), 1.58–1.50 (m, 6H, 3 × \O\CH 2 \ CH2\(CH2)n \, 3 × Ar\O\CH2 \CH2\(CH2)n \), 1.48–1.05 (m, ~ 84H, 3 × Ar\O\(CH2)2\CH2\, \CH2\(CH2)13\CH2\). 13 C NMR (100 MHz, CDCl3, TMS): δ (ppm) = 162.4 (Ar\CH_N\), 153.0 (Ar\O\), 139.0 (Ar\O\), 127.1 (Ar\CH_N\), 99.8 (Ar\H), 71.7–69.8 (mPEG backbone + Ar\O\CH2\), 58.8 (\CH3), 29.7–29.3 (C18 alkyl), 26.0 (Ar\O\(CH2)2\CH2\). IR (cm−1): 3412, 2918, 2851, 1739, 1643, 1467, 1343, 1278, 1240, 1146, 1116, 1059, 1046, 1015, 980, 941, 841. pH-CMS with mPEG1000: 1 H NMR (500 MHz, MeOH-d4, TMS): δ (ppm) = 8.3–7.7 (s broad, 1H, \CH_N\), 7.1–6.4 (s broad, 2H, Ar\H), 3.74\3.48 (m, ~160H, \(O\CH2\CH2)n\), 3.49–3.44 (m, ~ 4H, \O\CH2\(CH2)17\), 1.64–1.54 (m, ~ 6H, 3 × \O\CH2 \CH2 \(CH2 ) n \, 3 × Ar\O\ CH2\CH2\(CH2)n\), 1.50–1.15 (m, ~51H, 3× Ar\O\(CH2)2\CH2\, \CH2\(CH2)13\CH2\). 13 C NMR (125 MHz, MeOH-d4, TMS): δ (ppm) = 154.5 (Ar\O\), 114.1 (Ar\H), 73.0–71.4 (mPEG backbone + Ar\O\CH2\), 59.2 (PEG-OCH3), 31.5–31.1 (C18 alkyl), 27.6 (Ar\O\(CH2)2\CH2\). IR (cm−1): 3412, 2917, 2848, 1739, 1646, 1464, 1343, 1278, 1240, 1146, 1102, 1059, 1046, 1015, 980, 941, 841.

The pH-cleavable CMS architectures were synthesized by the six-step procedure shown in Fig. 2. Starting from 1,18-octadecanedioic acid, the acid groups were transformed into the corresponding methyl esters and reduced to hydroxyl groups in the next step. Subsequently, the diols were converted into a dibromide building block using the Appel reaction. The compound was reacted in a Williamson-ether reaction with mPEG to yield the poly(ethylene glycol) methyl ether octadecane18-bromides. This product was attached to trihydroxybenz-aldehyde (THBA) resulting in the combined inner and outer shell with a free aldehyde functionality. In the presence of potassium carbonate and potassium iodide the double shell building blocks underwent imine bond formation with dPG amine resulting in the pH-cleavable CMS nanocarriers. Final purification of the pH-CMS was achieved by ultrafiltration using a membrane with a MWCO of 10 kDa in methanol. Traces of triethylamine had to be added to avoid partial cleavage of the imine bond. The pH-CMS nanocarrier with mPEG350 were not soluble in water and have therefore not been further characterized concerning sizes and transport capacities as these measurements were performed in aqueous media. The results of the GPC measurements are summarized in Table 1. Both CMS nanocarriers showed equal Mw distributions and a similar number of arms. Since the pH-CMS nanocarriers is made out of a trivalent shell building block the number of arms for comparison with the CMS nanocarriers made out of monovalent shell building blocks should be multiplied by three. The higher dispersity of the CMS nanocarriers compared to the pH-CMS nanocarriers results as well from the different shell building blocks.

100

pH 4 pH 5 pH 6 pH 7.4

80 60 40 20 0 0

2

4

t4:1 t4:2 t4:3 t4:4 t4:5

NR

P

380

Transport capacity [mgguest/gcarrier]

D

379

Carrier conc. [mg/mL]

CMS

imine cleavge (%)

378

Nanocarrier

T

377

C

376

E

375

R

374

R

373

O

372

C

371

Table 4 Transport capacities of CMS and pH-CMS nanocarriers for the guest molecules Nile red (NR) and doxorubicin (DOX) at two different carrier concentrations determined by UV/Vis measurements. Transport capacities are corrected with respect to the inherent water solubility of the guest molecules.

N

370

30.2 (Ar\O\CH2\CH2\), 29.6–28.8 (\CH2\(CH2)12\CH2\, \OCH2\CH2\(CH2)n\), 26.0 (\O\(CH2)2\CH2\), 25.8 (\O\ (CH2)2\CH2\). IR (cm−1): 3412, 2917, 2850, 1739, 1693, 1467, 1343, 1278, 1240, 1146, 1102, 1059, 1046, 1013, 980, 941, 841, 772, 723. THBA-C18-mPEG1000: yield = 1.76 g, 86% 1 H NMR (500 MHz, CDCl3, TMS): δ (ppm) = 9.81 (s, 1H, (\CHO)), 7.06 (s, 2H, Ar\H), 4.04 (t, J = 6.4 Hz, 2H, Ar\O\CH2\), 4.01 (t, J = 5.9 Hz, 4H, 2 × Ar\O\CH2 \), 3.74–3.48 (m, ~ 250H, \(O\CH2\CH 2) n \), 3.44–3.40 (m, 6H, \O\CH2\(CH2 )17 \), 3.36 (s, 9H, \O\CH3), 1.85–1.77 (m, 4H, \CH2 \CH2\O\Ar), 1.76–1.69 (m, 2H, \CH2\CH2 \O\Ar), 1.59–1.51 (m, 12H, 3 × \O\CH2 \CH2 \(CH2 ) n \, 3 × Ar\O\CH2 \CH2 \(CH2 ) n \), 1.49–1.42 (m, 6H, 3 × Ar\O\(CH2)2\CH2\), 1.33–1.10 (m, 78H, \CH2\(CH2)13\CH2\). 13 C NMR (125 MHz, CDCl3, TMS): δ (ppm) = 197.1 (Ar\COH), 139.2 (Ar\O\), 99.9 (Ar\H), 77.2 (\O\CH2\(CH2)n\), 71.9 (\CH2 \OCH3 ), 71.5 (\O\CH2 \(CH2 )n \), 70.6–70.4 (\(O\ CH 2\CH 2) n \), 70.0 (\CH2 \O\(CH2) n \), 59.0 (\O\CH 3), 29.7–29.3 (\CH2 \(CH2 ) 12 \CH2 \, \OCH2 \CH2 \(CH2 ) n \), 26.1 (\O\(CH2)2\CH2\), 25.8 (\O\(CH2)2\CH2\). IR (cm−1): 3412, 2917, 2848, 1739, 1646, 1464, 1343, 1278, 1240, 1146, 1102, 1059, 1046, 1015, 980, 941, 841, 772, 723.

U

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E.F.K. Achazi et al. / Journal of Controlled Release xxx (2014) xxx–xxx

E

6

6

time (h) Fig. 3. pH-dependant cleavage of the imine bond of pH-responsive core–multishell nanocarriers (pH-CMS) monitored by the decreasing imine bond and the appearance of the aldehyde signal in 1H NMR spectra.

Please cite this article as: E.F.K. Achazi, et al., pH-responsive dendritic core–multishell nanocarriers, J. Control. Release (2014), http://dx.doi.org/ 10.1016/j.jconrel.2014.04.019

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pH-CMS pH5 pH-CMS pH 7.4

DOX Release (%)

60

40

20

7

were about 7 nm and 10 nm for the CMS and pH-CMS nanocarriers, respectively. The slight increase of the sizes compared to the single particles in MeOH most likely arose from a different conformation of the outer mPEG-shell. The stable CMS nanocarriers again showed bigger aggregates in the intensity distribution with radii of 90–100 nm. In the case of the pH-CMS nanocarriers, an aggregate peak of around 110 nm was only observable for the higher concentration. Again, no aggregate peak was observed for any CMS nanocarriers at any concentration in the volume distribution. This shows the tendency of CMS nanocarriers to form aggregates on their own which was already described earlier [13,14,45].

469 470

3.3. Size and aggregation behavior of loaded CMS nanocarriers

480

471 472 473 474 475 476 477 478 479

0 10

20

30

time (h)

40

50

F

0

R O

C E

467 468

R

465 466

R

463 464

N C O

461 462

U

459 460

P

In order to gain a better understanding of the stable CMS and pHresponsive CMS nanocarriers (see Fig. 1), the self-aggregation behavior of the different loaded and unloaded CMS nanoparticles was investigated with the help of DLS. Primarily, the unloaded CMS nanocarriers in methanol and in PBS with pH of 7.4 were studied. In methanol the single CMS nanocarriers had a hydrodynamic radius about 5 nm and the pHCMS had a radius about 9 nm (see Table 2). In all cases, the CMS nanocarriers showed bigger aggregates in the intensity distribution of the DLS measurements. These aggregates were not observed in the volume distribution and can therefore be considered as a minor pronounced species. The hydrodynamic radii of single particles in PBS

D

458

E

3.2. Size and aggregation behavior of unloaded CMS nanocarriers

T

457

As the size of a drug delivery system (DDS) has a big influence on its uptake into cells, and in order to see whether transport occurs via unimolecular carriers or via aggregates, the loaded CMS nanocarriers were also analyzed by DLS (see Table 3). Aggregates were formed by all NR-loaded CMS nanocarriers. For the stable CMS nanocarrier aggregates with a hydrodynamic radius of 130–140 nm were observed. The pH-CMS nanocarrier aggregates had a radius of about 115 nm at a concentration of 1 mg/mL but showed two peaks at a concentration of 5 mg/mL. The predominant size distribution with 7–13 nm indicates that mainly single particles are present in the sample. The aggregate peak is not observed in the volume-based size distribution. This result is in line with an earlier report, where we found that the aggregation of CMS nanocarriers is influenced by the concentration of NR used [14]. For the 5 mg/mL solution the amount of NR relative to the CMS nanocarriers is not sufficient to enhance aggregate formation. This further supports the observation that the slightly more branched outer shell decreases the tendency to form aggregates.

O

Fig. 4. Release of DOX from DOX-loaded pH-CMS nanocarriers at pH 7.4 and pH 5 over 48 h.

Fig. 5. Confocal fluorescence microscopy pictures of DOX-loaded CMS and DOX-loaded pH-CMS nanocarriers, as well as free DOX for comparison, taken up by A549 lung cancer cells after 24 h incubation. The cell nucleus is stained with DAPI (blue). The DOX fluorescence is shown in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: E.F.K. Achazi, et al., pH-responsive dendritic core–multishell nanocarriers, J. Control. Release (2014), http://dx.doi.org/ 10.1016/j.jconrel.2014.04.019

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8

3.4. Transport capacities

510 511

518 519

In order to investigate their suitability as DDS it is important to know the transport capacity of the different CMS nanocarriers. Therefore, they were loaded with the dye NR or the drug DOX and the transport capacities were determined by UV/Vis spectroscopy. The resulting transport capacities are given in Table 4. All carriers were able to slightly enhance the solubility of NR. Encapsulation of DOX was even more efficient as the transport capacities were one order of magnitude higher. This is astonishing as the size distributions show that the main species are single particles and we had assumed that the more efficient transport was occurring in aggregates [45].

520

3.5. Cleavage experiments

521 522

531 532

For the investigation of the pH-dependant shell cleavage, the decrease of imine and the appearance of aldehyde signal were monitored by 1H NMR spectroscopy at different pH values over time. At pH values between 6 and 7.4 the cleavage of the imine bond occurred rather slow. After 5 h about 20% of the imine bonds were cleaved (see Fig. 3). In contrast at a pH of 5 and lower rapid cleavage of the imine bond was observed. Within the first two hours about 75% of the pH-sensitive linkers were cleaved. This clearly demonstrates that the imine bond used in the pH-CMS cleaves at a pH between 5 and 6. This area is in particular of interest for the cleavage of pH-responsive DDS after cellular uptake as such pH values can be reached in the intracellular compartments like the late endosome or lysosome.

533

3.6. Stability and release

534 535

In order to test the stability of pH-CMS nanocarriers loaded with DOX under physiological and acidic conditions, DOX-loaded pH-CMS nanocarriers were kept at pH 7.4 and pH 5 at 37 °C for 48 h. Directly after sample preparation, after 1, 2, 4, 24 as well as 48 h the samples were applied to small SEC columns. The sample at pH 7.4 did only

523 524 525 526 527 528 529 530

536 537 538

T

C

E

R

516 517

R

514 515

O

512 513

539 540 541 542 543 544 545 546 547 548 549

3.7. Cellular uptake

C

506 507

N

504 505

U

502 503

P

509

500 501

release about 10 to 15% DOX over 48 h (see Fig. 4, Figure S5 only one colored band is visible, the upper part of the column is almost colorless). Even though, the NMR experiment showed partial cleavage of the imine bond at pH 7.4. This shows that the initial cleavage is not sufficient to release a high dose of DOX. Therefore, the loaded pH-CMS nanocarriers can be considered relatively stable under physiological conditions. The pH-CMS nanocarriers at pH 5 released DOX over time (see Fig. 4, Figure S5 two colored bands are visible, with the upper band increasing over time). This proves that DOX-loaded pH-CMS can efficiently release DOX under slightly acidic conditions.

D

508

In the intensity-based size distributions, the DOX-loaded CMS nanoparticles all show peaks corresponding to aggregate formation (Table 3) with radii of around 90–95 nm for the stable CMS and 100–105 nm for pH-CMS nanocarriers. However, based on the volume distributions, the single nanocarriers with radii of around 6–7 nm and 8 nm are obviously the predominant species for all carriers. The aggregation behavior of CMS nanocarriers is significantly influenced by the different guest molecules. NR seems to enhance the tendency of CMS to form aggregates, while the presence of DOX rather inhibits aggregation and single nanocarriers are the predominant species. These observations further strengthen our earlier findings [45].

E

498 499

R O

O

Fig. 6. Real time cell analysis of A549 lung cancer cells that were treated with different concentrations of unloaded CMS and pH-CMS after 48 h (left) and of DOX-loaded CMS nanocarriers after 48 h (right). Samples have been calibrated on the DOX concentration.

In order to evaluate if the CMS nanocarriers are taken up by cells we performed confocal microscopy measurements. As can be seen in Fig. 5 both carriers are taken up by A549 lung cancer cells. The DOX fluorescence is visible within the cells being localized either within the cytoplasm or in the nuclei.

550

3.8. In vitro studies

555

The advantage of pH-responsive CMS nanocarriers in comparison to the stable CMS nanocarriers lies in the possibility to control and trigger the release of guest molecules. Real time cell analysis (RTCA) experiments were performed to affirm this advantage in vitro. In order to exclude side effects of the different carriers we first investigated the toxicity of the unloaded CMS nanocarriers. Fig. 6 shows that unloaded CMS nanocarriers did not show any toxicity over a period of 48 h. The carriers showed no toxic effects even at very high concentrations (1 mg/mL, ten times higher than used for the loaded CMS nanocarriers). RTCA measurements of DOX-loaded CMS nanocarriers after 24 h only showed a slightly higher toxicity of pH-CMS nanocarriers in comparison to stable CMS nanocarriers. However, after 48 h the toxicity of the cleavable systems was significantly higher (see Fig. 6). The effect of the 0.1 mg/mL pH-CMS nanocarrier solution with 20 μM DOX after 48 h was comparable to that of free DOX. For a better comparison we calculated the IC50 values for the different formulations and summarized

556 557

Table 5 IC50 values for free DOX, DOX-loaded pH CMS, and DOX-loaded CMS nanocarriers after 48 h.

t5:1 t5:2 t5:3

Formulation

IC50 value [μM]

t5:4

DOX pH-CMS CMS

1.6 22.4 56.7

t5:5 t5:6 t5:7

Please cite this article as: E.F.K. Achazi, et al., pH-responsive dendritic core–multishell nanocarriers, J. Control. Release (2014), http://dx.doi.org/ 10.1016/j.jconrel.2014.04.019

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599

Acknowledgment

600 601

We are grateful to Dr. Juliane Keilitz and Dr. Pamela Winchester for proofreading the manuscript.

602

Appendix A. Supplementary data

603 604

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2014.04.019.

605

References

592 593 594 595 596 597

606 607 608 609 610 611 612 613 614 615 616 617

C

E

590 591

R

588 589

R

586 587

N C O

584 585

U

582 583

O

598

The introduction of the aromatic imine linkage into the CMS nanocarriers enabled the already highly versatile CMS nanocarriers to become pH-responsive. The imine linkage used was rapidly cleaved at a pH of 5 and lower. Doxorubicin-loaded pH-responsive CMS nanocarriers were stable at pH 7.4 and did not show any release. By real time cell analysis we were able to demonstrate that pH-responsive nanocarriers (pH-CMS) could release doxorubicin more efficiently under the acidic conditions of intracellular compartments and therefore showed higher cytotoxicity in comparison to the stable CMS nanocarrier. Interestingly, the transport of doxorubicin was achieved by unimolecular CMS and pH-CMS nanocarriers and not in aggregates as it was observed for other guest molecules, such as Nile red. This also resulted in higher transport capacities of up to 5 wt.-% for doxorubicin as compared to 0.3 wt.-% for Nile red. Hence, the pH-responsive CMS nanocarriers are highly potent unimolecular drug delivery systems. Due to their size, they should benefit from the EPR effect and can actively release their payload inside tumors. Funding sources The authors would like to thank the focus area nanoscale of the Freie Universität Berlin and the SFB 1112 for the financial support.

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4. Conclusion

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them in Table 5. The pH-CMS nanocarriers showed an IC50 value half as high as the CMS nanocarriers. The enhanced toxicity of the pH-cleavable CMS nanocarriers proves them to be superior over the pH-stable CMS system as it was able to actively release the encapsulated guest upon a pH-external stimulus, equivalent to the pH in the endosomal compartments [46] after cellular uptake (see Fig. 7).

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Fig. 7. DOX-loaded pH-responsive CMS nanocarriers get cleaved due to a drop in the pH inside of the late endosome or lysosome after cellular uptake. The transported DOX gets released and can induce cell death.

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