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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Macromol. Chem. Phys. 20

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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

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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


Scheme 1. Synthetic pathway leading to polymer 7a and 7b through microwave accelerated click-reaction.


www.mcp-journal.de

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).




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).

11, 212, 1080–1085


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



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

11, 212, 1080–1085

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Keywords: b-cyclodextrin (CD); click-chemistry; controllable coilsize; hyperbranched; self-assembly

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stimuli responsive size control of hyperbranched polymers

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