Dendrimer-based micelles as cyto-compatible nanocarriers

Article abstract of DOI:10.1039/c9nj02612f

A series of dendritic architectures have been synthesized using biocompatible materials such as glycerol, glucose, phloroglucinol, and alkyl chains of various sizes to give them aqueous solubility and to attain proper hydrophilic-hydrophobic balance, allo

Full text of DOI:10.1039/c9nj02612f

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Chechia Hu, Tzu-Jen Lin et al.
Yellowish and blue luminescent graphene oxide quantum dots
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Dendrimer-based Micelles as Cyto-compatible Nanocarriers
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Badri Parshad,a,b Preeti Yadav,a Yannic Kerkhoff,b Ayushi Mittal,a Katharina Achazi,b,* Rainer
Haag,b Sunil K. Sharmaa,*
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aDepartment of Chemistry, University of Delhi, Delhi 110 007, India
bInstitut für Chemie und Biochemie, Freie Universität Berlin, Takustraße 3, 14195 Berlin, Germany
Abstract
A series of dendritic architectures have been synthesized using biocompatible materials such as
glycerol, glucose, phloroglucinol, and alkyl chains of varied sizes to confer them aqueous
solubility and to attain proper hydrophilic-hydrophobic balance allowing them to form nano-sized
aggregates for the encapsulation and delivery of drugs/dyes for biomedical applications. The
aggregation behavior of these dendritic architectures as well as cytotoxicity and their cellular
uptake were studied using fluorescence measurements, dynamic light scattering (DLS),
transmission electron microscopy (TEM), live cell imaging by confocal laser scanning
microscopy (cLSM), and flow cytometry. Aggregation study has shown that dendrimers
proficiently self-assemble in aqueous solution with low micromolar concentration and form
uniformly spread micellar aggregates. The effective cellular uptake of Nile red loaded into the
dendrimers as well as the very low toxicity of the dendrimers points out their potential as carriers
for drugs and imaging agents.
Keywords: cellular internalization, cytotoxicity, dendrimers, micelles, self-assembly
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Introduction
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Reduced or limited drugs’ potency and therapeutic effects due to low aqueous solubility, non-
specific binding, poor cell membrane permeability, and partial degradation are the various hurdles
that the researchers across the world are attempting to address. To achieve the goal of a target-
specific delivery of drugs with improved pharmacokinetic profile, researchers are turning to
nanocarrier systems. In order to accomplish the discovery of new nanostructures and drug delivery
agents, numerous studies have been carried out.1-8 Among the many nanoparticulate drug delivery
systems being studied, the use of dendritic macromolecular systems as an efficient drug delivery
agent has attracted significant attention in recent years.9,10 Appropriate control over dendritic
architectures (size, shape, and solubility) and multiple functionalities at the surface make
dendrimers potential building blocks for the fabrication of efficient functional materials for drug
delivery applications.11 In comparison to linear amphiphiles, dendrimers lead to the formation of
stable micelles as they possess a well-defined shape with multiple branching that makes them
dense enough to prevent reverse micellization by inverting the branches. This stability enhances
the duration of their circulation in blood and reduces the risk of premature clearance of
encapsulated drug from the body with reproducible pharmacokinetic behavior.12 The use of
dendrimers for drug encapsulation and transport has extensively been reviewed by many research
groups.13-15 Encapsulation of small molecules including naphthalocyanine,16 pyrene,17 and
phenol blue18 within poly(aryl ether), polyester, or poly(amido amine) dendrimer has also been
reported and there is much more to explore in this field. To further explore the dendritic
architecture as drug delivery vehicles and their self-assembly in aqueous medium, herein, a series
of dendritic architectures were synthesized that employed the divergent approach and used the
symmetrical aromatic core of phloroglucinol, which not only reduced crowding but also imparted
π-π interactions, a driving force for the encapsulation of aromatic guest molecules. In order to
render aqueous solubility to the synthesized dendrimers, biocompatible materials such as glycerol
and D-glucose were used. Furthermore, to enhance the flexibility and hydrophobicity, longer alkyl
chains were incorporated that can facilitate the creation of interior hydrophobic cavity in the
aggregates formed in aqueous medium. In an attempt to attain micellar stability as well as to
maximize the transport potential of guest molecules, hydrophobic-hydrophilic balance was
optimized via the use of different types of hydrophilic/hydrophobic units. The aggregation of these
dendritic architectures in an aqueous medium to form micelles/micellar aggregates might be due to
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the formation of tripod like structures, allowing the efficient interaction between hydrophobic and
hydrophilic units in an individual manner. As a consequence of these interactions, the hydrophobic
core/branching are directed in the interior of the aggregates forming a hydrophobic cavity, with the
hydrophilic surface groups directed towards the exterior (Figure 1). The main cause of
encapsulation of hydrophobic entities is hydrophobic interactions between micellar core and
encapsulated molecules. Moreover, π-π stacking is also possible between the dendrimer aromatic
core and the π-electron cloud of the guest molecules. The self-assembling behavior of the resulting
dendrimers was studied by using dynamic light scattering (DLS), transmission electron microscopy
(TEM), and critical aggregation concentration (CAC) measurements, which facilitated a
comparison of the resulting D-glucosyl/glyceryl and alkylated dendritic architectures. The
synthesized dendritic systems were investigated for their transport potential using model
hydrophobic compounds like the dye Nile red and the biologically potent molecule curcumin. The
dendrimers exhibiting good encapsulation potential were further investigated for their cytotoxicity
and cellular internalization using adenocarcinomic human alveolar basal epithelial A549 cells.
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Figure 1. Proposed schematic representation of dendritic micellar aggregates in aqueous medium.
Experimental Section
NMR, IR, and Mass analysis
1H and 13C NMR spectra were recorded on Jeol-400/100.5 MHz spectrometer, respectively, using
the solvent residual peak as a reference, where the chemical shift values are on a δ scale and the
coupling constant values (J) are taken in Hertz. Infrared (IR) spectra and the HRMS data of the
compounds were recorded on a Perkin-Elmer 2000 FT-IR spectrometer and Agilent-6530, Q-TOF
LCMS, respectively.
Critical Aggregation Concentration (CAC) Measurement
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Nile red, a model hydrophobic dye, was used as a probe to determine the CAC of the dendrimers
by fluorescence measurements.19 A stock solution of 1 mg/mL of Nile red in THF (1 mL) was
prepared and 50 μL of this stock solution were taken in empty vials and THF was allowed to
evaporate completely. Different concentrations of dendrimers (3 mM to 0.73 μM) were prepared (2
mL) and added sequentially in the vials that had Nile red. The solution was stirred overnight, the
non-encapsulated dye was removed by a 0.45 μm polytetrafluoroethylene (PTFE) filter, and the
solution was subjected to fluorescence measurements. The CAC value was calculated by plotting
the fluorescence intensity maxima against the log[dendrimer] concentration.
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Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM)
The size of the nanoparticles was measured by DLS using Malvern Zetasizer Nano ZS analyzer
integrated with 4 mW He-Ne laser, λ = 633 nm, using backscattering detection (scattering angle θ
= 173°). An aqueous solution of dendrimers (3 mg/mL) transferred into disposable cuvettes was
used for measurements. Because of their limited solubility, dendrimers 13 and 16 formed turbid
solution. The nanoparticles formed were further analyzed by transmission electron microscopy
(TEM) using a TECNAI G2-30 U-TWIN TEM instrument (FEI, Eindhoven, The Netherlands)
with an acceleration voltage of 200 kV. The sonicated aqueous solution of the sample was drop-
coated and dried onto formvar-coated 200 mesh copper grids (Ted Pella, USA) and analyzed by
TEM.
Procedure for Nile red and curcumin encapsulation
For Nile red encapsulation, a stock solution of 1 mg/mL of Nile red in THF (1 mL) was prepared.
50 μL of the stock solution was transferred in a vial and evaporated to dryness to form a thin film
(film method)20 followed by the addition of 1 mL of an aqueous solution of dendrimer (3 mg/mL).
The resulting solution was stirred overnight. For quantification of Nile red, a known amount of the
sample was lyophilized and re-dissolved in methanol for absorbance measurement and the amount
of encapsulated dye was calculated employing Lambert-Beer’s law and using Nile red’s molar
extinction co-efficient (ε) of 45,000 M-1cm-1 at 552 nm.21
Curcumin encapsulation was carried out using a solid dispersion method,22 wherein, 1 mg of
curcumin dissolved in a minimum amount of methanol was mixed with the methanolic solution of
dendrimer (3 mg/mL). The mixture was evaporated to dryness under reduced pressure followed by
the addition of 1 mL of water and stirred for overnight. The quantification of encapsulated
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curcumin was done following the same method as used for Nile red and using the molar extinction
co-efficient (ε) of 55,000 M-1cm-1 at 425 nm for curcumin in methanol.
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Cellular uptake study
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Cellular uptake of Nile red-encapsulated dendritic systems in A549 cells (ACC 107, DSMZ, The
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH,
Braunschweig, Germany) was monitored by confocal laser scanning microscopy (cLSM) and flow
cytometry. The cells were routinely propagated in DMEM medium supplemented with 2%
glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (all from Gibco BRL, Eggenstein,
Germany), and 10% fetal calf serum (Biochrom AG, Berlin, Germany) at 37 °C with 5% CO2, and
subcultured twice a week. The Nile red encapsulated dendritic architectures were prepared by
incubating the dye in an aqueous solution of dendrimer using the film method at a concentration of
5 mg/mL.
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For cLSM, cells were seeded in 8-well ibidi µ-slides (27.000 cells/well) in colorless cell culture
medium. After 1 day, the compounds were added at a final test concentration of 0.5 mg/mL and
incubated for either 30 min, 5 h, or 24 h. For staining of endocytotic compartments, Cell Light
GFP reagents (Life Technologies GmbH, Darmstadt, Germany) were used according to the
manufacturer’s instructions to specifically label either early endosomes (CellLight™ Early
Endosomes-GFP, BacMam 2.0) or lysosomes (CellLight™ Lysosomes-GFP, BacMam 2.0) in cells
incubated for 5 h with the encapsulated dye. Non-treated either labeled or non-labeled cells served
as control to adjust the settings. Confocal images of the living cells were taken with an inverted
confocal laser scanning microscope Leica DMI6000CSB SP8 (Leica, Wetzlar, Germany) with a
63x/1.4 HC PL APO CS2 oil immersion objective using the manufacture-given LAS X software. If
not stated elsewhere, all the images were taken using the same settings to ensure that the
fluorescence intensity between the different images and times can be compared. Images analysis
was performed using a self-written ImageJ Macro. To determine the single-cell fluorescence
signal, the image stack was smoothed and then the background (threshold value > 5) was excluded
and the remaining pixel converted to a binary mask. On the resulting binary images, different
morphological operators were applied (despeckle, close, fill holes, dilate, erode) to reduce
remaining noise, fill the holes from the nucleus and reshape the cell borders. Then, a watershed
algorithm with a tolerance of 2 was applied to separate cells close to each other. The resulting
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binary mask was superimposed on the original image series to mask all the background noise and
separate the cells while preserving the original fluorescence signal. The mean fluorescence
intensity of each cell was automatically analyzed by ImageJ's Particle Analyzer function and the
mean intensity of all cells per frame was calculated. The data was then plotted using Origin.
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For flow cytometry, cells were seeded in 24-well plates (100.000 cells per well). After 1 day, the
Nile red loaded compounds were added at a final test concentration of 0.5 mg/mL and the cells
were incubated at either 37 °C or 4 °C for another 10 min or 30 min, respectively. Cells were
detached by trypsin, transferred to an Eppendorf tube, centrifuged at 140 xg and 4 °C for 4 min,
and resuspended in PBS. Fluorescence of the cells was measured in a BD Accuri C6 (Becton
Dickinson, Heidelberg, Germany) and analysis was done by the FlowJoe software. Data was
plotted with the Origin software.
The uptake profile of compound 14 loaded with Nile red (14/NR) at a final concentration of 0.5
mg/mL was compared to free Nile red (NR) by confocal laser scanning microscopy (cLSM and
flow cytometry) using A549 cells. The concentration of free NR was adjusted to the same
concentration loaded onto 14. cLSM images were taken from living A549 cells incubated for 30
min with 14/NR or free NR and the data was analyzed to generate the resulting time-dependent
uptake profile. For a quantitative analysis, ten images per sample were taken after 10 min and 30
min and the mean with SD was plotted for each sample. For the imaging and following time-
lapse analysis cells were placed under a confocal laser scanning microscope and excited at 651
nm with 0.1% laser intensity every 2.6 sec. Fluorescence was detected between 570 nm and 751
nm with a photomultiplier (PMT). In the images, Nile red is shown in red color. For the
quantitative analysis, laser intensity was increased for free NR to 1% and remained for 14/NR at
0.1% to prevent over saturation. By using images made at both laser intensities, the data was
normalized. For the time-dependent visualization and the quantitative analysis, the highest signal
was set to 1 and all other values were normalized accordingly. Flow cytometry was used
additionally for quantitative analysis of the uptake of 14/NR and NR in A549 cells and to
compare the uptake efficiency and at 4 °C and 37 °C. About 10,000 cells were counted per
experiment and analyzed plotting the mean with SD of two independent experiments. Cells were
excited at 633 nm and fluorescence was detected using a LP750 filter using Accuri C6 flow
cytometer (BD).
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Results and Discussion
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Synthesis and characterization
Initially the dendritic synthons like azido glycerol, glucose azide, alkyne, etc., were synthesized by
the already reported procedures using easily available biocompatible materials such as glycerol,
glucose, and phloroglucinol.23-26 The dendrimers were synthesized by following a divergent
approach using click and Williamson synthesis reactions (Schemes 1-4). All the synthesized
dendrimers were characterized by IR, 1H NMR, and 13C NMR, and the final dendrimers were also
characterized by their HRMS analysis. The characteristic peak of the three triazolyl protons (H-5'')
of dendrimer 3 appeared at δ 8.17 ppm as a singlet and the peak for the six methylene protons (H-
1') attached to the triazole moiety appeared as a singlet at δ 5.03 ppm (Figure S3, ESI). Similarly,
for dendrimer 4, the respective peaks for the triazolyl ring (H-5'') and the methylene protons (H-1')
appeared at δ 8.02 and δ 4.81 ppm, respectively (Figure S4, ESI). For the second-generation
dendrimer 6, the two singlets at δ 7.96 and 8.23 ppm for the six and three protons, respectively,
were from the external (H-c) and internal (H-5'') triazolyl ring, respectively (Figure S9, ESI). In
the case of dendrimer 7, both the triazolyl ring protons (H-5'' & H-c) appeared as a multiplet in
the range δ 7.81-7.80 ppm (Figure S10, ESI). For the modified G1 dendrimer 13, the peak at δ
8.00 ppm accounts for the triazole protons (H-5''') that appeared at δ 7.93 ppm for dendrimer 14.
Similarly, second-generation modified dendrimers 16 and 17 were characterized by their NMR and
mass spectra. The final deprotection of all the acetyl-protected dendrimers was also confirmed by the
disappearance of the ester carbonyl (-C=O) peak at ≈1740 cm-1 in the IR spectra.
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HO
HO
N
OH
N
N
5
6
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8
1'''
OH
5''
OAc OAc
O
O
N
N
4''
1'
2
1
2'''
N
3''
2''
O
iii
N3
3a
9
3
HO
OH
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N
N
d
OH
O
c
N
OH
f
e
i
b
ii
HO
a
O
OH
OH
HO
OH
O
O
OAc
N
2''
2
1
5
O
N
AcO
''
1'
N
3''
N3
AcO
4''
OAc
iii
4a
O
1
2
HO
N
N
N
O
O
OH
HO
HO
O
4
N
N
N
OH
O
OH
OH
HO
Scheme 1. Synthesis of first-generation (G1) dendrimers 3 and 4: (i) propargyl bromide, K2CO3, DMF;
(ii) CuSO4.5H2O, sodium ascorbate, THF:H2O (3:1), 45 oC; (iii) 1N NaOH in methanol, Dowex 50WX8.
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c
b
a
OAc OAc
N3
O
2'''
O
1'''
iii
2''
N
6
6a
N
5''
N
3''
4''
1'
O
1
i
ii
3
2
O
OAc
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O
O
N
O
O
N
N
N
AcO
AcO
N
N
N3
OAc
iii
7
7a
5
O
O
HO
HO
OH
OHOH
g
f
OH OH
O
e
HO
d
i
d
e
h
HO
OH
O
N
N
N
N
N
N
c
N
N
c
HO
HO
b
N
b
a
N
a
N
N
O
O
O
1'''
N
2'''
2'''
O
1'''
5''
2''
N
N
2''
N
5''
4''
1'
OH
N
3''
N
3''
4''
1'
OH
OH
O
1
O
1
N
HO
HO
N
N
2
O
N
O
N
2
O
N
O
O
OH
O
N
N
N
O
N
N
O
N
N
N
N
N
O
OH
OH
N
N
N
HO
N
N
OH
OH
O
N
N
N
N
N
O
N
O
O
O
OH
O
N
7
6
HO
OH
N
N
N
N
N
N
HO
HO
HO
HO
N
N
OH
O
OH
OH
HO
OH
OH
Scheme 2. Synthesis of second-generation (G2) dendrimers 6 and 7: (i) propargyl bromide, NaOH, TBAI,
o
H2O; (ii) CuSO4.5H2O, sodium ascorbate, THF:H2O (3:1), 45 C; (iii) 1N NaOH in methanol, Dowex
50WX8.
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HO
b
HO
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N
N
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N
5'''
4'''
1''
O
1'
5
OH
6'
2
O
O
O
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N
N
1
5
OH
N
OAc OAc
N3
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O
HO
OH
5
iii
d
c
O
13a
O
OH
f
e
b
N
13
HO
a
5
OH
N
O
O
O
N
O
i
5
N
ii
5'''
2'''
N
HO
OH
OH
OH
N
O
3'''
4'''
5
OAc
1''
O
O
O
6'
AcO
AcO
1
N3
12
5
OAc
iii
O
14a
1
2
HO
O
1'
N
N
O
O
O
HO
HO
N
5
5
O
OH
N
N
N
14
OH
O
OH
OH
HO
Scheme 3. Synthesis of alkylated first-generation (alkylated G1) dendrimers 13 and 14: (i) 1-
bromo-6-(prop-2-yn-1-yloxy)hexane (10), K2CO3, DMF; (ii) CuSO4.5H2O, sodium ascorbate,
THF:H2O (3:1), 45 oC; (iii) 1N NaOH in methanol, Dowex 50WX8.
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OAc OAc
O
O
O
iii
O
N3
O
16
16a
O
5
O
N
N
O
N
N
8
9
N
O
O
5
N
ii
O
5
O
OAc
i
13
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12
13
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O
O
AcO
AcO
N3
OAc
15
iii
17
17a
O
N
N
N
HO
OH
OH
OH
OH
O
O
5
OH
HO
HO
O
O
HO
OH
O
HO
O
OH
N
N
N
N
N
N
N
N
N
N
N
N
5
O
5
O
O
O
OH
OH
5
O
O
O
HO
HO
N
O
O
5
N
N
O
O
N
N
N
N
N
N
5
N
O
N
N
N
N
6'
O
5
O
O
N
O
2
5
5
O
1'
O
n
1
m
HO
l
OH
OH
o
N
O
N
N
i
1'
O
O
p
q
OH
O
5
1
6'
O
k
2
j
5
O
1''
4'''
O
OH
c
N
O
5
1''
4'''
N
N
5
b
5'''
O
3'''
N
N
N
O
HO
HO
N
N
O
N
2'''
a
O
N
3''' N
2'''
5
OH
5'''
N
a
O
O
5
O
b
O
c
5
O
O
5
N
N
HO
O
N
O
N
N
i
N
O
N
j
OH
OH
N
N
N
N
k
N
O
l
m
HO
OH
HO
OH
HO
16
17
OH
HO
HO
HO
Scheme 4. Synthesis of alkylated second-generation (alkylated G2) dendrimers 16 and 17: (i) 1-
bromo-6-(prop-2-yn-1-yloxy)hexane, NaOH, TBAI, H2O; (ii) CuSO4.5H2O, sodium ascorbate,
THF:H2O (3:1), 45 oC; (iii) 1N NaOH in methanol, Dowex 50WX8.
CAC measurement
The CAC of an aqueous solution of the self-assembled dendrimers (13, 14, 16, and 17) was determined
using Nile red as a fluorescent probe,27 wherein a fixed amount of dye was used for encapsulation in
different concentrations of dendrimers. The dye exhibited low fluorescence intensity in the absence of
the supramolecular structure and an increase observed at a specific concentration, which indicated its
encapsulation within the self-assembled nanostructures. The intensity maxima (λmax) of encapsulated
Nile red plotted versus log[dendrimer] was used to calculate the CAC value of the dendrimers. The
dendrimers 3, 4, 6, and 7 that lack a flexible C6 alkyl chain were found to exhibit an irregular
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pattern for Nile red encapsulation, hence their CAC analysis could not be satisfactorily performed.
However, the dendritic systems bearing C6 alkyl chain displayed systematic encapsulation
behavior, with CAC values observed to be in the order of 10-5 M (Figure 2 & Table S1, ESI). A
comparison of the CAC values of the modified G2 dendrimers (16 and 17) with their respective G1
analogs (13 and 14) revealed that the increase in dendrimer generation resulted in lower CAC
values. This might be attributed to higher hydrophobic and hydrophilic contents in the higher
generation dendrimers, facilitating them to aggregate at low concentration. Figure 1 shows the
proposed schematic representation of aggregation phenomenon in aqueous solution. Zhang et al.
also investigated the similar type of self-aggregation of amphiphilic dendrimers into various
morphologies including vesicular structures in aqueous medium by varying the alkyl chain length
on periphery.28
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13
14
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Figure 2. CAC measurements of (A) dendrimers 13 & 14 and (B) dendrimers 16 & 17.
DLS and TEM analysis
The aggregation phenomenon of the synthesized dendrimers (3, 4, 6, 7, 13, 14, 16, and 17) was
studied in aqueous medium by DLS measurements at a concentration of 3 mg/mL (Figure 3 &
Table S1, ESI). The dendrimers 3, 4, 6, and 7 that lack flexible alkyl chains were found to exhibit
an irregular pattern in DLS, hence their size could not be measured. The dendrimers 14, 16, and 17
exhibited size of 7-8 nm indicating defined micellar aggregates, however, the dendrimer 13
aggregated into larger sized particles of 0.7 µm.
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Figure 3. DLS size distribution plots (A) by intensity distribution and (B) by volume distribution, of
dendrimers 13, 14, 16, and 17.
According to literature reports, smaller nanoparticles in the range of 8-20 nm not only can easily
penetrate the deep regions of interstitial tissue but also circumvent the rapid renal clearance.29 Thus
the size of the self-assembled dendritic systems 14, 16, and 17 appear to be quite appropriate for
the transport of drugs. However, the dendrimers 3, 4, 6, and 7 did not exhibit the formation of
stable and particular sized aggregates as evidenced by DLS study. This might be the consequence
of a poor hydrophilic-lipophilic balance.30
In order to further investigate the morphology of the micellar aggregates, a TEM study was carried
out for two selected dendrimers 14 and 17 (Figure 4). The results obtained from TEM were found
to be consistent with that obtained from DLS with slight variation in size. In TEM study, different
sized particles can be seen with size in the range of 5 to 15 nm for the dendrimers 14 and 17, while
in DLS an average size of around 8 nm was observed for both.
Figure 4. TEM images of (A) dendrimer 14 and (B) dendrimer 17, showing spherical micellar assemblies (black
circles in both images indicate the size of around 15 nm and white circles indicate the size of around 5 nm).
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Transport potential of dendrimers for Nile red and curcumin
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Nile red, a fluorescent hydrophobic probe that is used for the recognition of the neutral lipid
deposit within the cells,31 was used to assess the transport behavior of the synthesized dendrimers.
The applicability of these new dendritic systems as nanocarriers was explored by performing
drug/dye solubilization experiments. The encapsulation potential of the dendrimers 13, 14, 16, and
17 was determined for two exemplary hydrophobic entities, the dye Nile red and the biologically
potent molecule curcumin. The fluorescence spectra of Nile red and curcumin loaded samples in
methanol are shown in Figure 5 while UV absorbance spectra for the same are shown in Figures
S23 and S24, ESI. The C6 alkylated G2 dendrimer 17 bearing glucosyl moiety at the periphery
showed the highest encapsulation of Nile red, with a transport efficiency of 4.56 mg/g as shown in
Figure 6 and Table 1. On the other hand, 1 g of dendrimer 13/14/16 was able to transport
0.59/1.32/3.93 mg of Nile red, respectively. The non-alkylated dendrimers (3, 4, 6, and 7) do not
exhibit any noticeable absorbance for Nile red in UV-Vis spectra up to the measured concentration.
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Figure 5. Fluorescence spectra in methanol for (A) Nile red and (B) curcumin loaded samples.
Curcumin, a hydrophobic drug, used as a dietary supplement in Southeast Asia and having
versatile biological and pharmacological activities such as anticancer, antioxidant, antimicrobial,
anti-parasitic, etc., but it has not been used clinically because of its poor aqueous solubility and low
permeability across the blood-brain barrier which cause poor oral and brain bioavailability.32-34
Nanocarriers can help to overcome these hurdles by making it water dispersible. Therefore, the
synthesized dendrimers (13, 14, 16, and 17) were used to investigate the encapsulation potential
using curcumin. Among the studied dendrimers, the second-generation modified dendrimers 16
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and 17 exhibited significant potential for the successful transportation of curcumin. On the other
hand, negligible curcumin encapsulation was observed for the C6 alkylated G1 dendrimers 13 and
14. The low encapsulation of curcumin by the dendrimers 13 and 14 might be due to the difficulty
of rigid and larger sized curcumin to fit in the small cavity/voids formed by these G1 dendrimers.
The transport efficiency of the dendrimers 16 and 17 for curcumin was found to be 8.78 and 9.0
mg/g, respectively (Figure 6 and Table 1). Jansen et al.35-38 studied the cavity size-based
encapsulation of hydrophobic molecules into dendrimers. They reported that large guest molecules
(Rose Bengal) were encapsulated in the larger interior cavities, while the smaller ones (p-
nitrobenzoic acid) were transported to the smaller outer cavity of the G5-PPI dendrimer.
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Figure 6. (A) Transport efficiency and (B) transport capacity of dendrimers 13, 14, 16, and 17 for Nile red
and curcumin.
Table 1. Transport behavior of dendrimer for the hydrophobic molecules Nile red and curcumin.
Transport Behavior
Dendrimer
Nile red/Polymer
Curcumin/Polymer
mg/g mmol/mol
mg/g
mmol/mol
1.65
13
14
16
17
0.59
1.32
3.93
4.56
-
-
4.78
-
-
29.9
8.78
9.0
57.84
72.19
42.6
Biological characterization
As the compounds showed only very low toxicity in A549 cells (Figure S26, ESI), live cell
imaging by confocal laser scanning microscopy (cLSM) as well as flow cytometry was used to
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analyze the uptake profile of Nile red loaded into dendrimers using adenocarcinomic human
alveolar basal epithelial (A549) cells.
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In a first approach, the uptake of two selected compounds 14 and 17 in A549 lung cancer cells was
analyzed by cLSM. Microscopy images revealed Nile red uptake in the cells and accumulation
over time (Figure S27, Video S1 and S2, ESI). In the cell, Nile red was located not only in
endocytotic compartments (lysosomes and early endosomes) but also in the cytoplasm around the
nucleus. No Nile red was seen in the nucleus. The results indicated a non-endocytotic continuous
uptake of the encapsulated Nile red as described earlier by Snipstad et al.39 To confirm passive
diffusion as a most relevant uptake mechanism for Nile red loaded in dendrimers, we further
analyzed compound 14 and compared it to free Nile red using time-lapse microscopy combined
with advanced image analysis and flow cytometry at 4 °C and 37 °C. Because active uptake, such
as endocytosis is energy driven, the cellular uptake should be decreased at 4 °C. The flow
cytometry data (Figure 7) revealed the passive diffusion mechanism as there was hardly any
difference between the Nile red uptake at 4 °C and 37 °C. Time-lapse analysis combined with
quantitative image analysis and the flow cytometry data further proved that the dendrimer was
responsible for the efficient uptake of Nile red.
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In summary, we assume that Nile red enters the cell by contact-mediated transfer as described
earlier by Snipstad et al.39 in which the carrier transports the guest to the target site (cells) by
interaction with the cells. By this uptake mechanism, the hydrophobic drugs avoid the uptake via
an endocytotic pathway and get direct access to intracellular targets.
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Figure 7. Uptake profile of compound 14 loaded with Nile red (14/NR) compared to free Nile red (NR)
using A549 cells. (A) cLSM images from living A549 cells incubated for 30 min with 14/NR or free NR;
(B) the resulting time dependent uptake profile and (C-D) the quantitative uptake profiles; (C) based on the
analysis of cLSM images and (D) based on flow cytometry measurements. In the images, Nile red is shown
in red color.
Conclusions
We have prepared a series of dendritic architectures of different generations using biocompatible
starting materials, i.e., D-glucose and glycerol. All of the dendritic architectures were characterized
on the basis of their physical and spectral data. Of all the synthesized dendritic architectures, the C6
alkyl chain containing dendrimers formed micellar aggregates in aqueous solution, with a CAC in
the order of 10-5 M. DLS and TEM measurements confirmed that the dendrimers 14 and 17 formed
stable and uniformly distributed micelles in the range of 5-15 nm. Furthermore, the modified G2
dendrimers having a larger hydrophobic cavity displayed the potential to solubilize curcumin and
Nile red in water. The uptake study revealed efficient internalization of encapsulated Nile red
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inside the cancer cells, ascertaining the potential of the dendritic micelles to deliver the
hydrophobic drug/dye by contact-mediated transfer inside the cells. The cytotoxicity profile
obtained from MTS assay unraveled that the synthesized dendrimers exhibited toxicity only at
higher concentrations, thus indicating their potential for drug delivery.
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Acknowledgements: Financial assistance from DST-DFG collaborative research grant is
gratefully acknowledged. Also, we are thankful to the Council of Scientific and Industrial Research
(CSIR), New Delhi, for providing Junior/Senior Research Fellowships to BP, PY, and AM. We
would like to acknowledge the assistance of the Core Facility BioSupraMol supported by the DFG.
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Dendrimer-based Micelles as Cyto-compatible Nanocarriers
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Badri Parshad,a,b Preeti Yadav,a Yannic Kerkhoff,b Ayushi Mittal,a Katharina Achazi,b,* Rainer
Haag,b Sunil K. Sharma,a,*
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aDepartment of Chemistry, University of Delhi, Delhi 110 007, India
bInstitut für Chemie und Biochemie, Freie Universität Berlin, Takustraße 3, 14195 Berlin, Germany
Graphical Abstract
The aim of present study is to compare the synthesized dendritic architectures for self-assembly
and transport potential for hydrophobic guest molecules.