Synthesis of non-ionic bolaamphiphiles and study of their self-assembly and transport behaviour for drug delivery applications

Article abstract of DOI:10.1039/c8ra05921g

A series of four bolaamphiphiles with different hydrophilic units has been synthesised. All the amphiphiles were well characterised from their physiochemical data. The aggregation tendency of newly synthesised amphiphiles was studied using fluorescence spectroscopy, dynamic light scattering (DLS), and cryogenic electron microscopy (cryo-TEM). Furthermore, their application as nanocarriers for hydrophobic guests was demonstrated by using two established standards, i.e. the dye Nile red and the drug nimodipine. A cytotoxicity and cellular uptake study has been carried out using A549 cells. Due to the presence of an ester linkage in PEG based bolaamphiphiles, a drug release study was performed in the presence of an immobilized enzyme Novozym-435 (a lipase).

Full text of DOI:10.1039/c8ra05921g

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PAPER
Synthesis of non-ionic bolaamphiphiles and study
of their self-assembly and transport behaviour for
drug delivery applications†
Cite this: RSC Adv., 2018, 8, 31777
a
a
b
c
Rashmi, Abhishek K. Singh, Katharina Achazi, Boris Schade,
c
b
a
Christoph Bottcher, Rainer Haag and Sunil K. Sharma
*
¨
A series of four bolaamphiphiles with different hydrophilic units has been synthesised. All the amphiphiles
were well characterised from their physiochemical data. The aggregation tendency of newly synthesised
amphiphiles was studied using fluorescence spectroscopy, dynamic light scattering (DLS), and cryogenic
electron microscopy (cryo-TEM). Furthermore, their application as nanocarriers for hydrophobic guests
was demonstrated by using two established standards, i.e. the dye Nile red and the drug nimodipine. A
cytotoxicity and cellular uptake study has been carried out using A549 cells. Due to the presence of an
ester linkage in PEG based bolaamphiphiles, a drug release study was performed in the presence of an
immobilized enzyme Novozym-435 (a lipase).
Received 11th July 2018
Accepted 27th August 2018
DOI: 10.1039/c8ra05921g
rsc.li/rsc-advances
that can be used as nano-carriers for encapsulation and release of
drug/dyes in a systematic manner. Among various amphiphilic
Introduction
Inspired by nature, the self-assembly of amphiphilic molecules architectures studied, bolaamphiphiles with two hydrophilic end
mimics various biological systems depending on their structure, groups separated by a hydrophobic spacer, exhibit unique hierar-
concentration, solution environment, and temperature. The chically self-assembled structures from micelles to large cylin-
spontaneous organization of sophisticated molecules like lipids, drical, vesical and monolayer formation at air–water interface.
proteins, saccharides and nucleic acids provides vital information Furthermore, compared to the conventional surfactants, bolaam-
to understand the factors responsible for their self-assembly. phiphiles have higher aqueous solubility and reduced critical
19,20
Various non-covalent interactions like hydrogen bonding, hydro- aggregation concentration.
Although a number of ionic and
phobic interactions, p–p stacking, etc. provide the momentum for non-ionic bolaamphiphiles constituted from carbohydrates and
self-aggregation. In recent years, this concept has largely been used amino acids have been reported for various pharmaceutical
1–8
21–27
for various applications in biomedical and material sciences. As applications,
however the bolaamphiphiles based on poly-
many drugs suffer from limited aqueous-solubility and shorter ethylene glycol (PEG) and dendritic polyglycerol (dPG) are not very
half-life in the bloodstream, resulting in their limited bioavail- common besides being biocompatible, nontoxic, stable, and
ability, the supramolecular nano-carriers have shown promising aqueous soluble. Furthermore, PEG and PG are known to evade
behaviour in the target specic drug delivery. For the enhanced recognition by the reticulo-endothelial system (RES), which in turn
28–35
therapeutic action of such drugs, these are encapsulated in the qualify them for use in drug delivery applications.
hydrophobic core of the amphiphiles and the hydrophilic groups
Herein we report the synthesis of four different pegylated
surfaced the assembly to keep the guest intact from aqueous and dendronized symmetrical bolaamphiphiles by coupling
9–18
medium and solubilize the whole molecule.
For the past together, a novel hydrophobic aryl–alkyne conjugate with
decades the amphiphilic architectures have earned considerable different hydrophilic PEG/dPG azides by following the CuAAC
attention to such delivery systems. To explore the usefulness of methodologies (Fig. 1). The synthesized bolaamphiphiles were
such systems, new amphiphilic architectures have been designed characterized using NMR, IR, HRMS and GPC. The aggregation
behaviour of amphiphiles was studied using DLS and cryo-TEM.
a
Their drug encapsulation potential and release prole was
studied using UV and uorescence spectroscopy.
Department of Chemistry, University of Delhi, Delhi 110 007, India. E-mail:
sksharma@chemistry.du.ac.in; Tel: +91-11-27666646
b
Institut f u¨ r Chemie und Biochemie, Freie Universit a¨ t Berlin, Takustraße 3, 14195
Berlin, Germany. E-mail: haag@zedat.fu-berlin.de; Fax: +49-30-838-452633; Tel:
Results and discussion
Synthesis and characterization
+
c
49-30-838-52633
Forschungszentrum f u¨ r Elektronenmikroskopie, Institut f u¨ r Chemie und Biochemie,
Freie Universit a¨ t Berlin, Fabeckstraße 36a, 14195 Berlin, Germany
The chemical and physical properties of amphiphilic systems
vary with respect to their hydrophobic and hydrophilic content.
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8ra05921g
1
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Critical aggregation concentration (CAC)
The CAC of bolaamphiphiles (10–13) was determined by uo-
rescence measurement using a hydrophobic dye Nile red. The
assembly was initially probed with Nile red by dissolving
respective bolaamphiphiles in water. The plot of uorescence
intensity of encapsulated Nile red versus log[amphiphile conc.]
gave the CAC value at the point of inection, and it was
À4
À5
observed to be in the range of 1.5 Â 10 to 9.2 Â 10 M (Table
38
1
). Employing the Griffin equation for calculating HLB
Hydrophilic–Lipophilic Balance) shows that in the pegylated
bolaamphiphiles Bola-PEG with low HLB exhibit higher CAC
value as compared to the Bola-PEG10 having an enhanced
Fig. 1 Polyethyleneglycol (PEG) and dendritic polyglycerol (dPGs)
based bolaamphiphiles.
(
6
To systematically explore the inuence of hydrophilic groups on hydrophilic character. A similar behaviour was observed with
aggregation behaviour, i.e. size, morphology, encapsulation and dendronized bolaamphiphiles i.e. Bola-[G2.0] having a higher
release prole of guests, etc., bolaamphiphiles with different HLB exhibit lower CAC as compared to Bola-[G1.0] (Fig. S16, see
polar head groups were synthesised by coupling together the ESI†).
hydrophobic and hydrophilic moiety using CuAAC methodolo-
gies. Four symmetrical bolaamphiphiles have been synthesised
Dynamic light scattering (DLS) and cryo-TEM measurement
by utilizing PEG (M : 350/550) and dendritic polyglycerol [G1.0/
n
The hydrodynamic size of the particles was determined by
G2.0] as hydrophilic moieties. The synthesis of hydrophilic
groups 6/7 involves acylation of azidodiol 5 with mono methoxy
dynamic light scattering (DLS) technique at a concentration of
À1
5
.0 mg mL . The particle size of the nanoparticles in the
n
PEG acid [(M : 350/550) 3/4 ], mPEG acids (3/4) and azidodiol
aqueous solution obtained from DLS was observed to be in the
range 4 to 7 nm (Fig. 2) for all the four bolaamphiphiles
synthesized. The size distribution was monomodal in volume
and number, but bimodal in intensity. In the case of bimodular
system, the two peaks were assigned for molecular aggregates
assemblies, whereas single peaks in volume and number
corresponds to simple molecular assembly in aqueous medium,
indicating that mainly small size particles are formed as
compared to larger molecular aggregates. Nanoparticles of up to
were in turn prepared by following literature procedure (Scheme
36
1
). G1.0 and G2.0 dendritic polyglycerol azides (dPGs azides)
37
were also synthesised by following literature procedure. For
the synthesis of the hydrophobic core, p-hydroxybenzoic acid
was rst converted quantitatively to its propargyl ester 8 by
using propargyl bromide in the presence of a mild base.
Compound 8 was then reacted with 1,10-dibromodecane on its
both ends to yield the desired diacetylinic product 9. The
hydrophobic alkyne and PEG/dPG azides were then subjected to
click reaction to obtain the target bolaamphiphiles (10–13)
2
00 nm size show prolonged delivery of therapeutic agents and
39
exhibit higher drug uptake compared to microparticles. The
nano sized aggregates formed by the bolaamphiphiles in this
study were found to be of appropriate size for studying their
biological applications. As the amphiphiles in aqueous medium
agglomerate, so the actual size and morphology cannot be
evaluated rather a hydrodynamic size is obtained from the DLS
study. For the precise measurement of particle size and to
visualize the actual morphology in aqueous medium cryo-TEM
technique was utilized for one of the representative amphiphile
Bola-[G1.0]. The cryo-TEM image for Bola-[G1.0] (Fig. 3) shows
small elongated aggregates with an average diameter of ꢀ3 nm,
which is in good agreement with the DLS data by considering
the fact that the hydrodynamic size is generally greater than the
(
Scheme 2). All the bolaamphiphiles synthesized were charac-
1
terized on the basis of their physical and spectral data i.e. H
and, C-NMR spectroscopy, HRMS and gel permeation chro-
matography (GPC).
1
3
40
visible assembly structure. Such smaller molecular aggregates
are of optimum size to be considered for therapeutic
applications.
Cytotoxicity study
Cytotoxicity of the bolaamphiphiles was assessed by an MTS
assay using A549 lung cancer cells. All of the amphiphiles
showed a similar trend i.e. low toxicity at the highest test
concentration of 1 mg mL (Fig. 4). However, at 0.1 mg mL ,
all the bolaamphiphiles were found to be non-cytotoxic and
thus suitable for biological applications.
ꢁ
Scheme 1 Synthesis of hydrophilic segments; (i) KMnO
4
, water, 80 C,
À1
À1
ꢁ
2
4 h; (ii) Novozym-435, vinyl acetate, THF, 37 C; (iii) mesylchloride,
ꢁ
triethylamine, DCM, 0–25 C, 2 h; (iv) NaN
3
, DMF, reflux, 12 h; (v) KOH,
ꢁ
EtOH, 12 h; (vi) EDC$HCl, DMAP, DCM, 35 C, 12 h.
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ꢁ
Scheme 2 Synthesis of target bolaamphiphiles; (i) 1,10-dibromodecane, K
2
CO
3
, 35 C, 15 h; (ii) compound 6/7, copper sulphate, sodium
ꢁ
ꢁ
ascorbate, THF : water (3 : 1), 50 C, 24 h; (iii) [G1.0] azide, copper sulphate, sodium ascorbate, THF : water, 50 C, 24 h; (iv) [G2.0] azide, copper
ꢁ
sulphate, sodium ascorbate, THF : water, 50 C, 24 h.
Table 1 Aggregation study of synthesized bolaamphiphiles
Bolaamphiphile HLB CAC (M) DLS (nm) Cryo-TEM (nm)
their absorbance by UV-vis spectroscopy. The transport capacity
of all the amphiphiles was calculated employing Beer–
Lambert's law and using a molar extinction coefficient of
À1
À1
À4
À4
À4
À5
45 000 M cm at 552 nm for the dye. In accordance with the
HLB values, the dendronized bolaamphiphiles constituted from
[G1.0] dendron (Bola-[G1.0]) exhibit higher encapsulation of
Bola-PEG
6
14.44 1.75 Â 10
15.85 1.50 Â 10
8.74 2.14 Â 10
7
6
5
4
—
—
2–3
—
Bola-PEG10
Bola-[G1.0]
Bola-[G2.0]
a
12.87
9.2 Â 10
À1
Nile red 5.68 mg g as compared to its analogue Bola-[G2.0].
a
Performed on one representative amphiphile.
Similarly, among the pegylated bolaamphiphiles, the one with
6
a higher lipophilic ratio, i.e. Bola-PEG encapsulated more
amount of Nile red as compared to Bola-PEG10 (Fig. 5).
Nimodipine encapsulation. Nimodipine, a potent calcium
channel blocker, is a hydrophobic drug with a limited aqueous
Encapsulation study
Nile red encapsulation. The impetus of self-organisation of
the non-ionic bolaamphiphiles in aqueous medium forming
a hydrophobic inner core renders the possibility to encapsulate
guest dye/drug in its hydrophobic core by non-covalent inter-
actions like p–p stacking, hydrogen bonding, hydrophobic
effects, etc. In the light of these effects, the encapsulation
potential of bolaamphiphiles was determined by employing the
À1
solubility of 3.86 mg mL , that renders low bioavailability of the
43
drug. To address this issue, Nimodipine was encapsulated in
the nanoaggregates fabricated from the bolaamphiphiles in
aqueous medium by following the thin lm method. The
quantication of nimodipine per gram of bolaamphiphile was
measured using UV spectrophotometer following Beer–
Lambert's law using an extinction coefficient of nimodipine as

lm method and taking Nile red as a model probe. Due to its
À1
À1
7
200 M cm at 365 nm in ethanol. Bola-PEG shows higher
6
limited aqueous solubility and high solvatochromic uores-
cence with strong emission in the lipophilic environment, Nile
red can be used to divulge useful information about the
encapsulation efficiency and site of encapsulation. Based on the
various literature reports and our own experience, 0.12 mg of
loading capacity than Bola-PEG , whereas in the case of
dendronized amphiphiles, Bola-[G2.0] shows higher loading
capacity of nimodipine as compared to Bola-[G1.0] (Fig. 5).
1
0
À1
Cellular uptake study
Nile red was encapsulated in 5 mg mL of amphiphiles to
avoid the possibility of aggregation of the dye and this results in Confocal laser scanning microscopy was used to analyse the
41,42
maximum encapsulation of the guest molecule.
The quan- cellular uptake behaviour of Nile red loaded bolaamphiphiles
tication of Nile red encapsulation was carried by lyophilisation Bola-PEG and Bola-[G1.0] to simulate drug transport by the
6
À1
of the dye encapsulated samples followed by the dissolution of amphiphiles. A test concentration of 0.1 mg mL
of the
contents in methanol (1 mL) with subsequent measurement of bolaamphiphiles was chosen considering the toxicity study
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Fig. 4 Cytotoxicity profile of the bolaamphiphiles after 24 h. Cyto-
toxicity of the amphiphiles was determined by an MTS assay using
A549 cells with 24 h post-treatment. Each bar represents the mean
value of triplicate study with SD.
Fig. 2 Plot of (a) intensity versus size; (b) volume versus size for
calculating hydrodynamic size (nm) of bolaamphiphiles obtained from
dynamic light scattering (DLS).
Fig. 5 Transport capacity of bolaamphiphiles.
Enzyme-triggered release of encapsulated guest
Besides encapsulation, the release of encapsulated therapeutic
entity is also signicantly important. We studied the enzyme
triggered release of the encapsulated guest from the inner
hydrophobic core of amphiphilic aggregates. Since the amphi-
philes Bola-PEG and Bola-PEG constitute from ester func-
6
10
tionality, we studied the enzyme mediated ester cleavage. Such
an act can be triggered in biological systems as a number of
hydrolytic enzymes are available over there.
Fig. 3 Cryo-TEM micrograph of Bola-[G1.0].
We performed the enzyme mediated release study using the
results. Both the amphiphiles exhibit a similar uptake pattern same hydrolytic enzyme that was used for the synthesis of
of Nile red inside the cell. Cellular uptake in the cell was seen bolaamphiphiles i.e. immobilized Candida antarctica lipase
even aer the rst hour of incubation (Fig. 6). Aer 24 h, the (Novozym-435). The release study was performed in dark and at
ꢁ
bolaamphiphiles were observed to be accumulated in the cells' 37 C. It was observed that within 5 h of enzyme exposure
cytosol. However, no Nile red was visible in the nucleus. The a complete release of encapsulated dye takes place, however the
results indicate that the amphiphiles are well suited to carry control experiment performed without the enzyme does not
drugs in the cytosol of cells.
exhibit any noticeable release of encapsulated dye (Fig. 7).
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amphiphile aggregation in aqueous medium. The size of the
aggregates was studied using DLS and cryo-TEM. Nile red as
a model dye and nimodipine as a model drug were used for
evaluating the encapsulation potential of amphiphiles.
6
Bola-PEG and Bola-[G1.0] exhibit a higher encapsulation
tendency than their larger pegylated and dendronized
analogues i.e. Bola-PEG10 and Bola-[G2.0] respectively. The
enzyme mediated release of the encapsulated dye was also
6
studied using Bola-PEG , which shows an efficient dye release
from the hydrophobic core of the amphiphile. Cell viability
study shows that all bolaamphiphiles are well tolerated upto
À1
a concentration of 0.5 mg mL . In conclusion the good
biocompatibility, the higher drug loading capacity and fast
enzymatic release shows that the synthesised bolaamphiphiles
can be used as a starting point to design and develop efficient
nanocarriers for drugs and dyes.
Conflicts of interest
There is no conicts of interest to be declare.
Fig. 6 Confocal laser scanning microscopy images of A549 cells after
1
hour (a–d and i–l) and 24 h (e–h and m–p) incubation with Nile red
encapsulated by bolaamphiphiles Bola-PEG
p). In the images, Nile red is shown in red color and the nucleus stained
with Hoechst 33 342 in blue. The bright field channel is shown in grey We gratefully acknowledge the DST, Government of India and
(a–h) and Bola-[G1.0] (i– Acknowledgements
6
scale. The scale bar equals 50 mm
DFG, Germany and the core facility BioSupraMol, FU-Berlin for
supporting a collaborative research project nancial assistance
and UGC-CSIR, New Delhi, for providing fellowship to Rashmi.
References
1
2
3
M. Ramanathan, L. K. Shrestha, T. Mori, Q. Ji, J. P. Hill and
K. Ariga, Phys. Chem. Chem. Phys., 2013, 15, 10580–10611.
A. Sorrenti, O. Illa and R. M. Ortun, Chem. Soc. Rev., 2013, 42,
8200–8219.
P. A. Albouy, S. Deville, A. Fulkar, K. Hakouk, M. Imperor-
Clerc, M. Klotz, Q. Liu, M. Marcellinib and J. Perezd, So
Matter, 2017, 13, 1759–1763.
4
5
6
7
8
9
P. Calandra, D. Caschera, V. T. Liveri and D. Lombardoc,
Colloids Surf., A, 2015, 484, 164–183.
M. Lee, B. K. Cho and W. C. Zin, Chem. Rev., 2001, 101, 3869–
3892.
J. A. A. W. Elemans, A. E. Rowan and R. J. M. Nolte, J. Mater.
Chem., 2003, 13, 2661–2670.
C. Wang, Z. Wang and X. Zang, Acc. Chem. Res., 2012, 45,
Fig. 7 Enzymatic mediated release of encapsulated dye Nile red from
Bola-PEG .
6
608–618.
Y. Tu, F. Peng, A. Adawy, Y. Men, L. K. E. A. Abdelmohsen
and D. A. Wilson, Chem. Rev., 2016, 116, 2023–2078.
R. Lin and H. Cui, Curr. Opin. Chem. Eng., 2015, 7, 75–83.
Conclusions
A set of four linear and dendronized bolaamphiphiles have
been synthesised in a chemo-enzymatic manner by employing
the immobilized enzyme Novozym 435 and following a CuAAC
methodologies. Furthermore, biocompatible materials like
polyethylene glycol (PEG) and polyglycerol dendrons (dPGs)
were used as building blocks. The bolaamphiphiles synthesised
were well characterized from their spectroscopic and physical
1
1
0 R. Haag, Angew. Chem., Int. Ed., 2004, 43, 278–282.
1 H. Chen, C. Khemtong, X. Yang, X. Cjang and J. Gao, Drug
Discovery Today, 2011, 16, 354–360.
1
2 P. Khadka, J. Ro, H. Kim, I. Kim, J. T. Kim, H. Kim, J. M. Cho,
G. Yun and J. Lee, Asian J. Pharm. Sci., 2014, 9, 304–316.
3 N. A. Kotov, Nanostruct. Mater., 1999, 12, 789–796.
4 S. Prasad, K. Achazi, C. Bottcher, R. Haag and S. K. Sharma,
RSC Adv., 2017, 7, 22121–22132.
1
1
1
13
data, e.g. H-NMR, C-NMR, HRMS, and GPC techniques.
Fabrication of nano-carrier has been achieved by allowing the
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 31777–31782 | 31781

View Article Online
RSC Advances
Paper
1
1
1
1
1
2
2
5 X. Hu, R. Liu, D. Zhang, J. Zhang, Z. Li and Y. Luan, ACS 30 T. M. Allen, C. Hansen, F. Martin, C. Redemann and A. Yau-
Biomater. Sci. Eng., 2018, 4, 973–980. Young, Biochim. Biophys. Acta, 1991, 1066, 29–36.
6 W. He, X. Hu, W. Jiang, R. Liu, D. Zhang, J. Zhang, Z. Li and 31 B. Obermeie, F. Wurm, C. Mangold and H. Frey, Angew.
Y. Luan, Adv. Healthcare Mater., 2017, 6, 1700829–1700842. Chem., Int. Ed., 2011, 50, 7988–7997.
7 J. Zhang, D. Zhang, X. Hu, R. Liu, Z. Lib and Y. Luan, RSC 32 S. Gupta, R. Tyagi, V. S. Parmar, S. K. Sharma and R. Haag,
Adv., 2018, 8, 13103–13111. Polymer, 2012, 53, 3053–3078.
8 P. Zhang, H. Zhang, W. He, D. Zhao, A. Song and Y. Luan, 33 B. N. S. Thota, L. H. Urner and R. Haag, Chem. Rev., 2016,
Biomacromolecules, 2016, 17, 1621–1632. 116, 2079–2102.
9 M. Fariya, A. Jain, V. Dhawan, S. Shah and 34 A. K. Singh, B. N. S. Thota, B. Schade, K. Achazi, A. Khan,
M. S. Nagarsenker, Adv. Pharm. Bull., 2014, 4, 483–491.
0 N. Nuraje, H. Bai and K. Su, Prog. Polym. Sci., 2013, 38, 302–
C. B ¨o ttcher, S. K. Sharma and R. Haag, Chem.–Asian J.,
2017, 12, 1796–1806.
3
43.
35 L. H. Urner, B. N. S. Thota, O. Nachtigall, S. Warnke,
G. Helden, R. Haag and K. Pagel, Chem. Commun., 2015,
51, 8801–8804.
36 S. Gupta, B. Schade, S. Kumar, C. B ¨o ttcher, S. K. Sharma and
R. Haag, Small, 2013, 9, 894–904.
1 S. Drescher, A. Meister, V. M. Garamus, G. Hause,
C. J. Garvey, B. Dobner and A. Blume, Eur. J. Lipid Sci.
Technol., 2014, 116, 1205–1216.
22 M. Popov, S. Grinberg, C. Linder, T. Waner, B. Levi-Hevroni,
R. J. Deckelbaum and E. Heldman, J. Controlled Release, 37 M. Wyszogrodzka and R. Haag, Chem.–Eur. J., 2008, 14,
012, 160, 306–314. 9202–9214.
3 Y. Jin, N. Qia, L. Tong and D. Chen, Int. J. Pharm., 2010, 386, 38 W. C. Griffin, J. Soc. Cosmet. Chem., 1949, 1, 311–326.
2
2
268–274.
39 R. Singh and J. W. Lillard Jr, Exp. Mol. Pathol., 2009, 86, 215–
2
2
4 C. Lee and S. Lee, RSC Adv., 2017, 7, 38989–38997.
223.
5 Z. Khan, S. A. Al-Thabaiti and M. A. Malik, RSC Adv., 2016, 6, 40 B. N. S. Thota, H. Berlepsch, C. B ¨o ttcher and R. Haag, Chem.
5993–46001. Commun., 2015, 51, 8648–8651.
6 A. M. Reddy, A. Sharma, Q. Maqbool and A. Srivastava, RSC 41 M. Kumari, S. Gupta, K. Achazi, C. B ¨o ttcher, J. Khandare,
4
2
2
2
2
Adv., 2013, 3, 18900–18907.
S. K. Sharma and R. Haag, Macromol. Rapid Commun.,
2015, 36, 254–261.
42 M. Kumari, M. Billamboz, E. Leonard, C. Len, C. B ¨o ttcher,
A. K. Prasad, R. Haag and S. K. Sharma, RSC Adv., 2015, 5,
48301–48310.
7 S. Schmid, D. Y. W. Ng, E. Mena-Osteritz, Y. Wu, T. Weil and
P. B ¨a uerle, Chem. Commun., 2016, 52, 3235–3238.
8 Y. Zhang, E. Mintzer and K. E. Uhrich, J. Colloid Interface Sci.,
2016, 482, 19–26.
9 K. J. Harrington, G. Rowlinson-Busza, K. N. Syrigos, 43 G. M. Soliman, R. Sharma, A. O. Choi, S. K. Varshney,
P. S. Uster, R. G. Vile and J. S. W. Stewar, Clin. Cancer Res.,
000, 6, 2528–2537.
F. M. Winnik, A. K. Kakkar and D. Maysinger, Biomaterials,
2010, 31, 8382–8392.
2
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