PH-responsive dendritic core-multishell nanocarriers

Article abstract of DOI:10.1016/j.jconrel.2014.04.019

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.

Full text of DOI:10.1016/j.jconrel.2014.04.019

COREL-07129; No of Pages 10
Journal of Controlled Release xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Controlled Release
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1
pH-responsive dendritic core–multishell nanocarriers
⁎
2Q1 Emanuel Fleige Katharina Achazi, Karolina Schaletzki, Therese Triemer, Rainer Haag
3
Freie Universität Berlin, Institut für Chemie und Biochemie, Takustraße 3, 14195 Berlin, Germany
4
a r t i c l e i n f o
a b s t r a c t
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Article history:
In this paper we describe novel pH-responsive core–multishell (CMS) nanocarrier (pH-CMS), obtained by intro- 16
Received 29 November 2013
Accepted 12 April 2014
Available online xxxx
ducing an aromatic imine linker between the shell and the core. At a pH of 5 and lower the used imine linker was 17
OOF
rapidly cleaved as demonstrated by NMR studies. The CMS nanocarriers were loaded with the dye Nile red (NR) 18
and the anticancer drug doxorubicin (DOX), respectively. The transport capacities were determined using UV/Vis 19
spectroscopy and the sizes of the loaded and unloaded CMS nanocarriers were investigated using dynamic light 20
scattering (DLS). We could show that CMS nanocarriers efficiently transported NR in supramolecular aggregates, 21
while DOX was transported in a unimolecular fashion. After cellular uptake the DOX-loaded pH-responsive 22
nanocarriers showed higher toxicities than the stable CMS nanocarriers. This is due to a more efficient DOX 23
release caused by the cleavage of the pH-labile imine bond at lower pH within the intracellular compartments. 24
© 2014 Published by Elsevier B.V.
9
Keywords:
Drug delivery
Nile red
Doxorubicin
Transport capacity
Dynamic light scattering
Real time cell analysis
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1. Introduction
multishell system which is based on a hydrophilic dPG core, a hydro- 47
phobic inner alkyl shell, and a hydrophilic outer poly(ethylene glycol) 48
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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], macromolecu-
lar 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]. Now-
adays, 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
methyl ether (mPEG) shell. This system is characterized by its high 49
solubility in a wide range of solvents and its ability to encapsulate 50
hydrophobic as well as hydrophilic guest molecules [13]. The transport 51
of guest molecules did not occur via unimolecular core–multishell 52
(CMS) nanocarriers but via the formation of CMS aggregates [14]. 53
Compared to polymer-drug conjugates, like for example PAMAM-based 54
(poly(amidoamine)) star HPMA (N-(2-hydroxypropyl)methacrylamide) 55
[15,16] or dPG-PEG polymer-drug conjugates [17], the loading of these 56
architectures with active agents does not require a synthetic step. On 57
the other hand the loading capacities of DDS using simple entrapment 58
as loading technique are usually lower than the ones for drug conjugates. 59
The CMS nanocarriers have already been used in biomedical applications, 60
e.g., for the modulation of the copper level in eukaryotic cells and the 61
in vivo targeting of a F9 teratocarcinoma tumor [18,19]. Additionally, 62
we were able to show that the nanocarriers can passively target tumors 63
based on the enhanced permeability and retention (EPR) effect [20,21] 64
which is one of the major benefits of macromolecular DDS. The CMS 65
nanocarriers specifically accumulated in tumor tissue and therefore 66
delivered their guest molecules more selectively to the desired site of 67
action [18]. Furthermore, the skin penetration of the hydrophilic dye 68
rhodamin B and the hydrophobic dye Nile red could be greatly improved 69
in comparison to other pharmaceutical formulations [22,23]. The CMS 70
nanocarriers were also able to enhance the delivery of an electron para- 71
magnetic resonance spin label into the upper layers of the stratum 72
Abbreviations: DDS, drug delivery system; dPG, dendritic polyglycerol; mPEG,
poly(ethylene glycol) methyl ether; PAMAM, poly(amido amine); HPMA, N-(2-
hydroxypropyl)methacrylamide; CMS, core–multishell; EPR, enhanced permeation
UNCORRECTED PR
and retention; PEI, poly(ethylene imine); pH-CMS, pH-responsive CMS nanoparti-
cles, C18diCOOH, 1,18-octadecanedioic acid; mPEG1000, mPEG with an average num-
ber averaged molecular weight of 1000 g/mol; GPC, gel permeation chromatography;
DLS, dynamic light scattering; DOX · HCl, doxorubicin hydrochloride, DOX, doxorubi-
cin, 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.
corneum [24].
73
One of the drawbacks of some DDS is their inability to release the en- 74
capsulated guest after reaching the desired site of action. For this reason, 75
many DDSs have been developed which are able to release their cargo 76
upon action of an external stimulus like light, ultrasound, magnetic 77
⁎
Corresponding author. Tel.: +49 30 83852633; fax: +49 30 83853357.
E-mail address: haag@chemie.fu-berlin.de (R. Haag).
URL: http://www.polytree.de (R. Haag).
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

2
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field, or a change in temperature, redox potential, or pH of the environ-
ment [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. de-
veloped a triple-shell DDS which is responsive to pH and temperature
[38].
molecular weight of 1000 g/mol) as the outer shell were synthesized 116
as described in the literature [13,23]. 117
NMR spectra were recorded on a Jeol ECX 400 or a Jeol Eclipse 500 118
MHz spectrometer. Proton and carbon NMR were recorded in ppm 119
and were referenced to the indicated solvents [41]. NMR data were re- 120
ported as follows: chemical shift, multiplicity (s = singlet, d = doublet, 121
t = triplet, m = multiplet), integration, and coupling constants (s) in 122
Hertz (Hz). Multiplets (m) were reported over the range (ppm) at 123
which they appear at the indicated field strength. Mass spectrometry 124
was performed on an Agilent 6210 ESI-TOF spectrometer. Gel perme- 125
ation chromatography (GPC) data was obtained by measurements 126
using an Agilent 1100 solvent delivery system with pump, manual in- 127
jector, and an Agilent differential refractometer. Three 30 cm Suprema 128
columns (PPS: Polymer Standards Service GmbH, Germany; Suprema 129
100 Å, 1000 Å, 3000 Å with 5 and 10 μm particle size) were used to 130
separate aqueous polymer samples using water with 0.05% NaN3 as 131
the mobile phase at a flow rate of 1 mL·min−1. The columns were 132
operated at ambient temperature with the RI detector at 50 °C. The cal- 133
ibration was performed by using linear pullulan calibration standard 134
(PPS GmbH, Germany). Measurements were carried out under highly 135
diluted conditions (10 mg/mL, injected volume 20 μL). WinGPC Unity 136
software from PSS was used for data acquirement and interpretation. 137
UV/Vis spectra were recorded on a Scinco S-3100 UV/Vis spectrometer. 138
The dynamic light scattering (DLS) measurements for the size determi- 139
nation were performed on a Malvern Zetasizer Nano equipped with a 140
He–Ne laser (633 nm) using backscattering mode (detector angle 141
173°). The samples were filtered through 0.2 μm regenerated cellulose 142
syringe filters prior to the DLS measurement and left for 24 h to equili- 143
brate. 100 μL of the solution to be analyzed was added to a disposable 144
microcuvette (Plastibrand) with a round aperture. The autocorrelation 145
functions of the backscattered light fluctuation were analyzed using 146
Zetasizer DTS software from Malvern to determine the size distribution 147
by intensity and volume. The measurements were performed at 25 °C, 148
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
100 shell (pH-CMS, Fig. 1). The aromatic imine bond can be hydrolyzed at
101 the required pH-value to achieve the cleavage of the carrier after cellu-
102 lar uptake. Additionally, it allows for further fine-tuning of the release
103 due to the possibility of introducing substituents at the aromatic ring
104 to adjust the pH to the required value for the cleavage of the shell [39].
105 2. Experimental section
106 2.1. General
107
Reactions requiring dry conditions were carried out in dried Schlenk
equilibrating the system on this temperature for 120 s.
149
108 glassware under argon. Analytical grade solvents and chemicals were
109 purchased from Acros or Sigma Aldrich and used as received. The
110 1,18-octadecanedioic acid (C18diCOOH) was a kind gift of Cognis.
111 Sephadex LH-20 was purchased from GE Healthcare. Dry solvents
112 were obtained from a MBraun SPS-800 solvent purification system.
113 dPG amine (Mn 10,000 g/mol) was prepared with a degree of amination
114 of 70% analogous to the published method [40]. Dendritic CMS
115 nanocarriers with mPEG1000 (mPEG with an average number averaged
2.2. Preparation of DOX
150
Doxorubicin hydrochloride (DOX · HCl) was transferred into the 151
free base doxorubicin (DOX) similar to the published method [42]. 152
DOX · HCl was dissolved in dimethylformamide (DMF) and stirred 153
with 2 eq. of triethylamine overnight.
154
Q4 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/
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E.F.K. Achazi et al. / Journal of Controlled Release xxx (2014) xxx–xxx
3
155 2.3. Loading of CMS nanocarriers
image processing a Leica (DMI6000CSB stand) confocal laser scan 212
microscope and the Leica LAS AF software were used.
213
156
Stock solutions of the stable CMS nanocarriers with concentrations
157 of 1 and 5 mg/mL in dist. water were prepared. The solution of the
158 pH-CMS nanocarriers with the same concentrations was always freshly
159 prepared before usage. A 5 mg/mL guest (Nile red (NR) or DOX) stock
160 solution in tetrahydrofuran (THF) or DMF in case of DOX was prepared.
161 200 μL of the guest stock solution was transferred into small sample
162 vials and the solvent was evaporated. Afterwards, 3 mL of the different
163 CMS nanocarrier stock solutions or dist. water as control was added. The
164 samples were stirred at 1200 rpm for 6 h. Subsequently, the NR samples
165 were filtered through 0.45 μm regenerated cellulose syringe filters to
166 remove unsolubilized NR. The DOX-loaded CMS nanocarriers were
167 freeze-dried, taken up with 0.5 mL dist. water, and purified by size ex-
168 clusion chromatography (SEC) using Sephadex LH-20 in dist. water.
169 After SEC the samples were freeze-dried and dissolved in phosphate
170 buffered saline (PBS) to obtain concentrations of 1 or 5 mg/mL CMS
171 nanocarriers.
2.8. Toxicity studies
214
The cell experiments were performed with an xCELLigence RTCA SP 215
(Roche) using A549 lung cancer cells. The xCELLigence RTCA SP was 216
used to dynamically monitor cell proliferation and viability in real 217
time, based on impedance. For background measurement 50 μL of 218
Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% 219
fetal calf serum and 1% penicillin, streptomycin and glutamine was 220
added to each well of a 96-well E-plate (Roche). Afterwards, a total of 221
1 × 104 A549 cells in 50 μL supplemented DMEM were seeded into 222
each well and the measurement started. On the next day, 20 μL of either 223
the unloaded or DOX-loaded CMS nanocarriers in PBS in different 224
concentrations plus 80 μL fresh medium were added to the wells in du- 225
plicates. All samples were calibrated on the DOX concentration using 226
UV/Vis spectroscopy. DOX and PBS treated cells served as controls. 227
The E-plate was incubated during the whole measurement with 5% 228
CO2 at 37 °C and monitored on the RTCA SP system with time intervals 229
172 2.4. Transport capacity determination
of at least 15 min for 72 h after treatment. Analysis was performed using 230
OOF
173
1 mL of the aqueous solution of the different nanocarriers with
the RTCA 1.2.1 software and GraphPad Prism 5. IC50 values were as 231
174 solubilized NR was freeze-dried and redissolved in 2 mL methanol.
175 The concentrations of NR in methanol (MeOH) were estimated using
176 the molar extinction coefficient (ε) of 45,000 M−1 cm−1 at 552 nm
177 [43]. The concentrations of DOX in PBS were estimated using the
1Q782 molar extinction coefficient (ε) of 10,645 M−1 cm−1 at 495 nm [44].
well calculated with GraphPad Prism (equation: log(inhibitor) vs. 232
response − variable slope (four parameters)) using end points gener- 233
ated by the RTCA software.
234
2.8.1. Octadecane-1,18-dimethylester (C18diCOOMe)
235
In a 1000 mL flask C18diCOOH (20.5 g, 63.7 mmol, 1.0 eq.) was added 236
to 600 mL of MeOH. After the addition of sulfuric acid (10 drops) the 237
mixture was stirred for 16 h under reflux. The C18diCOOH dissolved 238
upon heating. The reaction mixture was allowed to reach room temper- 239
ature (rt). The product crystallized as a colorless solid and was filtered 240
and washed with cold MeOH. The colorless solid was purified by flash 241
column chromatography (hexane:ethyl acetate = 9:1) to receive 21.7 242
179 2.5. Cleavage experiment
180
181
182
183
184
185
186
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 chloro-
form 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).
g of C18diCOOMe (99%) as colorless powder.
243
1H NMR (400 MHz, CDCl3, TMS): δ (ppm) = 3.65 (s, 6H,\O\CH3), 244
2.29 (t, 4H, J = 7.56 Hz,\CO\CH2\CH2\), 1.60 (m, 4H,\CH2\ 245
CH2\CH2\), 1.37–1.20 (m, 24H,\CH2\(CH2)12\CH2\).
246
187 2.6. Stability and release experiments
13C NMR (100 MHz, CDCl3, TMS): δ (ppm) = 174.3 (\CH2\CO\ 247
O\CH3), 51.4 (\O\CH3), 34.1 (\CH2\CO\), 29.6–29.2 (\CH2\ 248
(CH2)12\CH2\), 29.1 (\CH2\(CH2)2\CO\), 24.9 (\CH2\CH2\CO\). 249
IR (cm−1): 3451, 2916, 2847, 1738, 1472, 1463, 1435, 1412, 1382, 250
1370, 1350, 1316, 1273, 1233, 1191, 1163, 1117, 1091, 1033, 991, 968, 251
188
The stability of DOX-loaded pH-CMS nanocarriers as well as the re-
189 lease of DOX was evaluated via SEC combined with UV/Vis measure-
190 ments. DOX-loaded pH-CMS nanocarrier solutions were kept at pH
191 7.4, pH 5, and pH 4 at 37 °C for different time intervals. Directly after
192 preparation as well as after certain time intervals 200 μL of the sample
193 was applied to a small SEC column (Sephadex LH-20 equilibrated in
194 PBS pH 7.4). Additional PBS pH 7.4 was added to elute the sample and
195 achieve separation of encapsulated DOX from released DOX. The DOX
196 loaded pH-CMS nanocarrier fractions were collected and the absorption
197 of DOX at 495 nm was measured. The amount of released DOX was
198 calculated with the help of the remaining absorption in comparison to
199 the absorption at the beginning of the measurements.
882, 824, 769, 732, 719, 700.
252
MS (EI): m/z (%) calculated for C20H38O4Na: 365.2668; found: 253
365.2674.
2.8.2. Octadecane-1,18-diol (C18diol)
In a 1000 mL flask C18diCOOMe (14.8 g, 43.1 mmol, 1 eq.) was 256
dissolved in 700 mL dry THF and cooled to 0 °C. Then LiAlH4 (4.1 g, 257
107.8 mmol, 2.5 eq.) was added slowly. The resulting mixture was stirred 258
for 1 h at rt before refluxing it for 6 h. The mixture was cooled to 0 °C and 259
slowly quenched with a cold H2SO4/water mixture (4 eq.: 12 eq. in rela- 260
tion to LiAlH4) in THF. The precipitate was filtered off and washed with 261
254
255
200 2.7. Confocal laser scanning microscopy
UNCORRECTED PR
THF (3 × 400 mL). The resulting solution was concentrated under 262
reduced pressure yielding the product as a white solid (12.3 g, 98%).
263
201
Cellular uptake of DOX-loaded CMS nanocarriers and intracellular
202 release of DOX was monitored by confocal microscopy. For the uptake
203 study 50,000 A549 cells were seeded on 9 mm glass coverslips in each
204 well of a 24-well plate and cultured for 24 h before adding the DOX-
205 loaded CMS nanocarriers in a final concentration of 0.1 mg/mL and
206 0.01 mg/mL, respectively. PBS and DOX (20 μM and 2 μM) treated
207 cells served as controls. After 1, 4 or 24 h, respectively, cells were
208 washed three times with PBS and fixed with 4% paraformaldehyde for
209 20 min. Afterwards, cells were permeabilized with 0.1% TritonX for
210 5 min and washed 2 times with PBS. Cell nuclei were stained with
211 4′,6-diamidino-2-phenylindole (DAPI). For observation, imaging and
1H NMR (400 MHz, THF-d8, TMS): δ (ppm) = 3.45 (dd, J = 11.35, 264
6.15 Hz, 4H, HO\CH2\CH2\), 2.49 (s, 2H, HO\CH2\), 1.46 (m, 4H, 265
HO\CH2\CH2\), 1.39–1.25 (m, 24H,\CH2\(CH2)12\CH2\).
266
13C NMR (100 MHz, THF-d8, TMS): δ (ppm) = 62.7 (HO\CH2\), 267
34.3 (HO\CH2\CH2\), 30.9–30.7 (\CH2\(CH2)12\CH2\), 27.1 268
(HO\(CH2)2\CH2\).
IR (cm−1): 3414, 3355, 2917, 2846, 1468, 1352, 1334, 1059, 1046, 270
1015, 979, 723. 271
MS (EI): m/z (%) calculated for C18H38O2Na: 309.2770; found: 272
309.2770. 273
269
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/
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E.F.K. Achazi et al. / Journal of Controlled Release xxx (2014) xxx–xxx
Fig. 2. Synthesis of the pH-cleavable core–multishell architectures (pH-CMS).
274 2.8.3. Octadecane-1,18-dibromide (C18diBr)
2.8.4. Poly(ethylene glycol) methyl ether octadecane-18-bromides 295
(mPEGxC18Br) 296
275
In a 1000 mL Schlenk-flask under argon atmosphere C18diol (6.7 g,
276 23.4 mmol, 1 eq.) was dissolved in 700 mL dry THF. Then CBr4
277 (19.4 g, 58.5 mmol, 2.5 eq.) was added and the reaction mixture
278 was heated to 60 °C resulting in a yellow solution. Finally PPh3
279 (18.4 g, 70.2 mmol, 3 eq.) was added in small portions. After com-
280 plete addition the reaction was heated to reflux for 4 h, cooled to rt
281 and the formed precipitate was filtered off. The solvent was evapo-
282 rated and the remaining crude product was dissolved in a small
283 amount of CHCl3 and loaded onto silica gel. The final product was ob-
284 tained as white crystals after column chromatography using hexane
285 as the eluent (9.1 g, 94%).
In a 250 mL Schlenk-flask under argon atmosphere mPEGx (6.8 mmol, 297
1.0 eq.) was dissolved in 150 mL of dry THF and a catalytic amount (10 μL) 298
of 15-crown-5 ether was added. NaH (1.94 mmol, 1.5 eq.) was added 299
slowly and the mixture was stirred for 3 h. The resulting solution was 300
transferred with a transfer cannula into a 250 mL dropping funnel at- 301
tached to a 1000 mL Schlenk-flask filled with C18diBr (20.4 mmol, 302
3.0 eq.) in 500 mL dry THF. The solution in the dropping funnel was 303
added dropwise. The resulting mixture was stirred for 16 h before evapo- 304
rating the solvent. Purification was accomplished using dry column 305
chromatography (CHCl3 to CHCl3 + 10% MeOH), followed by a filtration 306
286
1H NMR (400 MHz, CDCl3, TMS): δ (ppm) = 3.41 (t, J = 6.88 Hz,
287 4H,\CH2\Br), 1.88–1.78 (m, 4H,\CH2\CH2\Br), 1.46–1.37 (m,
288 4H,\CH2\(CH2)2\Br), 1.34–1.20 (m, 24H,\CH2\(CH2)12\CH2\).
289
13C NMR (100 MHz, CDCl3, TMS): δ (ppm) = 34.0 (\CH2\Br),
Table 1
t1:1
290 32.8 (\CH2\CH2\Br), 29.7–29.4 (\CH2\(CH2)12\CH2\), 28.8
291 (\CH2\(CH2)3\Br), 28.2 (\CH2\(CH2)2\Br).
GPC data for the CMS nanocarriers used for the in vitro experiments.
t1:2
Nanocarrier
Mn [g/mol]
Mw [g/mol]
# of arms attached
Dispersity
t1:3
292
293 410.1177.
294
IR (cm−1): 2914, 2849, 1470, 1338, 1313, 1278, 1241, 1206, 716.
MS (ESI): m/z (%) calculated for C18H36Br2: 410.1184; found:
CMS
pH-CMS
43,800
76,700
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/
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E.F.K. Achazi et al. / Journal of Controlled Release xxx (2014) xxx–xxx
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t2:1
t2:2
Table 2
Hydrodynamic radii of unloaded CMS and pH-CMS nanocarriers in methanol and PBS at different nanocarrier concentrationsa.
t2:3
t2:4
Nanocarrier
Carrier conc. [mg/mL]
Size in MeOH [nm]
Size in PBS pH 7.4 [nm]
rH
rH
rH
rH
vol
int
vol
int
t2:5
CMS
1
5
1
5
73.5
5.1
74.7
6.0
9.9
96.2
9.1
3.4 (65%)
0.1 (35%)
2.7 (57%)
0.2 (43%)
0.4 (58%)
6.2 (42%)
0.2 (67%)
3.2 (33%)
4.4
4.0
6.9
5.9
0.1
0.1
0.2
0.1
89.9
6.9
98.7
6.7
8.9
115.1
9.8
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
5.2
7.4
7.7
0.2
0.2
0.2
0.1
t2:6
t2:7
t2:8
t2:9
pH-CMS
t2:10
t2:11
t2:12
93.6
109.8
a
t2:13
rH andrH 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
int
vol
abundance is given in brackets.
IR (cm−1): 2915, 2883, 1466, 1359, 1342, 1280, 1240, 1146, 1104, 338
1060, 958, 842, 715. 339
307
308
through a pad of RP silica gel (Chromabond® C18ec, MeOH + 60% water
to MeOH) to afford the desired product as colorless wax.
309
310
mPEG350C18Br: yield = 1.8 g, 80%
2.8.5. 3,4,5-Trioctadecane poly(ethylene glycol) methyl ether benzaldehyde 340
(THBA-C18-mPEGx) 341
1H 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\(CH2)13\CH2\).
311
312
313
314
315
316
317
OOF
In a 250 mL Schlenk-flask 3,4,5-trihydroxybenzaldehyde mono- 342
hydrate (THBA, 0.58 mmol, 1 eq.), mPEGxC18Br (2.03 mmol, 3.5 eq.), 343
potassium carbonate (0.32 g, 2,32 mmol, 4 eq.) and potassium iodine 344
(0.05 g, catalytic) were mixed in 160 mL DMF. The reaction mixture 345
was degassed by three consecutive cycles of applying vacuum and 346
bubbling argon through the DMF. The reaction was heated to 80 °C 347
and stirred for three days. After reaching rt, DMF was evaporated and 348
the residue taken up with CHCl3. The mixture was filtered through a 349
pad of silica gel (CHCl3 + 5% MeOH). The solvent was removed and 350
the residue dissolved in water and dialyzed (water, MWCO 2000 Da). 351
The product solution was freeze-dried yielding the product as slightly 352
13C 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\(CH2)2\CH2\).
318
319
320
321
322
323
324
325
326
yellow foam.
353
IR (cm−1): 2924, 2854, 1466, 1359, 1342, 1280, 1240, 1146, 1112,
1060, 958, 842, 715.
THBA-C18-mPEG350: yield = 0.6 g, 80%
354
1H NMR (500 MHz, CDCl3, TMS): δ (ppm) = 9.78 (s, 1H, (\CHO)), 355
7.03 (s, 2H, Ar\H), 4.02 (t, J = 6.4 Hz, 2H, Ar\O\CH2\), 3.98 356
(t, J = 5.9 Hz, 4H, 2× Ar\O\CH2\), 3.66–3.56 (m, ~72H, 357
\(O\CH2\CH2)n\), 3.39 (t, J = 6.8 Hz, 6H, \O\CH2\(CH2)17\), 358
3.33 (s, 9H, \O\CH3), 1.82–1.74 (m, 4H, \CH2\CH2\O\Ar), 359
1.74–1.66 (m, 2H, \CH2\CH2\O\Ar), 1.56–1.48 (m, 12H, 3× 360
\O\CH2\CH2\(CH2)n\, 3× Ar\O\CH2\CH2\(CH2)n\), 1.47– 361
1.38 (m, 6H, 3× Ar\O\(CH2)2\CH2\), 1.34–1.12 (m, 78H, 362
mPEG1000C18Br: yield = 3.5 g, 39%
1H 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, \CH2\CH2\Br), 1.59–1.49 (m, 2H, \O\
CH2\CH2\(CH2)n\), 1.44–1.34 (m, 2H, \CH2\(CH2)2\Br),
1.33–1.10 (m, 26H, \CH2\(CH2)13\CH2\).
327
328
329
330
331
332
13C 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\(CH2)2\CH2\).
\CH2\(CH2)13\CH2-).
363
13C NMR (100 MHz, CDCl3, TMS): δ (ppm) = 191.1 (Ar\COH), 364
153.4 (Ar\O\), 143.7 (Ar\O\)131.3 (Ar\COH), 107.7 365
(Ar\H), 73.5 (\O\CH2\(CH2)n\), 71.8 (\CH2\OCH3), 71.4 366
(\O\CH2\(CH2)n\), 70.4–70.3 (\(O\CH2\CH2)n\), 69.9 367
(\CH2\O\(CH2)n\), 69.1 (Ar\O\CH2\), 58.8 (\O\CH3), 368
333
334
335
336
337
t3:1
t3:2
Table 3
Hydrodynamic radii of loaded CMS and pH-CMS nanocarriers in PBS at different nanocarrier concentrationsa.
UNCORRECTED PR
t3:3
t3:4
b
Carrier conc. [mg/mL]
Size of NR-loaded CMS nanocarrier [nm]
rH rH
Size of DOX-loaded CMS nanocarrier [nm]
rH rH
int
vol
int
vol
t3:5
CMS
1
5
1
5
134.1
131.6
114.0
4.7
1.6
1.9
141.0
137.2
111.2
6.6
7.1
2.9
2.4
0.4
94.2
9.7
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
6.4
8.1
7.7
0.6
0.2
0.3
0.3
t3:6
t3:7
88.6
10.5
10.7
104.9
8.7
t3:8
t3:9
pH-CMS
t3:10
t3:11
t3:12
126.9
10.4
5.3 (73%)
1.0 (27%)
101.9
a
t3:13
rH andrH 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
int
vol
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/
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E.F.K. Achazi et al. / Journal of Controlled Release xxx (2014) xxx–xxx
Table 4
t4:1
t4:2
t4:3
t4:4
t4:5
369
370
371
372
373
374
375
30.2 (Ar\O\CH2\CH2\), 29.6–28.8 (\CH2\(CH2)12\CH2\,
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.
\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.
Nanocarrier
Carrier conc.
[mg/mL]
Transport capacity
[mgguest/gcarrier
]
t4:6
t4:7
THBA-C18-mPEG1000: yield = 1.76 g, 86%
1H 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,
\(O\CH2\CH2)n\), 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\(CH2)13\CH2\).
NR
DOX
376
377
378
379
380
381
382
383
384
CMS
1
5
1
5
3.0
1.1
2.6
2.2
41.7
12.6
51.2
19.4
t4:8
t4:9
pH-CMS
t4:10
t4:11
3. Results and discussion
428
429
13C 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 (\(O\
CH2\CH2)n\), 70.0 (\CH2\O\(CH2)n\), 59.0 (\O\CH3),
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.
3.1. Synthesis
385
386
387
388
389
390
391
The pH-cleavable CMS architectures were synthesized by the six-step 430
procedure shown in Fig. 2. Starting from 1,18-octadecanedioic acid, the 431
acid groups were transformed into the corresponding methyl esters 432
and reduced to hydroxyl groups in the next step. Subsequently, the 433
diols were converted into a dibromide building block using the Appel 434
reaction. The compound was reacted in a Williamson-ether reaction 435
with mPEG to yield the poly(ethylene glycol) methyl ether octadecane- 436
18-bromides. This product was attached to trihydroxybenz-aldehyde 437
(THBA) resulting in the combined inner and outer shell with a free 438
aldehyde functionality. In the presence of potassium carbonate and 439
potassium iodide the double shell building blocks underwent imine 440
bond formation with dPG amine resulting in the pH-cleavable CMS 441
nanocarriers. Final purification of the pH-CMS was achieved by ultrafil- 442
tration using a membrane with a MWCO of 10 kDa in methanol. Traces 443
of triethylamine had to be added to avoid partial cleavage of the imine 444
bond. The pH-CMS nanocarrier with mPEG350 were not soluble in 445
water and have therefore not been further characterized concerning 446
sizes and transport capacities as these measurements were performed 447
392 2.8.6. pH-responsive core–multishell nanocarrier (pH-CMS)
393
The functionalized benzaldehyde THBA-C18-mPEGx (1.0 eq.) dis-
394 solved in MeOH (1 mL per 0.1 mmol) was added dropwise to a mixture
395 of dPG amine (70% amine functionalization, 9.45 mmol (\NH2) per g)
396 in MeOH (1 mL per 0.1 mmol (\NH2)) and anhydrous sodium sulfate
397 (1 g per 1 mL MeOH). The mixture was refluxed for 24 h. After reaching
398 rt, the solution was filtered through Celite and concentrated under vac-
399 uum. The crude product was purified by ultrafiltration (MWCO 10 kDa)
400 in methanol with 0.1% triethylamine to give the final product with a
401 degree of functionalization of 31% (based on the amount of OH groups
402 of the dPG).
in aqueous media.
448
The results of the GPC measurements are summarized in Table 1. 449
Both CMS nanocarriers showed equal Mw distributions and a similar 450
number of arms. Since the pH-CMS nanocarriers is made out of a triva- 451
lent shell building block the number of arms for comparison with the 452
CMS nanocarriers made out of monovalent shell building blocks should 453
be multiplied by three. The higher dispersity of the CMS nanocarriers 454
compared to the pH-CMS nanocarriers results as well from the different 455
403
404
pH-CMS with mPEG350:
1H 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,\(O\CH2\CH2)n\), 3.33 (s, 9H,\O\CH3), 1.90–1.60
(m, 6H, 3× \O\CH2\CH2\), 1.58–1.50 (m, 6H, 3× \O\CH2\
CH2\(CH2)n\, 3× Ar\O\CH2\CH2\(CH2)n\), 1.48–1.05 (m,
~84H, 3× Ar\O\(CH2)2\CH2\, \CH2\(CH2)13\CH2\).
13C 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.
405
406
407
408
409
410
shell building blocks.
456
411
412
413
414
415
416
417
100
80
60
40
20
pH 4
pH 5
pH 6
pH 7.4
pH-CMS with mPEG1000
:
1H 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\(CH2)13\CH2\).
418
419
420
421
422
423
0
0
13C 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.
2
4
6
time (h)
424
425
426
427
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.
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E.F.K. Achazi et al. / Journal of Controlled Release xxx (2014) xxx–xxx
7
pH-CMS pH5
pH-CMS pH 7.4
were about 7 nm and 10 nm for the CMS and pH-CMS nanocarriers, 469
respectively. The slight increase of the sizes compared to the single 470
particles in MeOH most likely arose from a different conformation of 471
the outer mPEG-shell. The stable CMS nanocarriers again showed bigger 472
aggregates in the intensity distribution with radii of 90–100 nm. In the 473
case of the pH-CMS nanocarriers, an aggregate peak of around 110 nm 474
was only observable for the higher concentration. Again, no aggregate 475
peak was observed for any CMS nanocarriers at any concentration in 476
the volume distribution. This shows the tendency of CMS nanocarriers 477
to form aggregates on their own which was already described earlier 478
60
40
20
0
[13,14,45].
479
0
10
20
30
40
50
3.3. Size and aggregation behavior of loaded CMS nanocarriers
480
time (h)
As the size of a drug delivery system (DDS) has a big influence on its 481
uptake into cells, and in order to see whether transport occurs via 482
unimolecular carriers or via aggregates, the loaded CMS nanocarriers 483
were also analyzed by DLS (see Table 3). Aggregates were formed by 484
all NR-loaded CMS nanocarriers. For the stable CMS nanocarrier aggre- 485
gates with a hydrodynamic radius of 130–140 nm were observed. The 486
Fig. 4. Release of DOX from DOX-loaded pH-CMS nanocarriers at pH 7.4 and pH 5 over
48 h.
457 3.2. Size and aggregation behavior of unloaded CMS nanocarriers
458
In order to gain a better understanding of the stable CMS and pH-
pH-CMS nanocarrier aggregates had a radius of about 115 nm at a con- 487
centration of 1 mg/mL but showed two peaks at a concentration of 488
OOF
459 responsive CMS nanocarriers (see Fig. 1), the self-aggregation behavior
460 of the different loaded and unloaded CMS nanoparticles was investigat-
461 ed with the help of DLS. Primarily, the unloaded CMS nanocarriers in
462 methanol and in PBS with pH of 7.4 were studied. In methanol the single
463 CMS nanocarriers had a hydrodynamic radius about 5 nm and the pH-
464 CMS had a radius about 9 nm (see Table 2). In all cases, the CMS
465 nanocarriers showed bigger aggregates in the intensity distribution of
466 the DLS measurements. These aggregates were not observed in the
467 volume distribution and can therefore be considered as a minor pro-
468 nounced species. The hydrodynamic radii of single particles in PBS
5 mg/mL. The predominant size distribution with 7–13 nm indicates 489
that mainly single particles are present in the sample. The aggregate 490
peak is not observed in the volume-based size distribution. This result 491
is in line with an earlier report, where we found that the aggregation 492
of CMS nanocarriers is influenced by the concentration of NR used 493
[14]. For the 5 mg/mL solution the amount of NR relative to the CMS 494
nanocarriers is not sufficient to enhance aggregate formation. This fur- 495
ther supports the observation that the slightly more branched outer 496
shell decreases the tendency to form aggregates.
497
UNCORRECTED PR
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.)
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E.F.K. Achazi et al. / Journal of Controlled Release xxx (2014) xxx–xxx
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.
498
In the intensity-based size distributions, the DOX-loaded CMS nano-
release about 10 to 15% DOX over 48 h (see Fig. 4, Figure S5 only one 539
colored band is visible, the upper part of the column is almost colorless). 540
Even though, the NMR experiment showed partial cleavage of the imine 541
bond at pH 7.4. This shows that the initial cleavage is not sufficient to re- 542
lease a high dose of DOX. Therefore, the loaded pH-CMS nanocarriers 543
can be considered relatively stable under physiological conditions. The 544
pH-CMS nanocarriers at pH 5 released DOX over time (see Fig. 4, 545
Figure S5 two colored bands are visible, with the upper band increasing 546
over time). This proves that DOX-loaded pH-CMS can efficiently release 547
499 particles all show peaks corresponding to aggregate formation (Table 3)
500 with radii of around 90–95 nm for the stable CMS and 100–105 nm for
501 pH-CMS nanocarriers. However, based on the volume distributions, the
502 single nanocarriers with radii of around 6–7 nm and 8 nm are obviously
503 the predominant species for all carriers.
504
The aggregation behavior of CMS nanocarriers is significantly influ-
505 enced by the different guest molecules. NR seems to enhance the
506 tendency of CMS to form aggregates, while the presence of DOX rather
507 inhibits aggregation and single nanocarriers are the predominant
508 species. These observations further strengthen our earlier findings [45].
DOX under slightly acidic conditions.
548
3.7. Cellular uptake
549
509 3.4. Transport capacities
In order to evaluate if the CMS nanocarriers are taken up by cells 550
we performed confocal microscopy measurements. As can be seen in 551
Fig. 5 both carriers are taken up by A549 lung cancer cells. The DOX 552
fluorescence is visible within the cells being localized either within 553
510
In order to investigate their suitability as DDS it is important to know
511 the transport capacity of the different CMS nanocarriers. Therefore, they
512 were loaded with the dye NR or the drug DOX and the transport capac-
513 ities were determined by UV/Vis spectroscopy. The resulting transport
514 capacities are given in Table 4. All carriers were able to slightly enhance
515 the solubility of NR. Encapsulation of DOX was even more efficient as
516 the transport capacities were one order of magnitude higher. This is
517 astonishing as the size distributions show that the main species are
518 single particles and we had assumed that the more efficient transport
519 was occurring in aggregates [45].
the cytoplasm or in the nuclei.
554
3.8. In vitro studies
555
The advantage of pH-responsive CMS nanocarriers in comparison to 556
the stable CMS nanocarriers lies in the possibility to control and trigger 557
the release of guest molecules. Real time cell analysis (RTCA) experi- 558
ments were performed to affirm this advantage in vitro. In order to ex- 559
clude side effects of the different carriers we first investigated the 560
toxicity of the unloaded CMS nanocarriers. Fig. 6 shows that unloaded 561
CMS nanocarriers did not show any toxicity over a period of 48 h. The 562
carriers showed no toxic effects even at very high concentrations 563
(1 mg/mL, ten times higher than used for the loaded CMS nanocarriers). 564
RTCA measurements of DOX-loaded CMS nanocarriers after 24 h only 565
showed a slightly higher toxicity of pH-CMS nanocarriers in comparison 566
to stable CMS nanocarriers. However, after 48 h the toxicity of the cleav- 567
able systems was significantly higher (see Fig. 6). The effect of the 568
0.1 mg/mL pH-CMS nanocarrier solution with 20 μM DOX after 48 h 569
was comparable to that of free DOX. For a better comparison we calcu- 570
lated the IC50 values for the different formulations and summarized 571
520 3.5. Cleavage experiments
521
For the investigation of the pH-dependant shell cleavage, the de-
522 crease of imine and the appearance of aldehyde signal were monitored
523 by 1H NMR spectroscopy at different pH values over time. At pH values
524 between 6 and 7.4 the cleavage of the imine bond occurred rather slow.
525 After 5 h about 20% of the imine bonds were cleaved (see Fig. 3). In
526 contrast at a pH of 5 and lower rapid cleavage of the imine bond was ob-
527 served. Within the first two hours about 75% of the pH-sensitive linkers
528 were cleaved. This clearly demonstrates that the imine bond used in the
529 pH-CMS cleaves at a pH between 5 and 6. This area is in particular of
530 interest for the cleavage of pH-responsive DDS after cellular uptake as
531 such pH values can be reached in the intracellular compartments like
532 the late endosome or lysosome.
Table 5
t5:1
t5:2
t5:3
533 3.6. Stability and release
IC50 values for free DOX, DOX-loaded pH CMS, and DOX-loaded
CMS nanocarriers after 48 h.
534
In order to test the stability of pH-CMS nanocarriers loaded with
Formulation
IC50 value [μM]
t5:4
535 DOX under physiological and acidic conditions, DOX-loaded pH-CMS
536 nanocarriers were kept at pH 7.4 and pH 5 at 37 °C for 48 h. Directly
537 after sample preparation, after 1, 2, 4, 24 as well as 48 h the samples
538 were applied to small SEC columns. The sample at pH 7.4 did only
DOX
pH-CMS
CMS
1.6
22.4
56.7
t5:5
t5:6
t5:7
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E.F.K. Achazi et al. / Journal of Controlled Release xxx (2014) xxx–xxx
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The introduction of the aromatic imine linkage into the CMS
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598 Universität Berlin and the SFB 1112 for the financial support.
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