novel functional hyperbranched polyether polyols as prospective drug delivery systems

Novel Functional Hyperbranched Polyether

Polyols as Prospective Drug Delivery Systems

Leto-Aikaterini Tziveleka, Christina Kontoyianni, Zili Sideratou, Dimitris Tsiourvas, Constantinos M. Paleos*

Institute of Physical Chemistry, NCSR ‘‘Demokritos’’, 15310 Aghia Paraskevi, Attiki, GreeceFax: þ30-210-6529792; E-mail: [email protected]

Received: September 2, 2005; Accepted: November 11, 2005; DOI: 10.1002/mabi.200500181

Keywords: drug delivery systems; folate; hyperbranched; PEGylation; tamoxifen

Introduction

Functionalization of the surface groups of dendrimers[1] is a

very fruitful and convenient strategy for preparing novel

materials, including systems for drug delivery.[2] In this

connection, poly(ethylene glycol) (PEG) chains[3] have

been introduced at the external surface of dendrimers with

the aim of protecting the carrier from undesired attacks, as

has long been established with liposomes, and, therefore,

prolonging their circulation in biological milieu.[4] In

addition, cationic dendrimers have been employed as

gene-transfer agents,[5] due to the formation of DNA-

dendrimer complexes.

Analogous to this, but relatively limited, is the work

conducted into the functionalization of hyperbranched

polymers and, specifically, hyperbranched polyether poly-

ols.[6] Among the advantages exhibited by hyperbranched

polymers are their facile preparation and low polydispersity

in contrast to dendrimers which, although monodisperse,

are prepared under tedious synthetic routes. Additionally,

Summary: Multifunctional hyperbranched polyether poly-ols bearing protective poly(ethylene glycol) (PEG) chainswith or without the folate targeting ligand at their end havebeen prepared. Solubilization in these polymers of afluorescent probe, pyrene, and an anticancer drug, tamoxifen,was physicochemically investigated. It was found that PEGchains attached at the surface of these hyperbranchedpolymers, in addition to their well-established protective

role, enhance the encapsulation efficiency of the polymers.The release of pyrene and tamoxifen observed upon additionof sodium chloride is, in most of the cases, significant only atconcentrations exceeding the physiological extracellularconcentration. Thus, a significant amount of the probe ordrug remains solubilized inside the carriers, which is anencouraging result if the polymers are to be used for drugdelivery.

Schematic representation of the multifunctional hyperbranched polyetherpolyol bearing protective poly(ethylene glycol) (PEG) chains with folatetargeting ligand.

Macromol. Biosci. 2006, 6, 161–169 � 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper DOI: 10.1002/mabi.200500181 161

since structurally similar products, such as oligoglycerols,

present low toxicity,[7] hyperbranched polyether polyols are

most probably biocompatible. Low toxicity and biocom-

patibility of hyperbranched polyether polyols are promising

incentives for undertaking investigations to develop novel

functional derivatives based on these polymers that could be

employed as drug delivery systems.

The main objective of the functionalization of these pol-

yols is to simultaneously address the main issues encoun-

tered with drugs themselves, as well as with their carriers,

that is, water solubility, stability in biological milieu, and

targeting. For this purpose, one of the hyperbranched

polymeric derivatives prepared in this study bears on its

external surface a functional group combining protective

and targeting properties. Poly(ethylene glycol) chains

attached covalently at polyether polyol’s surface can

secure, as mentioned above, protection of the carrier from

undesired attacks in the biological media[3] and prolonged

circulation. Furthermore, introduction of the folate moiety

at the end of poly(ethylene glycol) chain can induce

endocytosis into folate-receptor-bearing cells.[8] The folate

receptor is known to be significantly overexpressed in a

wide variety of human cancers. Folate-mediated targeting

has already been applied with liposomes,[9] dendrimers,[10]

and various polymers and particles.[11] In addition, the

polyvalent effect[12] can be associated with the placement

of the folate group on the surface of these dendritic

molecules; this effect has a beneficial effect on bindingwith

the target.

In the present study, novel PEGylated and PEGylated-

folate functional hyperbranched polyether polyols have

been prepared and physicochemically characterized with

the prospect of being applied as drug delivery systems.

Specifically, functional polymers derived from hyper-

branched polyether polyols were prepared that bear at the

external surface poly(ethylene glycol) chains with or

without the folate group, as protective and targeting ligands,

respectively. For the investigation of the potential applica-

tions, experiments have been performed by employing the

well-known pyrene hydrophobic probe and also tamoxifen,

an anticancer hydrophobic drug, for the study of their

encapsulation and release properties.

Experimental Part

Materials

Hyperbranched polyether polyol (PG;Mn ¼ 5 000,Mw=Mn ¼1.5) was purchased from Hyperpolymers GmbH (Germany)and used after extensive drying under vacuum. The molecularweights of all other synthesized derivatives reported in thisstudy are based on thisMn by taking into account the degreeof functionalization of each derivative. This polyglycerol bears68 hydroxy groups according to the technical data sheet,which was also confirmed with IG 13C NMR experiments. Thepolyglycerol backbone consists[13] of linear (L), dendritic (D),

and terminal (T) units (Scheme 1). L13 and L14 describe the twodifferent glycerol-like units, in which either a primary/secondary or primary/primary ether linkage is formed,respectively. IG 13C NMR experiments allowed the estimationof the degree of branching, which was found to be 0.58, inaccordance with previously reported values for this type ofhyperbranched polyglycerols.[13b] Methoxy poly(ethyleneglycol)-amine (Mw ¼ 5 000, m-PEG-NH2) and poly(ethyleneglycol)-bis-amine (Mw ¼ 3 400, PEG-bis-NH2) were obtainedfrom Nektar (Huntsville, USA). Folic acid (FA) and succinicanhydride (SA) were purchased from Fluka.N-hydroxybenzo-triazole (HOBt) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium (HBTU) were purchased from Anaspec(San Jose, USA). Cellulose phosphate and tamoxifen (TAM)were obtained fromSigma. Dicyclohexylcarbodiimide (DCC),N,N-diisopropylethylamine (DIPEA), and pyrene (Py) werepurchased from Aldrich. Pyrene was purified by sublimationand recrystallization from ethanol.

Characterization

1H and 13C NMR spectra were recorded in D2O or DMSO-d6by employing a Bruker 500 spectrometer operating at 500 and125.1 MHz, respectively. 2D NMR experiments (COSY andHSQC)were also employed for the structural elucidation of thesynthesizedmaterials. FTIR studies were performed by using aNicolet Magna spectrometer at a resolution of 4 cm-1. For UV/Vis absorption spectroscopy a Helios a (ThermoSpectronic)spectrophotometer was employed. Fluorescence spectra wererecorded on a Perkin-Elmer LS-5B spectrophotometer.Dynamic light scattering (DLS) experiments were performedby employing an AXIOS-150/EX apparatus (Triton-Hellas),equipped with a 30 mW He-Ne laser source (658 nm) and anAvalanche photodiode detector at an angle of 908.

Synthesis

Succinate Functionalization of PG [PG-SA]

Polyether polyol (PG; 0.234 mmol) was treated with succinicanhydride (2.20 mmol) at 50 8C in the presence of triethyla-mine (TEA; 32.5 ml, 0.234 mmol), in anhydrous N,N-dimethylformamide (DMF; 19 ml; Scheme 1). After evapora-tion of the solvent, the residue was dissolved in distilled water,dialyzed by employing a 1 200 cut-off membrane, andlyophilized to afford compound PG-SA as a colorless paste(yield 74%).

IR (KBr): 1 732 (s, ester C O), 1 716 cm�1 (s, carboxylicacid C O).

1H NMR (D2O, 500 MHz): d¼ 5.25–4.95 (broad m,CHOCO, 1 H), 4.35–3.98 (broad m, CH2OCO, 2 H), 3.94(broad s, CHOH,L14), 3.82 (broad s, CHOH,T), 3.77–3.40 (m,CH, CH2 polyether backbone), 3.34 (broad m, CH3CH2

C(CH2O)3, 6 H), 2.63 (broad s, OCOCH2CH2COOH, 4 H),1.31 (broad s, CH3CH2C(CH2O)3, 2 H), 0.81 (broad s,CH3CH2C(CH2O)3, 3 H).

13C NMR (D2O, 125.1 MHz): d¼ 177.4 (COOH), 175.1(OCO), 80.5–79.6 (CH, L13), 79.2–78.0 (CH, D), 74.8–70.3(CH2CHOCO, T, CHOCO, L14, 2 CH2, 2 L14, CH2, T, 2 CH2,2 D, CHOH, T), 70.3–68.9 (CH2, L13, CHOH, L14), 68.9–64.0(CH2CH(OCO)CH2(OR), L14, CH2CH(OH)CH2OCO, T,

162 L.-A. Tziveleka, C. Kontoyianni, Z. Sideratou, D. Tsiourvas, C. M. Paleos

Macromol. Biosci. 2006, 6, 161–169 www.mbs-journal.de � 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CH(OR)CH2OCO, L13), 63.0 (CH2OH, T), 61.8–60.4 (CH2

OH, L13, OCOCHCH2OH, T), 43.6 (CH3CH2C(CH2O)3), 29.6(COOCH2CH2COOH), 22.5 (CH3CH2C(CH2O)3), 7.4 (CH3

CH2C(CH2O)3).

PEGylation of PG-SA [PG-PEG]

The previously prepared PG-SAwas treated with m-PEG-NH2

in dry DMSO and in the presence of DCC and pyridine(Scheme 1). Thus, DCC (0.105mmol) was used to activate PG-SA (0.017 mmol) dissolved in dry DMSO (3 ml) in thepresence of pyridine (8.3 ml, 0.103 mmol). Subsequently, m-PEG-NH2 (0.096 mmol) dissolved in dry DMSO (5 ml) wasadded. The mixture was allowed to react overnight, under anargon atmosphere at room temperature. After condensation ofthe reaction mixture, distilled water was added and centrifuga-tion was performed to remove dicyclohexylurea (DCU). Thesupernatant was dialyzed against a distilled water:methanolsolution (19:1 v/v)with a 12 400 cut-offmembrane. Finally, thesolution was lyophilized and dried under vacuum to affordcompound PG-PEG as a white solid (yield 22%).

IR (KBr): 1 649 (s, Amide I), 1 578 cm�1 (s, Amide II).1H NMR (D2O, 500 MHz): d¼ 5.25–4.95 (broad m,

CHOCO, 1 H), 4.31–3.98 (broad m, CH2OCO, 2 H), 3.98–3.39 (m, OCH2CH2O, CH, CH2 polyether backbone), 3.34(broad m, CH3CH2C(CH2O)3), 3.31 (s, CH3O, 3H), 3.14 (t,OCH2CH2NHCO, 2 H), 2.74–2.38 (m, OCOCH2CH2COOH,OCOCH2CH2CONH), 1.30 (broad s, CH3CH2C(CH2O)3,2 H), 0.81 (broad, s, CH3CH2C(CH2O)3, 3 H).

13C NMR (D2O, 125.1 MHz): d¼ 175.9 (COOH), 175.0(OCO), 171.7 (OCOCH2CH2CONH), 80.5–79.6 (CH, L13),79.5–77.8 (CH,D), 75.1–68.7 (CH2CHOCO,T,CHOCO,L14,2 CH2, 2 L14, CH2, T, 2 CH2, 2 D, CHOH, T, OCH2CH2, CH2,L13, CHOH, L14), 68.7–63.5 (CH2CH(OCO)CH2(OR), L14,CH2CH(OH)CH2OCO, T, CH(OR)CH2OCO, L13), 63.0 (CH2

OH, T), 61.9–60.5 (CH2OH, L13, OCOCHCH2OH, T), 58.5(OCH3), 43.6 (CH3CH2C(CH2O)3), 39.5 (OCH2CH2NHCO),32.6–28.5 (OCOCH2CH2CONH, OCOCH2CH2COOH), 22.5(CH3CH2C(CH2O)3), 7.4 (CH3CH2C(CH2O)3).

Multifunctionalization of PG [PG-PEG-Folate]

Preparation of Folate-PEG-NH2

This folate derivative was prepared by a method analogous tothe one described in the literature.[14] HOBt (0.387 mmol)dissolved in anhydrous DMF (2 ml) was added to PEG-bis-NH2 (0.294 mmol) dissolved in dry DMF (60 ml). Subse-quently, an equimolar quantity of HBTU (0.389 mmol) andfolic acid (0.385 mmol) dissolved in DMF were added to themixture; this was followed by the addition of DIPEA (0.672mmol). The mixture was allowed to react overnight, under anargon atmosphere. The volume of the solvent was reducedunder vacuum and the product was precipitated with diethylether. The crude product was dialyzed first against 5� 10�3

M

NaHCO3 (pH 9.0) and was then further dialyzed against waterand lyophilized. Subsequently, it was dissolved in 5� 10�3

M

phosphate buffer (pH 7.0) and subjected to column chromato-graphy, as previously described,[9a] with cellulose phosphatecation-exchange resin prewashed with an excess of 5� 10�3

M

Scheme 1. Functionalization steps leading to various polygly-cerol derivatives.

Novel Functional Hyperbranched Polyether Polyols as Prospective Drug Delivery Systems 163


phosphate buffer (pH 7.0) to remove the unreacted PEG-bis-NH2. After lyophilization and dialysis against water, theproduct Folate-PEG-NH2was obtained as a yellow solid (yield48%).

IR (KBr): 1 632 (s, Amide I), 1 510 cm�1 (w, Amide II).1H NMR (DMSO-d6, 500 MHz): d¼ 8.62 (s, H7, 1 H), 7.58

(d, J¼ 8.7, H13, H15, 2 H), 6.86 (t, J¼ 5.8 H10, 1 H), 6.63 (d,J¼ 8.7, H12, H16, 2 H), 4.46 (broad d, H9, 2 H), 4.18–4.25 (m,H19, 1H), 3.74–3.24 (broad, OCH2CH2O, OCH2CH2NH2,OCH2CH2NHCO), 3.18 (m, OCH2CH2NHCO), 2.77 (t,J¼ 5.5, OCH2CH2NH2), 2.39–2.13 (m, H22, 2 H), 1.98–1.72(m, H21, 2 H).

13C NMR (DMSO-d6, 125.1 MHz): d¼ 174.7 (CO), 173.8(CO), 165.0 (C17), 161.3 (C4), 156.3 (C2), 154.3 (C8a), 150.5(C11), 148.4, 148.2 (C7, C6), 128.4 (C13, C15), 127.9 (C4a),121.9 (C14), 111.2 (C12, C16), 69.7 (broad, OCH2CH2O,OCH2CH2NH2, OCH2CH2NHCO), 52.6 (C

19), 45.9 (C9), 39.8(OCH2CH2NH2), 38.0 (OCH2CH2NHCO), 32.7 (C22), 28.2(C21).

Multifunctionalization of HyperbranchedPolyglycerol [PG-PEG-Folate]

The preparation of the final PG-PEG-Folate derivative wasachieved by treating the Folate-PEG-NH2 with the succinic-modified polyether polyol, PG-SA, in dry DMSO and in thepresence of DCC and pyridine, as described above for PG-PEG. Thus, DCC (0.072 mmol) in dry solvent (1 ml) was usedto activate PG-SA (0.012 mmol) dissolved in dry DMSO(2 ml), in the presence of pyridine (5.8 ml, 0.072 mmol).Subsequently, Folate-PEG-NH2 (0.072 mmol) in dry solvent(1 ml) was added. The mixture was allowed to react overnight,under an argon atmosphere at room temperature. Afterevaporation of the solvent, distilled water was added to thesolid product; this was followed by centrifugation to removeDCU. The remaining material was dialyzed against a distilledwater:methanol solution (19:1 v/v) and lyophilized to affordPG-PEG-Folate as a pale yellow solid (yield 73%). Thedetermination of the folate content was also achieved byquantitative UV spectrometry of polymer solutions in phos-phate buffer (pH7.4): lmax (e)¼ 363 nm (6 564Lmol�1 cm�1).

IR (KBr): 1 633 (s, Amide I), 1 522 cm�1 (w, Amide II).1H NMR (D2O, 500 MHz): d¼ 8.70 (s, H7, 1 H), 7.61 (d,

H13, H15, 2 H), 6.74 (d, H12, H16, 2 H), 5.22–4.98 (broad m,CHOCO, 1 H), 4.55 (broad s, H9, 2 H), 4.34–3.23 (broad m,H19, CH2OCO, OCH2CH2O, OCH2CH2NHCO, CH, CH2

polyether backbone, CH3CH2C(CH2O)3), 3.15 (t, OCH2

CH2NHCO), 2.74–2.40 (m, OCOCH2CH2COOH, OCOCH2CH2CONH), 2.26 (t, H

22), 2.10–1.90 (m, H21), 1.31 (broad,CH3CH2C(CH2O)3), 0.81 (broad s, CH3CH2C(CH2O)3).

13C NMR (D2O, 125.1 MHz): d¼ 175.8 (CO), 174.8 (CO),173.0 (CO), 166.4 (C17), 154.2 (C8a), 151.0 (C11), 149.2 (C7,C6), 129.0 (C13, C15), 128.0 (C4a), 121.9 (C14), 112.0 (C12,C16), 80.6–79.8 (CH, L13), 79.2–78.0 (CH, D), 74.5–68.7(CH2CHOCO, T, CHOCO, L14, 2CH2, 2L14, CH2, T, 2CH2,2D, CHOH, T, OCH2CH2O, OCH2CH2NHCO CH2, L13,CHOH, L14), 68.9–63.6 (CH2CH(OCO)CH2(OR), L14,CH2CH(OH)CH2OCO, T, CH(OR)CH2OCO, L13), 63.2(CH2OH, T), 61.6–60.1 (CH2OH, L13, OCOCHCH2OH, T),50.1 (C19), 46.1 (C9), 39.3 (CH2NHCO), 32.1–28.0

(COOCH2CH2CONH, COOCH2CH2COOH, C22,C21), 22.4(CH3CH2C(CHO2)3), 7.9 (CH3CH2C(CH2O)3).

Encapsulation and Release of Pyrene and Tamoxifen

Pyrene and PG or PG-PEG were dissolved in methanol and,subsequently, the solventwas removed by distillation.Distilledwater was added to the dry film obtained, up to a final polymerconcentration of 1� 10�3

M. Pyrene was encapsulated bystirring overnight at room temperature (25 8C) and the non-solubilized Py was removed by centrifugation at 10 000 rpmfor 20 min. Effective removal of nonsolubilized pyrene wasevidenced byDLS experiments (see below). The concentrationof incorporated Py in the supernatant solution was determinedby UV absorption spectroscopy as previously described,[1k,1l]

with monitoring of the Py absorbance at lmax (e)¼ 334 nm(38 200 L mol�1 cm�1) after subtraction of the absorbance ofthe hyperbranched derivatives.

The same encapsulation procedure was followed for thehydrophobic drug tamoxifen in PG, PG-PEG, and PG-PEG-Folate. The concentration of incorporated TAM in the abovehyperbranched polymeric solutions was determined by UVabsorption spectroscopy, with monitoring of the TAMabsorbance at lmax (e)¼ 276 nm (10 820 L mol�1 cm�1) aftersubtraction of the absorbance of the hyperbranched polymericderivatives.

Release of the solubilized Py and TAM was conducted bytitrating the loaded hyperbranched polymeric derivatives withaqueous sodium chloride solution (5 M). After each addition ofNaCl solution, the suspension was centrifuged to remove theprecipitant. The encapsulation and release experiments wereconducted at least three times and the deviation of themeasurements was less than 5%.

Results and Discussion

Functionalization of HyperbranchedPolyether Polyol

The functionalization of hyperbranched polyether polyol,

PG, was achieved by employing experimental conditions

similar to those used for analogous compounds.[9a,13,14] Two

derivatives were prepared in which active drug ingredients

can, in principle, be encapsulated. In the first derivative,

poly(ethylene glycol) chains were introduced to afford PG-

PEG. As is well-established, PEG chains, in addition to

protecting the carrier and its encapsulated active ingredient

in the biological environment, can enhance the encapsulating

ability.[1l,3c] Subsequently, a second multifunctional deriva-

tive was prepared (PG-PEG-Folate) that combined both

targeting and protecting properties. The folate group was

placed at the end of the PEG chain and was, therefore,

accessible to exhibit its targeting properties. It should be

noted that in this case the PEG chain is not that derived from

m-PEG-NH2 (Mw ¼ 5 000) but that originated from PEG-

bis-NH2 (Mw ¼ 3 400) as described in the Experimental Part.

This may, in principle, affect the solubilization and release

properties of the folate-terminated derivative.



For preparing the two PG derivatives, a certain number of

the hydroxy groups of PG were interacted with succinic

anhydride, to afford PG-SA, as shown in Scheme 1. Ester

bond formation with both primary and secondary hydroxy

groups of PG was confirmed by the appearance in the 1H

NMR spectrum of characteristic broad signals at 4.95–

5.25 and 3.98–4.35 ppm, corresponding to the a-methine

and methylene protons of the ester bond, respectively, and

of one broad singlet at 2.63 ppm, attributed to the a- andb-methylene protons adjacent to the carboxy group.

Furthermore, the appearance of two signals in the 13C

NMR spectrum at 175.1 and 177.4 ppm confirmed the

presence of an ester group and a carboxylic acid group,

respectively. The degree of substitution was estimated from

the integral ratio in the 1H NMR spectrum of the signal at

2.63 ppm to the signal at 0.81 ppm, which corresponds to

the methyl group of the polyether polyol’s core moiety. The

average number of attached succinic acid moieties per

polymer was found to be 8.

Subsequently, partial PEGylation of PG-SA was per-

formed by interacting it with methoxy-terminated poly

(ethylene glycol)-amine, m-PEG-NH2, under analogous

experimental conditions to those previously reported.[9a]

The introduction of the PEG group was confirmed by 1H,13C, and 2D NMR (HSQC and COSY) experiments. The

appearance of a singlet at 3.33 ppm corresponding to the

terminal oxygenated methyl group of m-PEG-NH2, as well

as of a triplet at 3.16 ppm that corresponds to the a-CH2

protons relative to the newly formed amide bond (CON-

HCH2CH2O), along with the signal at 3.65 ppm corre-

sponding to the oxygenated methylene protons of poly

(ethylene glycol), confirmed the introduction of the PEG

moiety. The degree of substitution was estimated from the

integral ratio of the signal at 3.16 ppm, corresponding to

the poly(ethylene glycol) a-CH2 protons relative to the

amide bond, to the signal at 0.81 ppm, which corresponds to

the methyl group of the polyether polyol’s core moiety. The

average number of PEG moieties per polymer was 2.

PEGylation of PG-SA was also established by 13C NMR

experiments. The formation of the amide bond was

confirmed by the appearance in the 13C NMR spectrum of

a new signal at 171.7 ppm. Furthermore, a prominent signal

at 70.0 ppm appeared, which corresponds to the oxygenated

methylene groups of the poly(ethylene glycol) chain.

Additional signals appeared at 58.5 ppm, corresponding

to the oxygenated terminal methyl group of the poly

(ethylene glycol), and at 39.5 ppm, corresponding to the

a-CH2 group of the poly(ethylene glycol) next to the newly

formed amide bond.

For the preparation of the multifunctional PG derivative,

PG-PEG-Folate, a multistep process was used. Initially, the

Folate-PEG-NH2 intermediate was prepared by a method

analogous to that previously described.[14] The presence of

the folate group was confirmed by its characteristic 1H

NMR signals at 8.62 ppm, corresponding to the methine

group at position 7 of the pterin ring, and the two doublets

at 7.58 and 6.63 ppm, corresponding to the aromatic

ring.[15] The amide bond formation was confirmed by the

presence in the 1H NMR spectrum of a signal at 3.18 ppm,

which corresponds to the a-CH2 protons relative to the

newly formed amide bond (CONHCH2CH2O). Addition-

ally, in the 13C NMR spectrum, new signals appeared at

174.7 and 173.8 ppm, which were assigned to the unreacted

carboxylic acid group of the folate and to the amide

group, respectively, and at 38.0 ppm, which corresponded

with the a-CH2 relative to the amide bond. The presence

of only two peaks at 28.2 and 32.7 ppm, attributed to C21

and C22 of the folate moiety, respectively, suggests that

amide bond formation occurred in only one of the two

carboxylates, namely, the g-COOH which is the most

reactive.[10a]

The final multifunctional derivative was obtained by

treating PG-SAwith Folate-PEG-NH2. The structure of the

compoundwas confirmed by 1H and 13CNMR experiments

recorded in D2O. The presence of the Folate-PEG in the

final product was confirmed by the characteristic 1H NMR

signals of the folate moiety at 8.70, 7.61, and 6.74 ppm,

along with the signal at 3.63 ppm corresponding to the

poly(ethylene glycol) oxygenated methylene groups.

The formation of the amide bond was confirmed by the

appearance, in the 1H NMR spectrum, of a broad triplet at

3.15 ppm, which corresponds to the a-CH2 protons relative

to the newly formed amide bond (CONHCH2CH2O). Also

in the 13C NMR spectrum, a new signal appeared at 39.3

ppm, which corresponds to the a-CH2 relative to the amide

bond. The average number of folate groups per conjugate

was estimated, as exemplified above, from the integral

ratio of the signal at 8.70 ppm relative to the signal at

0.81 ppm and was found to be 3. This was in line with

the results from UV spectroscopy in phosphate buffer

(pH 7.4).

Encapsulation of Pyrene and Tamoxifen

Pyrene, a well-known hydrophobic probe, was first emplo-

yed for evaluating the loading capacity and release

properties of the parent hyperbranched polyether polyol,

PG, and its PEGylated derivative, PG-PEG. It was found

that PG-PEG encapsulated higher concentrations of Py

compared to the parent PG (Table 1). Specifically, the

solubility of Py in water (8� 10�7M) increased by a factor

of 2 in the parent PG and by a factor of 7 in the PEGylated

derivative. The observed increase in Py solubility may be

attributed to the presence of the poly(ethylene glycol)

moieties on the periphery of PG, which apparently

solubilize pyrene in addition to that incorporated in

the core. This is in linewith previous results[1l,3c] suggesting

that the presence of the poly(ethylene glycol) chains

enhances the solubilization efficiency of dendritic

polymers.



Based on these promising results on Py solubilization,

the loading capacity and release properties of the polyether

polyol derivatives for the hydrophobic anticancer drug

tamoxifen were also investigated. Tamoxifen is a non-

steroidal antiestrogen drug that is widely used in the

treatment and prevention of breast cancer.[16] It is clinically

administered in its protonated form, since its free-base form

is highly hydrophobic and is therefore likely to accumulate

in membrane lipid bilayers.[17] It would, therefore, be

interesting to prepare and investigate novel carriers that

would favor the encapsulation of the drug. For this purpose,

the encapsulation and release of TAM was comparatively

investigated for the parent hyperbranched polyether polyol,

PG, the PEGylated PG-PEG derivative, and the multifunc-

tional PG-PEG-Folate derivative.

The solubility of TAM in water was found to be

1.9� 10�6M. However, this value increases by a factor of

5 when TAM is solubilized in PG solution (Table 1). The

solubility of TAM is considerably further enhanced by a

factor of �12 in the presence of PG-PEG. This solubility

increase indicates that TAM is not only solubilized inside

the hyperbranched interior but also inside the covalently

bound poly(ethylene glycol) chains. This is in line with

previous results employing PEGylated dendrimeric deriv-

atives[1l,3c,18] from which it was established that the

introduction of the poly(ethylene glycol) chains in general

enhances the solubilization efficiency of dendritic poly-

mers. Finally, TAM was encapsulated in PG-PEG-Folate

bearing the folate targeting ligand at the end of the chain. It

is interesting to note that, in this case, an even greater�20-

fold increment of TAM solubility was observed compared

to that in PG-PEG.

With regard to the size of the PEGylated derivatives

dissolved in water, dynamic light scattering experiments

were performed. According to these studies, all the

polymeric carriers, that is, the PG and PEGylated

derivatives, both loaded and unloaded, show an extremely

weak scattering intensity, which is approximately equal to

the scattering intensity of water. This is clear evidence that

aggregates, that is, intermolecular micelles, were not

formed. This is not surprising since both the polyglycerol

scaffold and the PEG chains have almost the same structural

features and polarity and, therefore, segregation of these

segments cannot occur to justify the formation of

intermolecular micelles.

Release of Encapsulated Compounds

The use of increasing concentrations of NaCl for studying

the release of the encapsulated compounds was undertaken

for the following reasons: a) this salt is present in relatively

high concentrations (0.142 M) in extracellular fluids,[19] and

b) the sodium cation, as has been established in independent

studies[20] and as indicated by the structural features of

these carriers, can cationize the poly(ethylene glycol)

moieties of the carriers, thereby leading to the formation

of complexes. In this manner, the solubilized drug is

replaced by the metal ion and, therefore, release of the

drug can occur. It is thus necessary to investigate whether

sodium complexation can cause premature release of the

drug in the extracellular fluids, before the carrier reaches the

target cell.

The release of encapsulated active ingredients and the

way that it is triggered are the most important parameters

for the application of hyperbranched derivatives as drug

delivery systems. Effective release of the active ingredient

from the carrier when it reaches the target site enhances its

bioavailability. These requirements should generally be

taken into consideration in the design of novel drug delivery

systems, including those based on dendrimers or hyper-

branched polymers, in which various methods for inducing

drug release can be envisaged. Thus, diverse methods have

been reported for triggering drug release,[21] including pH-

or salt-triggered, enzymatic, thermal, or photochemically

induced processes. In this study, as mentioned, increasing

quantities of aqueous sodium chloride solution were used

for triggering pyrene and tamoxifen release.

The titration of the pyrene-loaded PG or PG-PEG

aqueous solutions with sodium chloride solution resulted

in the gradual release of the encapsulated compound as a

suspension in the bulk aqueous phase. By centrifugation of

the suspension after each NaCl solution addition, the

precipitate was collected; UV and 1H NMR spectroscopy

proved it to be pure pyrene. The amount of solubilized Py

inside the polymers in the supernatant solution was

determined by UV spectroscopy. The release of Py was

calculated after the subtraction of its solubility in water

(8� 10�7M).When PGwas used as the carrier, it was found

that at 0.142 M NaCl, that is, at the sodium cation

concentration in extracellular fluids,[19] 35% of the

solubilized Py was released, as shown in Figure 1A. For

PG-PEG, in which a higher solubilization of pyrene was

achieved compared to the parent hyperbranched polymer, a

smaller concentration of Py was released at the same

concentration of NaCl. Thus, when the concentration of

NaCl was 0.142 M, 13% of the solubilized Py was released,

as shown in Figure 1B. As will be discussed further below,

this is an encouraging result for when the polymer is used

for drug delivery, since at a sodium chloride concentration

equal to that of extracellular fluids, the greatest percentage

of the probe remains encapsulated inside the carrier.

Table 1. Solubility of pyrene and tamoxifen in PG, PG-PEG, andPG-PEG-Folate aqueous solutions.

Hyperbranchedpolymer

CPolymer CPyrene CTamoxifen

M M M

PG 1.0� 10�3 1.6� 10�6 9.6� 10�6

PG-PEG 1.0� 10�3 5.8� 10�6 1.23� 10�4

PG-PEG-Folate 1.0� 10�3 – 2.48� 10�3



Based on the previous results with pyrene, sodium

chloride was also employed for inducing TAM release. By

titrating polymeric solutions loadedwith TAMwith sodium

chloride solution, the drug was released and suspended in

the bulk aqueous phase. As in the case of pyrene, after

centrifugation, the isolated solid material proved to be

TAM, as established by 1H NMR and UV spectroscopy. By

monitoring the TAM concentration in the supernatant

solution, the release properties of PG, PG-PEG, and PG-

PEG-Folate were investigated. The release of TAM was

also calculated after the subtraction of its solubility in water

(1.9� 10�6M).

A gradual decrease of the TAMconcentration solubilized

in the solution of the hyperbranched polymeric derivatives

was observed as the amount of NaCl solution increased

(Figure 2). In the presence of 0.142 M NaCl, 40 and 25% of

the solubilized TAM in PG and PG-PEG, respectively

(Figure 2A and 2B), was released in to the aqueous media.

Under the same conditions and in the presence of PG-PEG-

Folate, only 6% of the solubilized TAM was released

(Figure 2C). It should, therefore, be noted that for the most

elaborated derivative prepared in this study, that is, the

multifunctional PG-PEG-Folate, most of the TAM remains

encapsulated in the polymer and it is not released in the

extracellular fluid at a concentration of 0.142 M NaCl.

Therefore, the carrier can reach target cells while still

appreciably loaded with TAM.

These results have to be considered before PEGylated

polyglycerols are to be applied as drug delivery systems in

experiments in vitro and in vivo. Sodium chloride in

extracellular fluids can form complexes with PEG chains,

thereby affecting the overall release profile of the drug. It is

Figure 1. Pyrene concentration in PG (A) and PG-PEG (B)aqueous solutions (1� 10�3

M) as a function of added NaClsolution.

Figure 2. Tamoxifen concentration in PG (A), PG-PEG (B), andPG-PEG-Folate (C) aqueous solutions (1� 10�3

M) as a functionof added NaCl solution.



therefore necessary, for designed PEGylated drug delivery

systems, to investigate whether premature drug release in

the extracellular fluid occurs.

Conclusion

Functional hyperbranched polymers based on a commer-

cially available polyether polyol and bearing either

protective PEG chains or PEG chains with folate targeting

ligands at their end were prepared. For a physicochemical

study of the encapsulation and release properties of the

system, pyrene and tamoxifen were employed. It was found

that PEG chains, in addition to their well-established

function as protective coating for drugs and their carriers,

enhance the encapsulation efficiency and control the release

of these compounds. In addition, it was established that Py

and TAM release is salt triggered and is enhanced as the

concentration of NaCl in the medium increases.

Acknowledgements: The work was partially supported byPAVET-NE-2004, 04BEN4, funded by GSRT, Greek Ministry ofDevelopment, Greece.

[1] [1a] G. R. Newkome, C. N. Moorefield, F. Vogtle,‘‘Dendrimers and Dendrons. Concepts, Syntheses, Perspec-tives’’, Wiley-VCH, Weinheim 2001, and references citedtherein; [1b] J. M. J. Frechet, D. A. Tomalia, ‘‘Dendrimersand Other Dendritic Polymers’’, in:Wiley Series in PolymerScience, 1st edition, J. Wiley & Sons, New York 2001, andreferences cited therein; [1c] A. W. Bosman, H. M. Janssen,E.W.Meijer,Chem.Rev. 1999, 99, 1665; [1d]A.D. Schluter,J. P. Rabe, Angew. Chem. Int. Ed. 2000, 39, 864; [1e] Top.Curr. Chem. 1998, 197; [1f]Top.Curr. Chem. 2000,210; [1g]Top. Curr. Chem. 2001, 212; [1h] Top. Curr. Chem. 2001,217; [1i] N. Ardoin, D. Astruc, Bull. Soc. Chim. Fr. 1995,132, 875; [1j] F. W. Zeng, S. C. Zimmerman, Chem. Rev.1997, 97, 1681; [1k] Z. Sideratou, D. Tsiourvas, C. M.Paleos, Langmuir 2000, 16, 1766; [1l] C. M. Paleos, D.Tsiourvas, Z. Sideratou, L. Tziveleka, Biomacromolecules2004, 5, 524; [1m] F. Vogtle, S. Gestermann, R. Hesse, H.Schwierz, B. Windisch, Prog. Polym. Sci. 2000, 25, 987.

[2] [2a] M. Liu, J. M. J. Frechet, Pharm. Sci. Technol. Today1999, 2, 393; [2b] N. Malik, R. Wiwattanapatapee, R.Klopsch, K. Lorenz, H. Frey, J. W.Weener, E. W.Meijer, W.Paulus, R. Duncan, J. Controlled Release 2000, 65, 133; [2c]S.-E. Stiriba, H. Frey, R. Haag, Angew. Chem. Int. Ed. 2002,41, 1329; [2d] O. L. Padilla De Jesus, H. R. Ihre, L. Cagne, J.M. J. Frechet, F. C. Szoka, Jr.,Bioconjugate Chem. 2002, 13,453; [2e] U. Boas, P. M. H. Heegaard,Chem. Soc. Rev. 2004,33, 43; [2f] A. E. Beezer, A. S. H. King, I. K. Martin, J. C.Mitchel, L. J. Twyman, C. F. Wain, Tetrahedron 2003, 59,3873; [2g] E. R. Gillies, J. M. J. Frechet, Drug DiscoveryToday 2005, 10, 35.

[3] [3a] M. Liu, K. Kono, J. M. J. Frechet, J. Polym. Sci., Part A:Polym. Chem. 1999, 37, 3492; [3b] M. Liu, K. Kono, J. M. J.Frechet, J. Controlled Release 2000, 65, 121; [3c] Z.

Sideratou, D. Tsiourvas, C. M. Paleos, J. Colloid InterfaceSci. 2001, 242, 272; [3d] A. Pantos, D. Tsiourvas, Z.Sideratou, C. M. Paleos, S. Giatrellis, G. Nounesis,Langmuir 2004, 20, 6165.

[4] [4a] D. D. Lasic, D. Needham, Chem. Rev. 1995, 95, 2601,and references cited therein; [4b] T. M. Allen, S. Zalipsky,Polym. Mater. Sci. Eng. 1997, 76, 497; [4c] D. Needham, D.H. Kim,Colloids Surf. B 2000, 18, 183; [4d]M. Silvander, P.Hansson, K. Edwards, Langmuir 2000, 16, 3696; [4e] P.Crosasso,M.Ceruti, P. Brusa, S.Arpicco, F. Dosio, L. Cattel,J. Controlled Release 2000, 63, 19; [4f] T. Kaasgaard, O. G.Mouritsen, K. Jørgensen, Int. J. Pharm. 2001, 214, 63.

[5] [5a] A. U. Bielinska, C. Chen, J. Johnson, J. R. Baker, Jr.,BioconjugateChem. 1999, 10, 843; [5b] J.D. Eichman,A.U.Bielinska, J. F. Kukowska-Latallo, J. R. Baker, Jr., Pharm.Sci. Technol. Today 2000, 3, 232; [5c] D. Luo, K. Haverstick,N. Belcheva, E. Han, W. M. Saltzman, Macromolecules2002, 35, 3456; [5d] M. Ohsaki, T. Okuda, A. Wada, T.Hirayama, T. Niidome, H. Aoyagi, Bioconjugate Chem.2002, 13, 510; [5e] B. H. Zinselmeyer, S. P. Mackay, A. G.Schatzlein, I. F. Uchegbu, Pharmaceut. Res. 2002, 19, 960.[5f] S. H. Kim, J. H. Jeong, K. C. Cho, S.W. Kim, T. G. Park,J. Controlled Release, 2005, 104, 223.

[6] [6a] A. Sunder, M.-F. Quincy, R. Mulhaupt, H. Frey, Angew.Chem. Int. Ed. 1999, 38, 2928; [6b] R. Haag, Chem. Eur. J.2001, 7, 327; [6c] A. Sunder, R. Mulhaupt, H. Frey,Macromolecules 2000, 33, 309; [6d] A. Sunder, R.Mulhaupt, R. Haag, H. Frey, Adv. Mater. 2000, 12, 235;[6e] M. Kramer, J.-F. Stumbe, H. Turk, S. Krause, A. Komp,L. Delineau, S. Prokhorova, H. Kautz, R. Haag, Angew.Chem. Int. Ed. 2002, 41, 4252; [6f] A. Sunder,M.Kramer, R.Hanselmann, R. Muhlaupt, H. Frey, Angew. Chem. Int. Ed.1999, 38, 3552; [6g] R. Haag, M. Kramer, J.-F. Stumbe, S.Krause, A. Komp, S. Prokhorova, Polym. Prepr. (Am. Chem.Soc., Div. Polym. Chem.) 2002, 43, 328; [6h] H. Frey, R.Haag, Rev. Mol. Biotech. 2002, 90, 257; [6i] C. Siegers, M.Biesalski, R. Haag, Chem. Eur. J. 2004, 10, 2831.

[7] According to theMaterial SafetyData Sheet: for structurallysimilar products (oligoglycerols): oral route, LD50, rat,> 2,000 mg kg�1; dermal route, LD50, rabbit, >2,000 mgkg�1.

[8] [8a] C. P. Leamon, P. S. Low,Drug Discovery Today 2001, 6,44; [8b] J. Sudimack, R. J. Lee, Adv. Drug Delivery Rev.2000, 41, 147; [8c] H. E. J. Hofland, C. Masson, S. Iginla, I.Osetinsky, J. A. Reddy, C. P. Leamon, D. Scherman, M.Bessodes, P. Wils, Mol. Ther. 2002, 5, 739; [8d] A. C.Antony; Adv. Drug Delivery Rev. 2004, 56, 1059; [8e] S.Sabharanjak, S. Mayor, Adv. Drug Delivery Rev. 2004, 56,1099.

[9] [9a] R. J. Lee, P. S. Low,Biochim. Biophys. Acta 1995, 1233,134; [9b]R. J. Lee, L.Huang, J. Biol. Chem. 1996,271, 8481;[9c] A. Gabizon, H. Shmeeda, A. T. Horowitz, S. Zalipsky,Adv. Drug Delivery Rev. 2004, 56, 1177.

[10] [10a] K. Kono, M. Liu, J. M. J. Frechet, Bioconjugate Chem.1999, 10, 1115; [10b] S. Shukla, G. Wu, M. Chatterjee, W.Yand, M. Sekido, L. A. Diop, R. Muller, J. Sudimack, R. J.Lee, R. J. Barth, W. Tjarks, Bioconjugate Chem. 2003, 14,158; [10c] S. D. Konda, M. Aref, S. Wang, M. Brechbiel,E. C. Wiener, Magn. Reson. Mater. Phys. Biol. Med. 2001,12, 104.

[11] [11a] D. Dube, M. Francis, J.-C. Leroux, F. M. Winnik,Bioconjugate Chem. 2002, 13, 685; [11b] C. P. Leamon, D.Weigl, R. W. Hendren, Bioconjugate Chem. 1999, 10, 947;[11c] O. Aronov, A. T. Horowitz, A. Gabizon, D. Gibson,Bioconjugate Chem. 2003, 14, 563; [11d] E. Dauty,



J.-S. Remy, G. Zuber, J. P. Behr, Bioconjugate Chem. 2002,13, 831; [11e] G. Zuber, L. Zammut-Italiano, E. Dauty, J. P.Behr,Angew. Chem. Int. Ed. 2003, 42, 2666; [11f] H. S. Yoo,T. G. Park, J. Controlled Release 2004, 96, 273; [11g] C.-H.Wang, G.-H. Hsiue, Bioconjugate Chem. 2005, 16, 391;[11h] M. Licciardi, Y. Tang, N. C. Billingham, S. P. Armes,Biomacromolecules 2005, 6, 1085; [11i] S. H. Kim, J. H.Jeong, C.O. Joe, T. G. Park, J. Controlled Release 2005, 103,625.

[12] [12a] M. Mammen, S. K. Choi, G. M. Whitesides, Angew.Chem. Int. Ed. 1998, 37, 2755; [12b] P. I. Kitov, D. R.Bundle, J. Am. Chem. Soc. 2003, 125, 16271.

[13] [13a] D. Holter, A. Burgath, H. Frey, Acta Polym. 1997, 48,30; [13b] A. Sunder, R. Hanselmann, H. Frey, R. Mulhaupt,Macromolecules 1999, 32, 4240; [13c] A. Sunder, H.Turk, R. Haag, H. Frey,Macromolecules 2000, 33, 7682.

[14] J. S. Choi, K.Nam, J.-y. Park, J.-B. Kim, J.-K. Lee, J.-s. Park,J. Controlled Release 2004, 99, 445.

[15] [15a] S. Wang, J. Luo, D. A. Lantrip, D. J. Waters, C. J.Mathias, M. A. Green, P. L. Fuchs, P. S. Low, Bioconjugate

Chem. 1997, 8, 673; [15b] K. Riebeseel, E. Biedermann, R.Loser, N. Breiter, R. Hanselmann, R.Mulhaupt, C. Unger, F.Kratz, Bioconjugate Chem. 2002, 13, 773.

[16] [16a] H. Wiseman, Trends Pharmacol. Sci. 1994, 15, 83;[16b] E. V. Mocanu, R. F. Harrison, Rev. Gynaecol. Pract.2004, 4, 37.

[17] J. B. A. Custodio, L. M. Almeida, V. M. Madeira, Biochim.Biophys. Acta 1991, 176, 1079.

[18] C. Kojima, K. Kono, K. Maruyama, T. Takagishi, Bioconju-gate Chem. 2000, 11, 910.

[19] A. C. Guyton, J. E. Hall, ‘‘Textbook ofMedical Physiology’’,10thedition, W. B. Saunders Company, Philadelphia 2000,p. 264.

[20] [20a] M. J. Bogan, G. R. Agnes, J. Am. Soc. Mass Spectrom.2002, 13, 177, and references cited therein; [20b] Y. Wand,H. Rashidzadeh, B. Guo, J. Am. Soc. Mass Spectrom. 2000,11, 639.

[21] J. A. Boomer, H. D. Inerowicz, Z. Y. Zhang, N. Bergstrand,K. Edwards, J. M. Kim, D. H. Thompson, Langmuir 2003,19, 6408, and references cited therein.



novel functional hyperbranched polyether polyols as prospective drug delivery systems

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