Journal of Colloid and Interface Science 338 (2009) 56–62

Contents lists available at ScienceDirect

Journal of Colloid and Interface Science

www.elsevier .com/locate / jc is

Preparation and characterization of nanoparticles based on dextran–drug conjugates

Stephanie Hornig a, Heike Bunjes b,c, Thomas Heinze a,*

a Center of Excellence for Polysaccharide Research, Member of the European Polysaccharide Network of Excellence, Friedrich-Schiller-Universität Jena, Humboldtstrasse 10,D-07743 Jena, Germanyb Department of Pharmaceutical Technology, Institute of Pharmacy, Friedrich-Schiller-Universität Jena, Lessingstr. 8, D-07743 Jena, Germanyc Institute of Pharmaceutical Technology, Technische Universität Carolo-Wilhelmina zu Braunschweig, Mendelssohnstrasse 1, D-38106 Braunschweig, Germany

a r t i c l e i n f o

Article history:Received 11 February 2009Accepted 5 May 2009Available online 18 May 2009

Keywords:DextranDrug deliveryPolymer–drug conjugatesNanoparticleIbuprofen

0021-9797/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.jcis.2009.05.025

* Corresponding author.E-mail address: thomas.heinze@uni-jena.de (T. He

a b s t r a c t

The presented concept combines the widely-established use of macromolecular prodrugs with nanopar-ticulate drug delivery devices. For this purpose, the water-soluble biopolymer dextran was functionalizedwith poorly water-soluble drugs (ibuprofen, naproxen) via in situ activation of the carboxylic groups withN,N

0-carbonyldiimidazole (CDI). The resulting hydrophobic derivatives self-assemble into nanoparticles

with high loading efficiency during nanoprecipitation. The degree of substitution (DS) and the prepara-tion technique strongly influence the size and the size distribution of the resulting nanoparticles. The par-ticle suspensions remained stable over months in a pH value range between 4 and 11. Derivatives withhigh DS values are more stable against hydrolysis and after the addition of electrolytes than lowly substi-tuted ones. Therefore, a defined tuning of the DS value may allow the adjustment of the pH-dependenthydrolysis rate and, hence, the release of the drugs.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

Nanoparticles containing therapeutic agents have been the sub-ject of increasing interest in scientific and commercial fields becauseof their potential for site-specific drug delivery, and thus the optimi-zation of drug therapy [1]. In particular, polymers play a significantrole as drug carrier devices. Pharmacologically active agents caneither be covalently bound to the polymer backbone, physicallyincorporated into a polymeric matrix, e.g. polymeric micelles or por-ous particles, or form polyelectrolyte complexes of oppositelycharged polymer/drug systems [2]. Important objectives achievedusing such polymeric carriers include stabilization of the therapeu-tic agent, enhancement of drug solubility, improvement of thecirculation life time and the therapeutic index, and reduction ofside-effects [3]. In particular, the polyglucan dextran is widely usedfor polymer–drug conjugates in combination with bioactive mole-cules such as pharmaceutics (anti-cancer agents, anti-inflammatorydrugs, antibiotics, immunosuppressants), enzymes, proteins, pep-tides, and hormones [4,5]. For instance, the use of a paclitaxel–car-boxymethyl dextran conjugate increased the efficiency in anantitumor study more than twice comparing to the parent drug[6]. However, the toxicity of the drug is still a concern, not only forpaclitaxel. Consequently, the drugs should be addressed specificallyand protected on their way to the targeted tissue. Nanoparticles canbe used to provide such a protecting matrix and may also be

ll rights reserved.

inze).

functionalized with targeting ligands. In addition, nanoparticlescontaining biologically active substances can penetrate deeply intotissues through fine capillaries and can be taken up efficiently bythe cells due to their sub-cellular and sub-micron size. Drugs, whichare physically incorporated drugs in a particle matrix, may, however,diffuse in an uncontrolled way so that the drug leaks out of the car-rier and is not completely transported to and released at the targetedside. Alternatively, the drug may be covalently bound to the poly-mer. In the case of hydrophobic drugs, however, the achievable load-ing efficiencies of the resulting polymer prodrugs are comparativelylow because the water solubility of the polymer derivatives needs tobe maintained. In contrast, the concept of nanoprecipitation ofhydrophobic macromolecular polymer–drug conjugates presentedin the following work allows the preparation of aqueous suspensionsof highly loaded nanoparticles.

One essential feature of the polymers to be employed in drugdelivery is their compatibility with biological tissues. Besidessynthetic biodegradable polymers, dextran is a promising, physio-logically evaluated carrier [7]. Dextran is composed of a-(1 ? 6)linked D-glucose units with varying branches depending on thedextran-producing bacterial strain. Its biocompatibility, biodegrad-ability, non-immunogenic and non-antigenic properties qualifydextran as a physiologically harmless biopolymer [8]. Dextran pro-pionate nanoparticles were easily be incorporated in human fibro-blasts in remarkable amounts, and the cells show no changes over22 days as observed by confocal fluorescence microscopy [9].

Dextran can be depolymerized by different a-1-glycosidases(dextranases) occurring in liver, spleen, kidney, and lower part of

S. Hornig et al. / Journal of Colloid and Interface Science 338 (2009) 56–62 57

the gastrointestinal tract. The high amount of hydroxyl groupsfacilitates the introduction of, e.g., proteins, aptamers, or drugs intothe polymer backbone. In recent decades, dextran was functional-ized with various pharmaceutical agents, like naproxen [10], dau-norubicin [11], mitomycin C [12], and cisplatin [13] yieldingefficient prodrugs. In the case of water-insoluble drugs, the degreeof substitution (DS), and hence the loading capacity, was, however,rather low. This limitation can be overcome by using the concept ofnanoprecipitation of hydrophobic dextran derivatives leading towater-dispersible systems [14,15]. The hydrophobic dextran deriv-atives can easily be obtained by an efficient esterification with car-boxylic acids via in situ activation with N,N

0-carbonyldiimidazol

(CDI). In the present study, this reaction was used to immobilizeibuprofen and naproxen as model drugs on dextran. The resultingdextran derivatives with varying degrees of substitution were sub-sequently precipitated from dilute solutions yielding aqueous sus-pensions with particles in the nanometer scale, which wereanalyzed concerning particle size and surface characteristics aswell as their stability.

2. Materials and methods

2.1. Materials

CDI, ibuprofen-Na salt ((RS)-2-[4-(2-methylpropyl)phenyl]pro-pionic acid sodium salt), Tween 80, and dextranase from penicil-lium sp. (12.9 U/mg) were purchased from Sigma–Aldrich.Naproxen ((S)-2-(6-methoxy-2-naphthyl)propionic acid), dextran,NaCl, DMSO-d6 and other solvents were received from Fluka. Dex-tran was produced by Leuconostoc mesenteroides strain no. NRRLB-512(F) and possessed a Mw of 54,400 g/mol. All chemicals wereused without further treatment.

2.2. Synthesis of ibuprofen dextran ester, sample 5

To a solution of 10.2 g (49.4 mmol) ibuprofen (obtained byacidic treatment of an aqueous solution of ibuprofen-Na salt) in20 mL DMSO, 8.0 g (49.4 mmol) CDI were added. After 24 h stirringat room temperature, 2.0 g (12.34 mmol) dextran were added. Themixture was allowed to react for 24 h at 80 �C under stirring. Theproduct was isolated by precipitation in 500 mL water and washedseveral times with water. The product was dried at 60 �C undervacuum. Yield: 5.4 g (78.3%). DSIbu 2.08 (determined by means of1H NMR spectroscopy after perpropionylation). In order to revealthe DS of the ibuprofen ester (because of a limited separation ofthe peaks of the perpropionylated samples in the 1H NMR spectra),the sample was additionally pernitrobenzoylated and analyzed by1H NMR spectroscopy. The resulting DS value (DSIbu 2.18) is in therange of the one determined after perpropionylation. 1H NMR(250 MHz, DMSO-d6): d = 7.2, 7.1 (10–13), 5.5–3.2 (1–6), 3.7 (8),2.4 (14), 1.8 (15), 1.5 (9), 0.9 ppm (16). 13C NMR (400 MHz,DMSO-d6): d = 173.8 (7), 140.5 (13), 137.4 (10), 129.3 (12), 127.2(11), 95.4 (1

0), 73.4–66.5 (2–6), 45.0 (8,14), 30.1 (15), 22.4 (16),

18.5 ppm (9). The atoms are numbered according to Fig. 1a. Theyield was calculated with respect to the value of a dextran ibupro-fen ester with DS 2.08. Therefore, a yield of 100% of dextran ibupro-fen ester with DS 2.08 would be 6.9 g.

The preparation of samples 1–4 with different DS values wascarried out by variation of the molar ratio of dextran/CDI/ibuprofen(1/1/1, 1/3/3, and 1/4/4).

2.3. Synthesis of naproxen dextran ester, sample 7

To a solution of dextran (0.3 g, 1.9 mmol) in 5 mL DMSO, 1.28 g(5.6 mmol) naproxen and 0.9 g (5.6 mmol) CDI were added. The

mixture was allowed to react for 24 h at 80 �C under stirring. Theproduct was isolated by precipitation in 150 mL isopropanol andwashed two times with 50 mL isopropanol. The resulting whitepowder was dried at 60 �C under vacuum. Yield: 0.7 g (70.5%).DSNap 1.62 (determined by means of 1H NMR spectroscopy afterperpropionylation). 1H NMR (250 MHz, DMSO-d6): d = 7.7–7.0(10–19), 5.6–3.3 (1–6), 3.8 (8,20), 1.4 ppm (9). 13C NMR(400 MHz, DMSO-d6): d = 173.4 (7), 157.6 (15), 135.9 (10), 133.7(17), 129.6, 128.8, 127.3, 126.1 (11-13,18,19), 119 (14), 106.1(16), 99.0 (1), 96.2 (1

0), 71.7–65.7 (2–6), 55.5 (20), 45.0 (8),

19.0 ppm (9). The atoms are numbered according to Fig. 1b.The preparation of sample 6 with different DS value was carried

out by variation of the molar ratio of dextran/CDI/naproxen (1/1/1).

2.4. Perpropionylation

As a typical procedure, a total of 0.2 g of the sample dissolved in6 mL of pyridine was allowed to react with 6 mL of propionic anhy-dride in the presence of 50 mg of N,N-dimethylaminopyridine ascatalyst for 24 h at 80 �C. The polymer was precipitated in ethanol,washed with ethanol (250 mL) two times, and dried at 60 �C. Inaddition, the sample was reprecipitated from 3 mL of chloroformin 100 mL of ethanol, followed by filtration and drying at 60 �C un-der vacuum.

2.5. Pernitrobenzoylation

As a typical procedure, a total of 0.2 g of the sample dissolved in3 mL of pyridine and 5 mL of N,N-dimethylformamide was allowedto react with 0.6 g of 4-nitrobenzoyl chloride for 24 h at 60 �C. Thepolymer was precipitated in water, washed with ethanol (250 mL)two times, and dried at 60 �C. In addition, the sample was reprecip-itated from 3 mL of chloroform in 100 mL of ethanol, followed byfiltration and drying at 60 �C under vacuum.

2.6. Nanoparticle preparation by dialysis

As a typical example, the dextran ester (20 mg) was dissolved in5 mL purified N,N-dimethylacetamide (DMAc) and dialyzedagainst 500 mL distilled water. The water was exchanged fivetimes after at least 3 h of dialysis.

2.7. Nanoparticle preparation by dropping technique

As a typical example, 20 mg of the dextran ester was dissolvedin 5 mL acetone. The solution was either added dropwise to 15 mLdistilled water or vice versa, i.e. water was added dropwise to thepolymer solution. The resulting nanoparticle suspensions werestirred at 60 �C for 10 h until the acetone was completely removedfrom the aqueous suspension.

2.8. Characterization of polymers and nanoparticles

NMR spectra were acquired on a Bruker AMX 250 and DRX 400spectrometer with 16 scans for 1H NMR (room temperature) andup to 21,000 scans for 13C NMR (45 �C) measurements (25 mg sam-ple/mL for 1H NMR and 100 mg sample/mL for 13C NMR studies).The number average molar mass was estimated by gel permeationchromatography (GPC) on a JASCO system equipped with a NOV-EMA 300 column and DMAc/LiCl as eluent with a flow rate of1 mL/min at 40 �C. For SEM studies, one droplet of nanoparticle sus-pension was lyophilized on a mica surface and covered with gold.The images were obtained with the scanning electron microscope(SEM) equipment LEO-1530 VP Gemini (LEO, Oberkochen, Ger-many) operating at 10 kV. The particle size, polydispersity and zetapotential of the nanoparticles were determined by dynamic light

RORO

O

O

O

O

O

RORO

O

O

O

O

50100150[ppm]

~ ~ CHCl3

1‘AGU(2-6)

7

8,14

910

15

16

11

12

13

50100150[ppm]

3

5 6

7

8 91011

12 13

14

15 16

1 2

4

7

8 9 10 11

1213

14

3

56

12

4

1516

17 18

19

20AGU(2-6)

89

10

14

7

11-13,18,19

1‘

15

16

17

1

~ DMSO 20

a)

b)

Fig. 1. 13C NMR spectra of (a) dextran ibuprofen ester (DSIbu 1.50, sample 4) in CDCl3 and (b) naproxen dextran ester (DSNap 1.62, sample 7) in DMSO-d6; the structure of thedextran esters are schematically represented (1

0indicates C1 adjacent to a C2 bearing an ester function).

58 S. Hornig et al. / Journal of Colloid and Interface Science 338 (2009) 56–62

scattering using a Zetasizer Nano ZS (Malvern Instruments, Mal-vern, UK). The suspensions were diluted with demineralised, fil-tered water to a concentration of about 0.005%. The mean particlesize was approximated as the z-average diameter and the width ofthe distribution as the polydispersity index (PDI) obtained by thecumulants method assuming spherical shape [16]. Each samplewas measured for 10 min (corresponding to 4 runs over 150 s). Zetapotential measurements were made with the same apparatus at25 �C. The dispersions were diluted with NaCl-solution with varyingconcentrations and phosphate buffered saline (PBS; c = 2 mmol/L)with varying pH values. In the experiments on the effect of the pres-ence of surfactant, Tween 80 was added to the particle suspensionwith a concentration of 0.1 lmol/L before the dilution. The zeta po-tential values were calculated by the DTS Dispersion TechnologySoftware (Version 3.30 2002, Malvern instruments) using theSmoluchowski model. The values given are averages of three mea-surements with 30 single runs each.

2.9. Hydrolysis

The nanoparticle suspensions (1 mg/mL) were diluted with PBSsolution and brought to the desired pH value by addition of aque-ous NaOH (1 mol/L). The hydrolysis was examined by UV–Vis spec-troscopy. For this purpose, the particles (if existing) were filteredoff, and the residual solution was analyzed for amounts of ibupro-fen- and naproxen carboxylates.

2.10. Enzymatic degradation

The dextranase (final concentration 0.034 wt%) was added tothe nanoparticle suspension containing 0.0083 wt% polymer in

McIlvan buffer (pH 5.5, 200 mM disodium hydrogen phosphate,100 mM citric acid) and stirred at 37 �C for 4.5 h. The solid partswere filtered off and analyzed by GPC measurements.

3. Results and discussion

3.1. Synthesis and characterization of the dextran esters

A suitable method for the immobilization of bioactive com-pounds at polysaccharides is a direct linkage with the polymerbackbone by esterification of hydroxyl groups, e.g. with carboxylicacids. An efficient esterification of dextran can be carried out viain situ activation of the carboxylic acid with N,N

0-carbonyldiimid-

azole (CDI). High degrees of substitution (DS) are reached in com-bination with the formation solely of non-toxic, easily removablebyproducts that are beneficial for the conversion of dextran witheven complex and sensitive acids [17]. The previous formation ofreactive intermediates like acid chlorides or anhydrides is not re-quired. Moreover, CDI allows the use of DMSO, which is a good sol-vent for dextran and most of the complex carboxylic acids.

The non-steroidal anti-inflammatory drugs ibuprofen and na-proxen were chosen as biologically active carboxylic acids, whichare hardly soluble in water, to evaluate the potential of the result-ing dextran derivatives as nanoparticulate polymer–drug conju-gates. By varying the ratio of drug and CDI to anhydroglucoseunit (AGU), different DS values in the range from 0.50 to 2.08 areaccessible. Selected samples are summarized in Table 1. The drugcontent of the dextran esters varies from 37 to 71 wt% and isremarkably high compared to the loading capacity of known ibu-profen and naproxen release materials like molecular sieves (e.g.30 wt% ibuprofen in polymer coated SiO2 particles) [18], lipid

Table 1z-Average mean diameter and polydispersity index (PDI) of nanoparticle suspensions of ibuprofen- and naproxen dextran esters prepared by different methods.

No. Drug DSa Drug content (wt%) Solvent/technique Size (nm) PDI

1 Ibuprofen 0.50 37.0 DMAc/dialysisb 102 0.1172 Ibuprofen 0.62 42.1 DMAc/dialysisb 173 0.0653 Ibuprofen 0.74 46.5 DMAc/dialysisb 198 0.1663 Ibuprofen 0.74 46.5 Acetone/dialysisb 344 0.2293 Ibuprofen 0.74 46.5 Acetone/droppingc 171 0.0753 Ibuprofen 0.74 46.5 Acetone/droppingd 69 0.1784 Ibuprofen 1.50 63.9 DMAc/dialysisb 453 0.1935 Ibuprofen 2.08 71.1 DMAc/dialysisb 287 0.1905 Ibuprofen 2.08 71.1 Acetone/dialysisb 309 0.2335 Ibuprofen 2.08 71.1 Acetone/droppingc 279 0.1295 Ibuprofen 2.08 71.1 Acetone/droppingd 77 0.0946 Naproxen 0.41 35.1 DMAc/dialysisb 177 0.0627 Naproxen 1.62 68.3 DMAc/dialysisb 387 0.209

a Determined by 1H NMR spectroscopy of perpropionylated samples.b Dialysis of the dissolved polymers (4 mg/mL) against water.c Dropwise addition of water to the polymer dissolved in acetone (4 mg/mL).d Dropwise addition of polymer dissolved in acetone (4 mg/mL) to water.

S. Hornig et al. / Journal of Colloid and Interface Science 338 (2009) 56–62 59

nanoparticles (e.g. 10 wt% ibuprofen in smectic cholesterol esternanoparticles) [19], polymeric nanoparticles (e.g. 9 wt% ibuprofenin Eudragit� nanoparticles) [20], and water-soluble polymeric pro-drugs (e.g. 7 wt% naproxen in dextran naproxen ester) [7]. Evencomplex coacervation of N,N-diethylaminoethyl dextran and thedrug itself, i.e. ibuprofen sodium salt, results in core-shell nanopar-ticles containing only up to 32 wt% ibuprofen [21].

The dextran derivatives prepared are soluble in dimethylsulfox-ide (DMSO), N,N-dimethylacetamide (DMAc), N,N-dimethylform-amide (DMF), and acetone. The structure of the dextran esters wasexamined by 13C NMR spectroscopy. Fig. 1 shows 13C NMR spectraincluding assignments of dextran ibuprofen (Fig. 1a, DSIbu1.50) anddextran naproxen ester (Fig. 1b, DSNap1.62). The determination ofthe DS values was carried out via 1H NMR spectroscopy of the per-propionylated samples, in which the hydroxyl groups of the dextranderivatives are completely functionalized [22]. The DS of propionateand, thus, the DS prior to perpropionylation can be determined fromthe 1H NMR spectra using the integral intensities of the AGU and thepropionyl proton signals. Pernitrobenzoylated samples were alsoanalyzed to confirm the DS yielding comparable values.

3.2. Nanoparticle formation

Hydrophobic polymers may self-assemble into nanoparticulatesystems during precipitation from dilute solutions in water[11,23,24]. Therefore, water-miscible solvents should be used forthe nanoprecipitation. Furthermore, low concentrations typicallybelow the critical overlap concentration are necessary, ensuringthat the molecules are in a dispersed state and can separate intonanodomains after adding the non-solvent water. A concentrationof 4 mg/mL is recommended for the formation of small particleswith a uniform size distribution.

An efficient approach for the controlled and ideally completeexchange of the solvents against water is dialysis. The nanoparti-cles formed in aqueous suspensions were analyzed for size andshape by dynamic light scattering (DLS) and scanning electronmicroscopy (SEM). By applying nanoprecipitation via dialysis of aDMAc solution against water, ibuprofen- and naproxen dextran es-ter nanoparticles in the range from 102 to 287 nm, and 177 to387 nm, respectively, are accessible (Table 1). The formation ofthe particles strongly depends on the solvent used, the concentra-tion of the polymer solution, and the degree and character of thesubstituents. With increasing DS, the particles tend to become lar-ger and less uniform as indicated by the comparatively high poly-dispersity index (PDI). The preparation of the particles is notlimited to dialysis of DMAc solutions. The dialysis of an acetone

solution of ibuprofen dextran esters (samples 3 and 5) yields largerparticles with a less uniform size distribution (Table 1).

An alternative technique of nanoprecipitation is the introduc-tion of the polymer solution into water (or vice versa; water intothe polymer solution) in droplets, while stirring. The use of acetoneas solvent is favored because it can be removed completely fromthe aqueous suspension by evaporation. The dropping techniqueis obviously a less time- and materials-consuming process in addi-tion to the lower toxicity of acetone compared to DMAc. The drop-wise addition of water to an acetone solution results in particleswith sizes in the same range as obtained by dialysis of DMAc solu-tion at the same concentration, whereas the reverse procedureleads to small particles below 100 nm but possessing a higherPDI as exemplified for sample 3 in Table 1. These findings seemto be a general phenomenon found for the preparation of nanopar-ticles based not only on dextran esters but also for cellulose deriv-atives [21]. An exception that was found is an ibuprofen dextranester (DSIbu 2.08, sample 5) whose nanoparticles have a low PDIeven if the acetone solution is dropped into water. Fig. 2 illustratesthe dependency of the particle sizes and the PDI on the concentra-tion and the path of nanoprecipitation. Dropping of acetone solu-tion into water while stirring usually yields small and uniformlysized particles (size < 100 nm, PDI < 0.100) whereas the additionof water to the acetone solution leads to larger particles with aslightly higher polydispersity. Only at a polymer concentration of1 mg/mL, dropping water into the acetone solution results in moreuniformly sized particles (189 nm, PDI 0.060) compared to the re-verse procedure (62 nm, PDI 0.126).

3.3. Nanoparticle characterization

It is remarkable that nanospheres from dextran derivatives canbe prepared without any addition of surfactants which are com-monly required for the preparation of polymeric nanoparticles.By varying the preparation conditions, the particle size and theirdistribution can be precisely tuned. The nanoparticles, which areprepared either by dialysis or dropping technique, do not differin their structure. SEM studies reveal the regular shape of the nan-ospheres obtained by dialysis (Fig. 3a) and dropping technique(Fig. 3b). It can be assumed that the hydrophobic ibuprofen moie-ties are commonly located in the interior of the particles because itis known that nanoparticles based on dextran esters possess ahydrophobic core [25].

The nanoparticle suspensions are stable at physiological pHvalues and temperatures around 37 �C. In deionized water, the par-ticle suspensions are constant in size over 1.5 years after prepara-

0 2 4 6 80

100

200

300

400

500

600a)

polymer concentration [mg/mL]

part

icle

siz

e [n

m]

0 2 4 6 80.00

0.05

0.10

0.15

0.20b)

polymer concentration [mg/mL]

poly

disp

ersi

ty in

dex

Fig. 2. Particle size (a) and polydispersity index (b) of ibuprofen dextran esternanoparticles (DSIbu 2.08, sample 5) prepared by dropping of acetone solution intowater (j) or water into acetone solution (h) with varying polymer concentrations.

Fig. 3. SEM images of nanoparticles of ibuprofen dextran ester prepared (a) bydialysis of polymer (DSIbu 0.62, sample 2) dissolved in DMAc (c = 4 mg/mL) and (b)by dropwise addition of water into an acetone solution of the polymer (DSIbu 0.74,sample 3) containing 4 mg/mL.

Table 2Hydrolysis of ibuprofen dextran esters at different pH values (37 �C) over timedetermined by UV/Vis spectroscopy (+ indicates hydrolysis, � indicates nohydrolysis).

Time (h) DSIbu 0.50 (sample 1) DSIbu 2.08 (sample 5)

pH 10 pH 11 pH 12 pH 13 pH 10 pH 11 pH 12 pH 13

0 � � � + � � � �4 � � + + � � � �

24 � + + + � � � +48 � + + + � � � +72 � + + + � � � +96 + + + + � � � +

60 S. Hornig et al. / Journal of Colloid and Interface Science 338 (2009) 56–62

tion as proven for selected ibuprofen esters (e.g., DSIbu 0.62, sample2: after 21 months size of 177 nm and PDI of 0.096). The concentra-tion of the nanoparticle suspension, which is usually 0.1% afterpreparation, can be increased up to 1% by rotary evaporation ofwater without changing the particle size and distribution. Concen-trations from 0.1% to 1% should be sufficient because the describeddextran–drug conjugates possess very high loading efficiencies andsimilar particle concentrations are used for pharmacokinetic stud-ies and other nanoparticular systems [17,26]. For biological exper-iments, it is important to note that the nanoparticle suspensionsbased on dextran esters are stable under standard sterilizing proce-dures in an autoclave at 121 �C and 2 bar for removing germs [6].However, the stability of the nanoparticle suspensions strongly de-pends on the pH value and the electrolyte concentration of the sus-pending medium. The suspensions are constant in their size frompH 4 to 11. At a pH value above �11 (depending on time andDS), clear solutions are obtained due to the saponification of thedextran ester, which leads to dissolution of pure dextran. Althoughsuch a high pH value is not relevant for the controlled release ofdrugs in biological media, it is of interest to determine the stabilityof the suspensions. The studies have shown that more highly-substituted dextran esters are more stable against hydrolysis(Table 2). The hydrolysis of the ibuprofen dextran derivative withDSIbu 0.50 (1) at 37 �C proceeds faster and at lower pH values com-pared to a dextran ibuprofen ester with DSIbu 2.08 (5) as examinedby ultraviolet–visible (UV–Vis) spectroscopy (determination of thefree ibuprofen content at 265 nm). The hindered hydrolysis of dex-tran ester 5 might be due to the limited accessibility of the basetowards the hydrophobic particle. Furthermore, the surface-to-vol-ume ratio of the dextran ester nanoparticles with DSIbu 2.08 is low-

er because of their larger size. Consequently, the volume-relatedsurface accessible for hydrolysis is smaller.

Below pH 4, agglomeration of the particles occurs, which can beexplained by the results of zeta potential measurements (Fig. 4).Generally, suspensions with a zeta potential above ±20 mV are sta-ble [27]. In the present example, the electrostatic repulsion below±10 mV is not sufficient and, hence, the particles aggregate. Themajority of colloidal systems dispersed in water acquire a negativesurface charge, probably due to preferential adsorption of hydroxylions [28]. With decreasing pH, the surface charge is neutralized byH+, the zeta potential decreases and, thus, repulsion between theparticles becomes less intensive.

The results of zeta potential measurements of ibuprofendextran ester suspensions (DS values of 0.50, 1; 2.08, 5) are dis-played in Fig. 4a and b, respectively. In the range of pH values from7 to 10, the particles of the lowly-substituted derivative possess azeta potential of about �30 mV, whereas the derivative bearing a

2 4 6 8 10 12-80

-60

-40

-20

0

20a)

pH

zeta

pot

entia

l [m

V]

0

500

1000

1500

2000

2500

3000

particle size [nm]

2 4 6 8 10 12-80

-60

-40

-20

0

20

pH

zeta

pot

entia

l [m

V]

0

500

1000

1500

2000

2500

3000b)

particle size [nm]

Fig. 4. Dependency of the zeta potential (j) and particle size (w) on pH value ofibuprofen dextran ester nanoparticle suspensions prepared by dialysis of a DMAc-solution (4 mg/mL) against water; (a) DSIbu 0.50 (1) and (b) DSIbu2.08 (5). Pleasenote that the particle sizing for particles above 1 lm is not very reliable (highvariability was observed between measurements at the same conditions, and PDI’sover 0.3). They are given here only as an indication for aggregation, not to reflect thereal size of the particles. The same applies for the values in Fig. 5.

10-5 10-4 10-3 10-2 10-1-80

-70

-60

-50

-40

-30

-20

-10

0

NaCl concentration [mol/L]

zeta

pot

entia

l [m

V]

a)

0

500

1000

1500

2000

2500

particle size [nm]

10-5 10-4 10-3 10-2 10-1-80

-70

-60

-50

-40

-30

-20

-10

0

NaCl concentration [mol/L]

zeta

pot

entia

l [m

V]

b)

0

500

1000

1500

2000

2500

particle size [nm]

Fig. 5. Zeta potential (j) and particle size (w) of ibuprofen dextran ester particlesuspensions with varying NaCl concentrations: (a) DSIbu 0.50 (1) and (b) DSIbu2.08(5).

S. Hornig et al. / Journal of Colloid and Interface Science 338 (2009) 56–62 61

higher amount of ibuprofen groups has a zeta potential of about�70 mV, which indicates a stronger electrostatic repulsionbetween the particles, and therefore a higher stability of the sus-pensions. Under acidic conditions, the zeta potential decreaseswith decreasing pH. Below pH 4, the particles aggregate due to areduced repulsion between the particles.

Furthermore, nanoparticles of ibuprofen dextran ester with ahigh DS (sample 5) are stable in 0.1 M NaCl/LiCl- and 0.01 M CaCl2

solution whereas the particles with low DS (sample 1) aggregate atthese electrolyte concentrations due to the lower zetapotentialeven in buffer solutions. Fig. 5 reveals the dependency of the zetapotential and the particle size on the NaCl-concentration of dex-tran ibuprofen ester suspensions (samples 1 and 5). Again, the par-ticles of the higher substituted derivative exhibit a higher zetapotential. The particle suspension of sample 1 remains stable untila NaCl concentration of 0.01 mol/L and a zeta potential of�15.2 mV (Fig. 5a). Aggregation occurs at higher NaCl concentra-tion. The zeta potential of sample 5 has a maximum value of�56.9 mV at 0.001 mol/L NaCl-solution (Fig. 5b). The particles donot aggregate even at a concentration of 0.1 mol/L, and thus, theyshow improved stability against environmental changes. As aconsequence, the stability of the nanoparticles based on dextranibuprofen- and naproxen esters can directly be tuned via the DS va-lue – the higher the content of hydrophobic groups in the moleculethe higher is the stability in electrolyte containing media.

The stability of the particle suspensions only at low electrolyteconcentrations might limit their pharmaceutical applications. This

drawback can be overcome by using surfactants as stabilizers. Theaddition of a surfactant, such as polymeric non-ionic Tween 80(polyoxyethylene (20) sorbitan monooleate), avoids aggregationdue to steric repulsion of the nanospheres. Although the zeta po-tential is only �11.3 mV (�5.2 mV) at a NaCl– (CaCl2–) concentra-tion of 0.1 mol/L, the particle suspension of 1 is stable and exhibitsno change in particle size. A similar effect can be observed in thepresence of Tween 80 (c = 0.1 lmol/L) at acidic conditions. Despitea pH value of 1.7 and a zeta potential of 0.7, the particle suspensionis stable in the nanoscale range.

For the use of the dextran derivative nanoparticles prepared aspotential prodrugs in drug delivery systems, the feasibility of estercleavage of the dextran derivatives under physiological conditionsis an essential prerequisite. An enzyme, which is responsible forbiodegradation of dextran and its derivatives in living organisms,is dextranase[5]. After degradation of the polymer into low molarmass fractions, esterases and other hydrolases can attack easily[29]. In a preliminary experiment, the number average molar massin a particle suspension (sample 1) was reduced from 38,300 to1300 g/mol upon treatment with dextranase for 4.5 h. The degra-dation of the dextran ibuprofen ester particles into lower molarmass fractions thus seems to be possible.

4. Conclusion

The biopolymer dextran was functionalized with ibuprofen andnaproxen applying in situ activation with CDI, which led to hydro-phobic dextran derivatives. The molecules self-assemble into

62 S. Hornig et al. / Journal of Colloid and Interface Science 338 (2009) 56–62

nanoparticles, which are interesting novel potential drug deliverysystems, during nanoprecipitation via dialysis and dropping tech-nique without the need of additional surfactants. The degrees ofsubstitution, the character of the substituents as well as the prep-aration conditions strongly influence the size and the size distribu-tion of the nanoparticles. The nanoparticle suspensions are stableover months in a pH value range between 4 and 11 and at low elec-trolyte concentrations. Highly substituted derivatives form nano-particles that are more stable against hydrolysis and aggregationand exhibit a higher zeta potential. Improved stability can furtherbe obtained after addition of a non-ionic polymeric surfactant.

By choosing the appropriate DS, dextran based nanoparticles,which are also easily incorporated into cells [6], can be used as alter-native nanoparticulate polymer–drug conjugates. Because of thestability of the particles under physiological conditions, their sus-ceptibility of enzymatic degradation via dextranase is a basic requi-site for its use as macromolecular prodrug. As a further perspective,either natural molecular recognition of the polymer matrix due tospecific receptors in certain cells, or its functionalization with bio-specific ligands could be used to direct the particles to a target tissueor organ [30].

Acknowledgments

The authors gratefully acknowledge F. Steiniger (EMZ Jena) fortechnical assistance with the SEM equipment. T.H. thanks the‘‘Fonds der Chemischen Industrie”.

References

[1] R. Haag, Angew. Chem. Int. Ed. 43 (2003) 278.[2] R. Duncan, Nat. Rev. 2 (2003) 347.[3] J. Panyam, V. Labhasetwar, Adv. Drug Delivery Rev. 55 (2003) 329.

[4] C. Larsen, Adv. Drug. Delivery Rev. 3 (1989) 103.[5] R. Mehvar, J. Control. Release 69 (2000) 1.[6] S. Sugahara, M. Kajikia, H. Kuriyama, T. Kobayashi, J. Control. Release 117

(2007) 40.[7] T. Heinze, T. Liebert, B. Heublein, S. Hornig, in: D. Klemm (Ed.), Polysaccharide

II, Advances in Polymer Science, vol. 205, Springer, Berlin, 2006, p. 199.[8] J.A.M. Cadée, J.A. Van Luyn, L.A. Brouwer, J.A. Plantinga, P.B. Van Wachem, C.J.

De Groot, W. Den Otter, W.E. Hennink, J. Biomed. Mater. Res. 50 (2000) 397.[9] S. Hornig, C. Biskup, A. Gräfe, J. Wotschadlo, T. Liebert, G.J. Mohr, T. Heinze, Soft

Matter 4 (2008) 1169.[10] E. Harboe, C. Larsen, M. Johansen, H.P. Olesen, Pharm. Res. 6 (1989) 919.[11] F. Levi-Schaffer, A. Bernstein, A. Meshorer, R. Arnon, Cancer Treat Rep. 66

(1982) 107.[12] T. Kojima, M. Hashida, S. Muranishi, J. Sezaki, J. Pharm. Pharmacol. 32 (1980)

30.[13] Y. Ohya, H. Oue, K. Nagatomi, T. Ouchi, Biomacromolecules 2 (2001) 927.[14] T. Liebert, S. Hornig, S. Hesse, T. Heinze, J. Am. Chem. Soc. 127 (2005)

10484.[15] A. Aumelas, A. Serrero, A. Durand, E. Dellacherie, M. Leonard, Colloids Surf. B

59 (2007) 74.[16] D.E. Koppel, J. Chem. Phys. 57 (1972) 4814.[17] M. Vallet-Regi, A. Ramila, R.P. del Real, J. Perez-Pariente, Chem. Mater. 13

(2001) 308.[18] J. Kuntsche, K. Westesen, M. Drechsler, M.H.J. Koch, H. Bunjes, Pharm. Res. 21

(2004) 1834.[19] S.A. Galindo-Rodriguez, F. Puel, S. Briancon, E. Allemann, E. Doelker, H. Fessi,

Eur. J. Pharm. Sci. 25 (2005) 357.[20] B. Jiang, L. Hu, C. Gao, J. Shen, J. Int. J. Pharm. 304 (2005) 220.[21] D. Quintanar-Guerrero, E. Allémann, H. Fessi, E. Doelker, Drug Dev. Ind. Pharm.

24 (1998) 1113.[22] S. Hornig, T. Liebert, T. Heinze, Macromol. Biosci. 7 (2007) 297.[23] H.C. Fessi, J.-P. Devissaguet, F. Puisieux, C. Thies, US patent 5118528, 1992.[24] S. Hornig, T. Heinze, Biomacromolecules 9 (2008) 1487.[25] S. Hornig, T. Heinze, Carbohydr. Polym. 68 (2007) 280.[26] H. Cheng, J.D. Rogers, J.L. Demetriades, S.D. Holland, J.R. Seibold, E. Depui,

Pharm. Res. 11 (1994) 824.[27] T.M. Riddick, in: Control of Colloid Stability Through Zeta Potential, Zeta Meter,

New York, 1968.[28] K.G. Marinova, R.G. Alargova, N.D. Denkov, O.D. Velev, D.N. Petsev, I.B. Ivanov,

R.P. Borwankar, Langmuir 12 (1996) 2045.[29] R. Vercauteren, D. Bruneel, E. Schacht, R. Duncan, J. Bioact. Compat. Polym. 5

(1990) 4.[30] I. Brigger, C. Dubernet, P. Couvreur, Adv. Drug Delivery Rev. 54 (2002) 631.