stimuli responsive size control of hyperbranched polymers
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1080
Stimuli Responsive Size Control ofHyperbranched Polymers
Indra Bohm, Helmut Ritter*
The synthesis of stimuli–responsive hyperbranched polymers is reported. Through polymer-analogous amidation of polyethylenimine (PEI) with 5-hexynoic acid and 10-undecynoic acidtwo polymers were obtained, with different spacer lengths between the hyperbranched PEI-and the triple bond. In a further step, these triple bonds were clicked with mono-(6-azido-6-desoxy)-b-cyclodextrin (CD-N3). The resulting polymers PEI-(CH2)3-CD and PEI-(CH2)8-CD showstimuli–responsive contractions and expansions, which was observed by use of dynamic lightscattering (DLS). These size effects depend on the formation of inter- and intramolecularaggregates due to the different spacer length between the triazol and the CD. Furthermore, thesize of the polymer with the longer spacer (PEI-(CH2)8-CD) could be influenced with adamantylcarboxylate through host–guest-complexation resulting in a reassemble of the aggregates.Moreover, the pH dependent size control was studied by adding sodium hydroxide. Thedeprotonation of the ammonium groups from the PEI scaffold leads to a further decrease in thehydrodynamic diameters of PEI-(CH2)3-CD and PEI-(CH2)8-CD. Thus, the different polymersystems exhibit a wide range (4–170 nm)of mean size. Furthermore, successful com-plexation experiments of the obtainedpolymer with phenolphthalein were car-ried out.
Introduction
In recent years, hyperbranched polymers have become an
attractive field of research due to their special character-
istics. Among them, hyperbranched polyethylenimine (PEI)
is technical available. As a special example, PEI is employed
in gene and drug delivery systems because of their high
biocompatibility.[1] The linear version of PEI is synthesized
from oxazolines, while the corresponding hyperbranched
structure is easily accessible through ring opening poly-
merization of aziridine. This hyperbranched PEI offers a
H. Ritter, I. BohmInstitute of Organic Chemistry and Macromolecular Chemistry II,Heinrich-Heine-University of Duesseldorf, Universitaetsstrasse 1,D-40225 Duesseldorf, GermanyFax: þ49 211 81 15840; E-mail: [email protected]
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great variety of chemical modifications. That allows for
example an efficient functionalization with cyclodextrin
(CD) which is in focus of the present work. Modified CDs
have recently attracted considerable attention as gene
delivery vectors due to their excellent biocompatibility and
uniquemolecular architecture.[2] CD is well known to form
inclusion complexes with various organic molecules of
appropriate size and hydrophobic segments.[3] In this
context, we recently described host–guest interactions
and supramolecular complexes in aqueous solution.[4]
Some results about modifying PEI with 6-monotosyl-b-
cyclodextrin has been recently reported.[5] However, these
structures do not contain any spacer groups between PEI
and CD. Spacer groups allow a more flexible behavior for
better host–guest complex stabilities. The inclusion of
spacers via click chemistry is one of the aims of the present
work. The original click criteria by Sharpless and co-
library.com DOI: 10.1002/macp.201100006
Stimuli Responsive Size Control of Hyperbranched Polymers
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workers[6] has been re-defined for polymer specific
chemistry.[7] The microwave-assisted copper-catalyzed
cycloaddition allows a fast, complete, and regioselective
conversion under equimolar conditions and large-scale
purification.[8] The CuI-cycloaddition of azides and alkynes
has already been utilized successfully to functionalize
surfaces[9] and to synthesize dendritic polymers.[10]
In this paper, we describe the synthesis of multivalent
polymers consisting of covalently attached CDand PEI. This
work also focuses on pH-induced contraction and expan-
sion of the modified PEI.
Experimental Section
Materials
All reagents used were commercially available and were used
without further purification. Cyclodextrin (b-CD) were obtained
fromWacker-ChemieGmbH,Burghausen,Germany,andwereused
after drying overnight in vacuum oil pump over P4O10. 2-Propynyl
2-methacrylate (98%) and 10-undecynoic acid (96%) were pur-
chased from Alfa Aesar GmbH & CoKG, Germany. Sodium azide
(99%) and hyperbranched polyethylenimine (100%, Mn ¼10.000)
(PEI) were obtained from Aldrich Chemicals, Germany.
Copper(II)sulfate pentahydrate (99%) was purchased from Carl
Roth GmbH & Co., sodium L(þ)-ascorbate (99%) from AppliChem,
Germany. N,N-dimethylformamide (DMF) from Fluka, Germany,
dimethyl-d6 sulfoxide (99.9 atom% D and DMSO) from Deutero
GmbH, Germany and 5-hexynoic acid (95%) was obtained from
Wako Chemicals, Germany.
Measurements
FT-IR (Fourier transform infrared) spectra were recorded with a
Nicolet 5 SXB spectrometer equipped with an ATR unit. The
measurements were performed in the range of 4 000–300 cm�1 at
room temperature. 1H NMR and 13C NMR were performed using a
Bruker Advance DRX 500 spectrometer at 500.13MHz for proton
and 125.77MHz for carbon in DMSO-d6 as solvent. The d scale
relative to tetramethylsilane (TMS) was calibrated to the solvent
value d¼2.51ppm for DMSO-d6. Matrix-assisted laser desorption/
ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) was
performed on a Bruker Ultraflex TOF mass spectrometer. Ions
formed with a pulsed nitrogen laser (25Hz, 337nm) were
accelerated to 25 kV, the molecular masses being recorded in
linear mode. 2,5-Dihydroxybenzoic acid (DBH) in acetonitrile/
water (25mg �mL�1) was used as a matrix. The samples
(1mg �mL�1 in water) were mixed with the matrix solution at
volumetric ratios of 1:2. Dynamic light scattering (DLS) experi-
ments were carried out with a Malvern Nano ZS ZEN 3600 in a
temperature range from 25 to 45 8C. The particle size distribution
was derived by deconvolution of the measured intensity auto-
correlation function of the sample by the NNLS general purpose
mode algorithm included in the DTS software. Each experiment
was performed at least five times to obtain statistical information.
Microwave-assisted synthesis was performed using a CEM Dis-
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cover Synthesis Unit (monomode system). The temperature was
measured by infrared detection with continuous feedback
temperature control andmaintained at a constant value by power
modulation. Reactions were performed in closed vessels under
controlledpressure aswell as in standardopenvessels under reflux
conditions. UV/VIS spectra were recorded in water using the UV
540 system of the company Unicam. Elemental analysis was
carried outwith a Perkin–Elmer Analyzer 2400with an accuracy of
measurement of �0.3%.
Synthesis of the Monomers
Mono-(6-O-(p-tolylsulfonyl))-b-cyclodextrin (2) and Mono-(6-
azido-6-desoxy)-b-cyclodextrin (3) were synthesized according to
the known procedure.[3c]
Synthesis of the Polymers
Synthesis of the Polymer 7a
For the present study, commercially available hyperbranched PEI
(Mw ¼10.000 g �mol�1) was employed. The polymer-analogous
amidation with 5-hexynoic acid was implemented under micro-
wave conditions. A solutionof 5-hexynoic acid (5a) (1 g, 0.89mmol)
andPEI (4) (1.12 g) in8mLDMFwasfilled inapressure-resistant test
tube. The tube was sealed and placed in the CEM monomode
microwave and irradiated at 85 8C and 60W for 30min. The
following reaction was implemented, without isolation of the
product6a, by adding sodiumascorbate (36mg, 0.02mmol), copper
(II) sulfate pentahydrate (14mg, 0.01mmol) and 3 (2.12 g,
0.18mmol) to the solution. The tube was sealed and placed again
in theCEMmonomodemicrowave and irradiated at 85 8Cand60W
for 60min. The product 7awas precipitatedwith acetone (100mL)
and dialyzed (MWCO 3500) to separate the polymer 7a from
catalysts and monomers. The product was freeze-dried to afford
92% of pure product as a brownish powder.
UV (water): lmax (e)¼300nm (1200 L �mol�1 � cm�1). 1H NMR
(DMSO-d6): d¼0.93 (3H, C CH), 1.13–1.27 (12H,�CH2�), 1.35–1.41
(4H, �CH2�), 1.46 (8H, �CH2�), 1.62–1.65 (4H, �CH2�), 1.78–1.81
(8H,�CH2�), 1.35–1.41 (4H,�CH2�), 2.05–2.08 (16H,�CH2�), 2.10–
2.17 (16H,�CH2�), 2.22–2.25 (8H,�CH2�), 2.33–2.35 (8H,�CH2�),
2.68 (2H, �CH2�), 2.84 (2H, �CH2�), 2.78 (12H, �CH2�), 2.80 (4H,
�CH2�), 2.93 (12H,�CH2�), 3.34 (14H,H-2, 4), 3.56–3.63 (Br, 28H,H-
3, 5, 6), 4.49 (Br, 6H, OH-6), 4.82 (d, 7H, H-1), 5.76 (14H, OH-2, 3), 7.79
(1H, �CH�), 8.02 (1H, �NH). IR: 3 304 (OH), 2 927 (CH), 2 825 (CH),
1 651 (Amide I), 1 546 (Amide II), 1 401 (OH), 1 151 (CN), 1 079 (OH),
1 028 (CH), 939, 853, 753 (NH), 703 cm�1 (CH). Elemental analysis:
Calcd. C 51, H 8, N 6, O 35; Found C 50.32, H 7.84, N 10.93, O 30.91.
Synthesis of the Polymer 7b
The synthesis of product 7b follows the same route as polymer 7a.
The difference between these two polymers is the length of the
alkyl chain. To obtain polymer 7b, we modified 4 with 10-
undecynoic acid (5b). Yield: 91%.1H NMR (DMSO-d6): d¼1.23 (16H, �CH2�), 1.45 (4H, �CH2�),
1.56 (4H, �CH2�), 2.11–2.15 (4H, �CH2�), 2.30 (2H, �CH2�), 2.35
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Figure 1. Proposed structure of polymer 7a.
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I. Bohm, H. Ritter
(2H,�CH2�), 2.76–2.84 (4H,�CH2�), 2.93–2.98 (4H,�CH2�), 3.38–
3.42 (14H, H-2, 4), 3.49–3.76 (Br, 28H, H-3, 5, 6), 4.43 (Br, 6H, OH-6),
4.82 (d, 7H, H-1), 5.72 (14H, OH-2, 3), 7.74 (1H, �CH�), 7.99 (2H,
�NH). IR: 3 290 (OH), 2 923 (CH), 2 850 (CH), 1 650 (Amide I), 1 554
(Amide II), 1 387 (OH), 1 151 (CN), 1 079 (OH), 1 027 (CH), 944, 853,
753 (NH),703 cm�1 (CH). Elementalanalysis:Calcd.C52,H8,N11,O
29; Found C 46.31, H 7.58, N 10.14, O 35.97.
Figure 2. UV–Vis spectra of the unmodified PEI 4 (- -) and 7a (-)with lmax (e)¼ 300 nm.
Results and Discussion
Synthetic Route to Prepare the CD Modified PEI
Characterization of the hyperbranched PEI (4) was further
accomplished by inverse gated 13C NMR spectroscopy
which showed a 1:2.5:1.3 ratio of primary, secondary, and
tertiaryamineunits. Longetal. suggestedavariation for the
calculation of the DB:[12]
DB ¼ Dþ Tð Þ= Dþ T þ Lð Þ
Where D is dendric units, T is terminal units, and L is linear
units. In comparison to a linear polymer with no branches
and 100% of a perfect dendrimer our polymer shows an
intermediate branching of about 48%.
Macromol. Chem. Phys. 2011, 212, 1080–1085
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In thispaper,hyperbranched
PEI (4) was modified with
5-hexynoic acid (5a) under
MW-irradiation to obtain 6a.
After that amidation reaction,
the alkyne functionality of 6a
was used to covalently attach
mono-(6-azido-6-desoxy)-b-
cyclodextrin (CD-N3,3) via click
chemistry.[11]
The obtained product 7a
(Figure 1) was characterized
by elemental analysis,1H NMR, IR, and UV–Vis spec-
troscopy to confirm the pro-
posed structures. The chemical
shift of the triazolic proton, for
example, can be observed at
7.79 ppm. The CD modification
of PEI was studied in more
detail and quantitatively by1H NMR. Hence, around 4% of
the ethyl units are clicked to
one CD.
The completion of the
cycloaddition-reaction was also
proven by the absence of the
specific alkyne vibration at
2110 cm�1 in IR spectra.
Furthermore the UV–Vis spec-
trum of 7a shows a maximum absorption at lmax (e)¼300nm whereas the unmodified PEI does not show any
absorption (Figure 2). By elemental analysis the ratio of
0.6 mmol �CD/g PEI was calculated. It becomes obvious
that only the primary amines are functionalized.
Accordingly, IR spectroscopy showed absorption bands at
1 651 (Amide I) and 1 546 (Amide II).
Polymer 7b was synthesized analogously. The polymer-
analogous amidation of 4 was accomplished with
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Scheme 1. Synthetic pathway leading to polymer 7a and 7b through microwave accelerated click-reaction.
Stimuli Responsive Size Control of Hyperbranched Polymers
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10-undecynoic acid (5b) (Scheme 1) in this case. The
successful reaction was proven by 1H NMR and IR. The
ratio of CD is 0.6 mmol � g�1 PEI, calculated by elemental
analysis.
Size Controllable Behavior Studied by Dynamic LightScattering (DLS)
DLS measurements were employed to investigate the
hydrodynamic diameter (Dh) of 7a (C3H6-spacer) and 7b
(C8H16-spacer). Product7a showsahydrodynamic diameter
of 8nm as expected for a single molecule. Surprisingly,
polymer 7b exhibits a significantly higher diameter of
Figure 3. Comparison of Dh of polymer 7a and 7b (Cp ¼0.5 g � L�1,in H2O, 25 8C, pH 7).
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170nm (Figure 3). This indicates intermolecular aggrega-
tion due to a higher hydrophobicity through the relatively
long spacer groups. The addition of adamantyl carboxylate
(8) to 7a does not influence the Dh of the polymer
significantly. In contrast, the inclusion of 8 into the
moieties of 7b induces a remarkable shift in Dh from
170 to 6nm (Figure 4). This can be ascribed to a
disaggregation of the CDs by the repellent forces of the
negatively charged adamantyl carboxylates (8).
Furthermore, the pH dependent size control of 7a was
studied because the amino groups, such as NHþ3 of the PEI
scaffold, cause cationic repulsive forces in a pH range of
acidic to neutral. The proof of the existence of these ionic
Figure 4. Comparison of Dh of polymer 7b and the inclusioncomplex of 8 into the moieties of 7b (Cp ¼0.5 g � L�1,in H2O, 25 8C, pH 7).
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Figure 5. Comparison of Dh of polymer 7a and the solution of 7awith sodium hydroxide (Cp ¼0.5 g � L�1, in H2O, 25 8C, pH 7and pH 14).
Figure 6. Comparison of Dh of polymer 7b and the solution of 7bwith sodium hydroxide (Cp¼0.5 g � L�1, in H2O, 25 8C, pH 7 and pH14).
Figure 7. A solution (in buffer pH 9) of phenolphthalein (a), acomplex formation by adding 7a (b) and decomplexation throughthe competitive guest 8 (c).
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I. Bohm, H. Ritter
effects can be carried out by adding sodium hydroxide
causingadecrease inDh (Figure5). This reductionof7a from
8 to 6nm is clearly due to the neutralization of the
ammoniumgroups to amineswhile repulsive forces can no
longer occur. Furthermore, a strong decrease inDh of 7b can
be induced changing the pH-value to the range of alkaline
(Figure 6). Therefore, after reversing the agglomeration and
the repulsive forces both polymers (7a and 7b) show the
same Dh.
Complexation with Phenolphthalein
The complexation process of the CDs, which are covalently
attached to the hyperbranched polymer 7a, could be
visualized utilizing a solution of phenolphthalein
(Figure 7).[13] In basic media, phenolphthalein exhibits its
characteristic pink color, caused by its planar configuration
and electron delocalization. The addition of 7a leads to a
complexation of phenolphthalein and the solution
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becomes colorless. The hydrogen bond formation is
accompanied with conformational change as the planar
state of phenolphthalein is reversed which causes decolor-
ization. A simple color change due to a pH change can be
excluded since the solution remains alkaline. Accordingly,
the addition of8 to the complex of7awithphenolphthalein
displaces the included phenolphthalein and the solution
regains its characteristic pink color.
Conclusion
In this paper, we described modified hyperbranched PEI
which contains covalently attached b-CD via a spacer
group. The polymer PEI-(CH2)8-CD with a spacer of eight
CH2-groups aggregates, as a result of intermolecular
interactions which lead to a significant size expansion. In
contrast, the polymer PEI-(CH2)3-CD does not aggregate
since the bond of the CD component to the hyperbranched
system is relatively stiff. Through the addition of adaman-
tyl carboxylate theaggregatesofPEI-(CH2)8-CDdisassemble
due to the carboxylates repellent forces, thus the hydro-
dynamic diameter decreases. By adding adamantyl
carboxylate to PEI-(CH2)3-CD no further contraction can
beobserved.However, adecrease inDh is inducedbyadding
sodium hydroxide to PEI-(CH2)3-CD, thus the size of the
polymer is controllable through pH changes.
Furthermore successful complexation experiments with
phenolphthalein could be visualized inclosing phe-
nolphthalein into the cavity of the CDs.
These obtained polymers PEI-(CH2)3-CD and PEI-(CH2)8-
CD may find potential application, e.g., in drug and gene
delivery systems due to their complexation and decom-
plexation capabilities as well as their controllable contrac-
tion and expansion.
Received: January 3, 2011; Revised: February 17, 2011; Publishedonline: April 5, 2011; DOI: 10.1002/macp.201100006
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Stimuli Responsive Size Control of Hyperbranched Polymers
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Keywords: b-cyclodextrin (CD); click-chemistry; controllable coilsize; hyperbranched; self-assembly
[1] A. V. Kabanov, P. L. Felgner, L. W. Seymour, Self-AssemblingComplexes for Gene Delivery: From Laboratory to ClinicalTrial, John Wiley and Sons, New York 1998.
[2] [2a] X. Lu, Y. Ping, F. J. XubZ, H. Li, Q. Q. Wang, J. H. Chen, W. T.Yang, G. P. Tang, Bioconjugate Chem. 2010, 21, 1855; [2b] M. L.Forrest, N. Gabrielson, D.W. Pack, Biotechnol. Bioeng. 2005, 89,416.
[3] [3a] N. Micali, V. Villari, A. Mazzaglia, L. Mons’u Scolaro,A. Valerio, A. Rencurosi, L. Lay, Nanotechnology 2006, 17,3239; [3b] R. Haag, Angew. Chem. Int. Ed. 2004, 43, 278;[3c] S. W. Choi, H. Ritter, J. Macromol. Sci. A 2004, 42, 321;[3d] H. Ritter, O. Sadowski, E. Tepper, Angew. Chem. Int. Ed.2003, 42, 3171; Corrigendum:H. Ritter, O. Sadowski, E. Tepper,Angew. Chem. Int. Ed. 2005, 44, 6099.
[4] [4a] S. Schmitz, H. Ritter, Angew. Chem. Int. Ed. 2005, 44, 5658;[4b] O. Kretschmann, S. W. Choi, M. Miyauchi, I. Tomatsu,A. Harada, H. Ritter, Angew. Chem. Int. Ed. 2006, 45, 4361;[4c] C. Koopmans, H. Ritter, Macromolecules 2008, 41, 7418.
[5] S. H. Pun, N. C. Bellocq, A. Liu, G. Jensen, T. Machemer,E. Quijano, T. Schluep, S. Wen, H. Engler, J. Heidel, M. E. Davis,Bioconjugate Chem. 2004, 15, 831.
[6] [6a] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001,113, 2056; [6b] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew.Chem. Int. Ed. 2001, 40, 2004.
www.MaterialsViews.com
Macromol. Chem. Phys. 20
� 2011 WILEY-VCH Verlag Gmb
[7] C. Barner-Kowollik, F. E. Du Prez, P. Espeel, C. J. Hawker,T. Junkers, H. Schlaad, W. Van Camp, Angew. Chem. Int. Ed.2011, 50, 60.
[8] [8a] S. W. Choi, M. Munteanu, H. Ritter, J. Polym. Res. 2009, 16,389; [8b] P. Eckstein, H. Ritter, Design. Monomer Polym. 2005,8, 601; [8c] S. Sinnwell, H. Ritter, Macromol. Rapid Commun.2005, 26, 160; [8d] S. Sinnwell, A. M. Schmidt, H. Ritter, J.Macromol. Sci. Pure Appl. Chem. 2006, A43, 469; [8e] M. Klink,U. Kolb, H. Ritter, e-Polymers 2005, 69, 1. [8f] F. Wiesbrock,R. Hoogenboom, U. S. Schubert, Macromol. Rapid. Commun.2004, 25, 739; [8g] M. Iannelli, H. Ritter, Macromol. Chem.Phys. 2005, 206, 349; [8h] C. Goretzki, A. Krlej, C. Steffens,H. Ritter, Macromol. Rapid. Commun. 2004, 25, 513;[8i] M. Iannelli, V. Alupei, H. Ritter, Tetrahedron 2005, 61,1509.
[9] [9a] M. Munteanu, S. W. Choi, H. Ritter,Macromolecules 2008,41, 9619; [9b] F. Fazio, M. C. Bryan, O. Blixt, J. C. Paulson, C. H.Wong, J. Am. Chem. Soc. 2002, 124, 14397; [9c] M. C. Bryan,F. Fazio, H. K. Lee, C. Y. Huang, A. Chang, M. D. Best, D. A.Calarese, O. Blixt, J. C. Paulson, D. Burton, I. A. Wilson, C. H.Wong, J. Am. Chem. Soc. 2004, 126, 8640.
[10] [10a] J. P. Collman, N. K. Devaraj, C. E. D. Chidsey, Langmuir2004, 20, 1051; [10b] P. Wu, A. K. Feldman, A. K. Nugent, C. J.Hawker, A. Scheel, B. Voit, J. Pyun, J. M. J. Frechet, K. B.Sharpless, V. V. Fokin, Angew. Chem. Int. Ed. 2004, 43,3928; [10c] B. Helms, J. L. Mynar, C. J. Hawker, J. M. J. Frechet,J. Am. Chem. Soc. 2004, 126, 15020.
[11] K. Ohga, Y. Takashima, H. Takahashi, Y. Kawaguchi,H. Yamaguchi, A. Harada, Macromolecules 2005, 38, 5897.
[12] C. J. Hawker, R. Lee, J. M. J. Frechet, J. Am. Chem. Soc. 1991, 113,4583.
[13] T. Trellenkamp, H. Ritter, Macromolecules 2010, 43, 5538.
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