2013

http://informahealthcare.com/mncISSN: 0265-2048 (print), 1464-5246 (electronic)

J Microencapsul, Early Online: 1–10! 2013 Informa UK Ltd. DOI: 10.3109/02652048.2013.814727

ORIGINAL ARTICLE

Cytotoxicity and in vitro characterization studies of synthesizedJeffamine-cored PAMAM dendrimers

K|v|lc|m Ozturk1, Ali Serol Erturk2, Can Sar|sozen1, Metin Tulu2, and Sema Cal|s� 1

1Department of Pharmaceutical Technology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey and 2Department of Chemistry, Faculty of

Arts and Sciences, Yıldız Technical University, Istanbul, Turkey

Abstract

The objective of this study is to make comprehensive cytotoxicity evaluation and in vitrocharacterization of Jeffamine-cored polyamidoamine (PAMAM) dendrimers on L929 cell linesfor oral drug delivery purposes. Ester-, amine- and carboxylic acid-terminated PAMAMs wereinvestigated for their cytotoxicity on L929 cells at different generations and concentrations.Cationic surface charge caused highest cytotoxicity on L929 cells, while ester-terminatedPAMAMs showed generation- and concentration-dependent toxicity. Anionic dendrimers weredetermined as the lowest cytotoxic group, and highest generation number presented lowestcellular toxicity. Encapsulation studies were performed with anionic PAMAMs at 2.5, 3.5 and 4.5generations and different concentrations. Increasing generation number provides greaterloaded naproxen amounts and larger particle size. Moreover, formulations provide controlledrelease at simulated terminal ileum conditions. Consequently, Jeffamine-cored carboxylic acid-terminated PAMAMs can be a promising option for oral drug delivery of poorly water-solubledrugs.

Keywords

Cell culture, dendrimer, Jeffamine, naproxen,oral drug delivery

History

Received 19 December 2012Revised 21 May 2013Accepted 10 June 2013Published online 17 July 2013

Introduction

Oral route is the major route of administration of dosage formsand provides various advantages over alternative administrationroutes such as possessing non-invasive nature, patient complianceand cost-effectiveness (Sastry et al., 2000; El-Sayed et al., 2002;Lin et al., 2011). On the other hand, it also has severaldisadvantages such as low bioavailability, low stability, insuffi-cient controlled release in some cases and gastrointestinal tractirritation (Felt et al., 1998). Nonsteroidal anti-inflammatory drugs(NSAIDs) are good representatives for the mentioned side effects.They are used to treat rheumatic disease, osteoarthritis and otherchronic musculoskeletal conditions, also have gastrointestinaland renal side effects, and hypersensitivity reactions (Cheng &Xu, 2005). Naproxen, presumably the most studied NSAIDs, ispractically insoluble in water and a nonspecific anti-inflammatorydrug that is usually used in water-soluble sodium salt form toformulate (Mura et al., 2001; Mitchell et al., 2003).

To overcome these low-numbered but important disadvantagesabout oral administration, several attempts have been made usingnanotechnological approaches. To name a few, drug-encapsulatednano-sized carriers like cyclodextrins, micelles and nanoparticleswere used to increase the solubility, stability and, at the end,bioavailability of the active substances (Mathot et al., 2006;Sajeesh & Sharma, 2006; Muchow et al., 2008). Among thesecarriers, dendrimers are hyperbranched, monodisperse, three-dimensional macromolecules, having defined molecular weight

and host–guest entrapment properties (Esfand & Tomalia, 2001;Cheng & Xu, 2005). Due to their well-defined structure, easilymodified surface groups, monodispersity, specific size and shapecharacteristics, dendrimers are excellent candidates for drugdelivery systems (Tomalia et al., 1985; Wong et al., 2012) andthus have been used for various pharmaceutical applicationsincluding, but not limited to targeted drug delivery, gene therapy,imagining, oral drug delivery, solubility and permeabilityenhancement (Najlah et al., 2007; Tsai & Imae, 2011).

Polyamidoamine (PAMAM) dendrimers are the first completedendrimer family to be synthesized, characterized and commer-cialized (Tomalia et al., 1985). They comprise repeated branchesthat are organized in concentric layers, called ‘‘generations’’,around a central core. Amine-terminated PAMAM dendrimers arecalled full generation (G0, G1, G2, G3, etc.), whereas carboxylate-terminated dendrimers are half-generation (G1.5, G2.5, G3.5,etc.). PAMAM dendrimers can exist as positively chargedamine-terminated, neutral hydroxyl-terminated or negativelycharged carboxyl-terminated (Lin et al., 2011), all three havingdifferent advantages in terms of functionalization and surfaceproperties (Perumal et al., 2008).

Drug–dendrimer interactions can be subdivided into theentrapment of drugs within the dendritic architecture (involvingelectrostatic, hydrophobic and hydrogen bond interactions) andthe interaction between a drug and the surface of a dendrimer(electrostatic and covalent interactions). Entrapment of drugswithin the dendritic structure is defined as encapsulation andobtained product can be termed as inclusion complex.Electrostatic interactions between the dendrimer surface groupsand drug is defined as complexation. Chemical conjugationprovides more stable structure than electrostatical interactions andthus favourable for targeted drug delivery systems, due to their

Address for correspondence: Prof. Sema Calis, Department of Pharma-ceutical Technology, Faculty of Pharmacy, Hacettepe University, 06100,Ankara, Turkey. Tel: + 90 312 305 12 41. Fax: + 90 312 310 09 06.E-mail: scalis@hacettepe.edu.tr

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ability to carry the drug to the target site without significant lossand early release. Although electrostatic complexation of drugs tothe dendrimer surface can provide controlled release, hydro-lysable or biodegradable linkages give a greater control over therelease. On the other hand, inclusion complexes may also haveadvantages like enhancing the stability and improving thesolubility of labile or poorly soluble drugs and overall increaseof their bioavailability too (D’Emanuele & Attwood, 2005;Patri et al., 2005).

Drug–dendrimer inclusion complexes are used to provideenhanced water solubility and transepithelial permeability ofpoorly water-soluble and/or low permeable drugs (Morgan et al.,2006; Najlah et al., 2007; Svenson & Chauhan, 2008). In thisstudy, synthesized JEFFAMINE T series (T-3000) core PAMAMdendrimers were used to evaluate cytotoxicity, to obtain inclusioncomplexes with practically insoluble drug naproxen base andto perform in vitro characterization of inclusion complexes.The cytotoxicity evaluations of amine-, ester- and acid-modifiedPAMAM dendrimers which were synthesized starting fromcommercial JEFFAMINE� T series (T-3000) core were per-formed. Naturally, not only the functional terminal groups but alsothe effect of generations and dendrimer concentration on cellulartoxicity were investigated. Although there are several studiesinvolving the cytotoxicity of PAMAM-based dendrimers ondifferent cell lines, the lack of a study systematically comparingdifferent end groups and generation numbers is needed to have abetter understanding of toxicity and oral use of dendrimers (Chenet al., 2004; Agashe et al., 2006; Hong et al., 2006). Ester- andcarboxylic acid-terminated dendrimers were synthesized at threedifferent generation numbers, 2.5, 3.5 and 4.5. Third-generationamine-terminated dendrimers were included in the study due totheir known cytotoxicity. A representative ester-, acid- and amine-terminated PAMAM dendrimers are illustrated in Figure 1.

Materials and methods

Materials

PAMAM dendrimers having ester, acid and amine terminalgroups with JEFFAMINE� T-3000 (provided from Huntsman,Europe) core were synthesized at the Department of Chemistry,Y|ld|z Technical University. Methanol and Cell Counting Kit-8(CCK-8) were purchased from Sigma (St Louis, MO). FetalBovine Serum (FBS), Dulbecco’s Modified Eagle’s Medium(DMEM) with high glucose, Penicillin–Streptomycin mixture(10 000 U Penicillin and 10 mg/mL Streptomycin), L-glutamineand Trypsin-EDTA solutions were from AppliChem (Darmstadt,

Germany). Naproxen base (NAP) and soluble Naproxen HCl saltwere gifts from Abdi _Ibrahim Pharmaceutical Company, Turkey.

Synthesis of PAMAM dendrimers

The compounds are prepared by slight modification of literatureprocedures (Tomalia et al., 1986; Newkome et al., 1992; Beezeret al., 2003). Full general procedures for preparation of thesecompounds are given in the experimental and the reactionsequence is shown in Figure 2. The physical and spectroscopicdata from FT-IR, 13C, 1H NMR and GPC do provide usefulinformation for their formation and structural characterization.These data and their prominent band assignments are reported inTables 1 and 2. Table 2 contains the molecular weightsdistribution of the first-generation dendrimers (P0, P0.5e, P0.5aand P1). These values were obtained from GPC versus polystyr-ene (PS) standard. Required analytical data and physical proper-ties are summarized at the end of each experimental.

Starting from P0 (Jeffamine�) polymer, P0.5e, P0.5a, P1dendrimers with three different surface functional groups weresynthesized as described below. By repeating the cycle in Figure2, higher generation of PAMAM dendrimers was synthesized (upto G4.0, G4.5e and G4.5 a). Purity of PAMAM dendrimers wascharacterized via FT-IR, 1H and 13C NMR and elemental analysis.The results agreed well with our previous report (Tulu et al.,2009).

Amine esterification (P0.5 e)

Methanolic solution (20 mL) of methylacrylate (20 g, 230 mmol)and a methanolic solution (100 mL) of (P0) (100 g, 33 mmol) weremixed slowly with constant stirring under nitrogen for 48 h atambient temperature. After this period of time, the solutionmixture was heated at 50 �C for further 1 h. The excessmethylacrylate and the solvent were removed under reducedpressure to dryness. The residue was purified by dialysis usingmembrane filter (3 kDa) in methanol–water (1:1), resulting ayellowish gel product (yield 100%). Elemental analyses found(calculated): C, 59.42 (62.18); H, 9.65 (10.23); N, 1.15 (1.20)[C182H357N3O58].

Ester hydrolysis (P0.5 a)

A solution of (P0.5 e) (20 g, 5.70 mmol) in formic acid (30 mL)was stirred for 12 h. After removing the excess formic acid andhydrolysed esteric groups under reduced pressure, gel product(P0.5 a) was obtained (yield 100%). Elemental analyses found(calculated) C, 58.72 (61.60); H, 8.65 (10.13); N, 1.17 (1.22)[C176H345N3O58].

Figure 1. Illustration of ester, acid and amine-terminated JEFFAMINE� cored PAMAM dendrimers (Tulu et al., 2009).

2 K. Ozturk et al. J Microencapsul, Early Online: 1–10

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Ester aminolysis (P1)

Methanolic solution (100 mL) of Hexaester (P0.5 e) (10 g,2.85 mmol) was added dropwise to a stirred methanolic solution(5 mL) of ethylenediamine (2.0 g, 33.33 mmol). The resultingsolution was stirred at room temperature for 7 days. The excessethylenediamine and solvent were removed under vacuum. Finaltraces of ethylenediamine was removed by dissolving the residuein 50 mL of n-butanol (a competitive hydrogen bonding solvent),the butanol was then removed under vacuum. The crude productwas dialyzed (MWCO of 3.0 kDa) in water. After evaporation ofwater, the yellowish gel product (P1) was obtained (9.20 g, 83%).Elemental analyses found (calculated) C, 59.32 (61.29); H, 9.67(10.42); N, 5.66 (5.70) [C188H381N15O52].

Cytotoxicity assay

The cytotoxicity of the dendrimer formulations, which areintended to be used orally, with different end groups andgenerations was tested using L929 mouse fibroblast cell line.Studied generations for ester- and carboxylic acid-terminateddendrimers were 2.5, 3.5 and 4.5, while the amine-terminateddendrimers were third generation. The medium for cell cultivatingwas prepared by adding FBS, penicillin-streptomycin andL-glutamine to DMEM at final concentrations of 10% v/v,50 U/mL–50 mg/mL and 2 mM, respectively. Cells were grown in25 cm2 cell culture flasks in a cell culture incubator (5% CO2,37 �C) until approximately 80% confluence. Then, cells weretrypsinized and 100 mL of cell suspension containing 5�103 cells

Figure 2. Synthetic route of ester, acid andamine-terminated dendrimers�.

DOI: 10.3109/02652048.2013.814727 Cytotoxicity and in vitro characterization of Jeffamine-cored PAMAM dendrimers 3

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was seeded into each well of the 96-well, flat-bottomed plates andwere kept overnight in the incubator to allow them to attach to thewells. The following day, medium was removed, and 100mL ofester-, amine- or carboxylic acid-terminated dendrimer formula-tions with different dilutions in culture medium was added intothe wells (n¼ 3). After 24 h of continuous incubation, mediumcontaining dendrimer formulations was removed and replacedwith 100mL of fresh medium to prevent possible interactionsbetween excess dendrimers and cell counting kit reagent. Tenmicrolitre of CCK-8 reagent was then added into the wells andtests were carried on according to the manufacturer’s protocol.Wells that were not treated with dendrimer formulations wereused as the control group. Mann–Whitney U non-parametric testwas used for statistical investigation of differences betweenthe groups.

Preparation of naproxen–dendrimer inclusion complexes

Carboxylic acid-terminated dendrimers were selected for nextstep studies according to cytotoxicity assay results. The initialNAP solution concentrations were 5 mg/mL and 10 mg/mL forcomplexation of drug with dendrimer. Encapsulation efficienciesaccording to drug concentration were also determined. Drugcontents were evaluated for each generation number and dendri-mer concentration. For the preparation of complexes, NAP anddendrimers were dissolved in methanol separately. Drug solutionof 1 mL and dendrimer solution of 1 mL were used for eachformulation.

In brief, NAP solution was added onto dendrimer solution onmagnetic stirrer drop by drop. Vials were covered and protectedfrom light due to photosensitivity of the active compound. Then,the solutions were stirred at 500 rpm for 24 h at room temperature.After 24 h, methanol was removed under nitrogen gas andfollowed by addition of 1 mL distilled water. At the last step ofpreparation of inclusion complexes, naproxen-loaded dendrimersand non-encapsulated free naproxen are separated via centrifuga-tion (5000 rpm, 20 min). Free naproxen in the aqueous mediumforms crystals and precipitates immediately, and at the bottom ofthe centrifuge tube unloaded free naproxen is obtained. Particlesizes of the untreated naproxen crystals were approximately10 mm, while loaded dendrimers have a hydrodynamic diameter

Table 1. Prominent FT-IR, 1H and 13C band assignment for the (P0, P0.5 e, P0.5 a, P1) compounds*.

Com. IR (cm�1) 13C NMR (ppm) 1H NMR (ppm)

P0 3296, 1110 CNH2¼ 46.17 CNH2¼ 3.54 (6H, br, s)

P0.5 e 1739 COOCH3¼ 173.06 CH2CH2COOCH3¼ 2.39 (12H, t)1108 COOCH3¼ 51.35 CH2CH2COOCH3¼ 2.75 (12H, t)

CNR2¼ 55.19 COOCH3¼ 3.63 (18H, s)CH2CH2COOCH3¼ 46.38CH2CH2COOCH3¼ 34.46

P0.5 a 3300–2650 COOH¼ 171.95 CH2CH2COOH¼ 3.40 (12H, t)1671 CNR2¼ 56.90 CH2CH2COOH¼ 3.55 (12H, t)1110 CH2CH2COOH¼ 46.59 COOH¼ 8.05 (6H, br, s)

CH2CH2COOH¼ 31.28

P1 3296 CONCH2CH2NH2¼ 173.02 CNH2¼ 4.79 (12H, br, s)1653 CONCH2CH2NH2¼ 57.51 CH2CH2NH2¼ 3.38 (12H, m)1551 CONCH2CH2NH2¼ 46.20 CH2CH2NH2¼ 3.54 (12H, m)1108 CONHR¼ 4.79 (6H, br, s)

P1.5 e 3450 COOCH3¼ 172.91 CH2CH2COOCH3¼ 2.40 (24H, t)1731 COOCH3¼ 75.25 CH2CH2COOCH3¼ 2.72 (24H, t)1666 CH2CH2NR2¼ 51.49 COOCH3¼ 3.63 (36H, s)1105 CH2CH2COOCH3¼ 49.69

CH2CH2COOCH3¼ 32.56

P1.5 a 3300–2550 COOH¼ 172.06 CH2CH2COOH¼ 2.63 (24H, t)1667 CNR2¼ 52.15 CH2CH2COOH¼ 3.11 (24H, t)1105 CH2CH2COOH¼ 48.26 COOH¼ 9.97 (12H, br, s)

CH2CH2COOH¼ 29.55

P2 3421, 3349 CONCH2CH2NH2¼ 172.88 CNH2¼ 5.10–4.60 (24H, br, s)3289, 1643 CONCH2CH2NH2¼ 55.67 CH2CH2NH2¼ 3.39 (24H, m)1567, 1112 CONCH2CH2NH2¼ 46.29 CH2CH2NH2¼ 3.55 (24H, m)

CONHR¼NO

P2.5 e 3309 COO CH3¼ 172.86 CH2CH2COOCH3¼ 2.42 (48H, t)1737 COO CH3¼ 75.41 CH2CH2COOCH3¼ 2.74 (48H, t)1650 CH2CH2NR2¼ 51.48 COO CH3¼ 3.65 (72H, s)1544 CH2CH2COOCH3¼ 49.15

CH2CH2COOCH3¼ 32.63

P2.5 a 3442 COOH¼ 172.67 CH2CH2COOH¼ 2.50 (48H, t)1656 CNR2¼ 52.14 CH2CH2COOH¼ 2.85 (48H, t)1590 CH2CH2COOH¼ 48.58 COOH¼ 8.31 (24H, br, s)1101 CH2CH2COOH¼ 30.22

*Spectral deconvolution of higher generations (up to P4.5) is very close to the data assigned in Table 4.

Table 2. Molar mass average (Mn, Mw) and polydispersity index(Mw/Mn) values of dendrimer samples.

Sample Expected Mw Mn (g/mol) Mw (g/mol) Mw/Mn

P0 3000 469 505 1.07P0.5 e 3511 470 512 1.09P0.5 a 3427 418 451 1.08P1 3679 462 498 1.08

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of 290 nm max (Nokhodchi & Maghsoodi, 2008). Supernatantcomprises suspension of the naproxen–dendrimer complexes andcan be called as complex suspension. Characterized dendrimerformulations were encoded according to generation number andconcentration of dendrimer with carboxylic acid terminal groupsas seen in Table 3.

Characterization of naproxen–dendrimer inclusioncomplexes

Particle size and zeta potential measurements

Mean particle diameter and polydispersity index were determinedby dynamic light scattering using a Malvern Instruments ZetasizerNano Series (Malvern, UK) at 173� backscattering. For eachsample, the mean diameter of six determinations was calculated.Nanoparticles were also characterized with respect to zetapotential using the same instrument. Samples were diluted indeionized water and placed in disposable measurement cells andthen measured in triplicate. Mann–Whitney U non-parametrictest was used for statistical investigation of differences betweenthe groups.

Differential scanning calorimetry analysis

Differential Scanning Calorimetry (DSC) thermograms wererecorded on a Q-200 DSC instrument (TA instrument, NewCastle, DE) calibrated with indium. Samples of precisely weighted5 mg lyophilized powders were packed in an aluminium pan andthen heated from 25 �C to 300 �C at a scanning rate of 10 �C/min.

Determination of naproxen content in the inclusion complexes

UV-Vis spectrometer was used to estimate the amount of drugincorporated in and released from dendrimers. NAP in methanolgives maximum absorbance in UV region at 231 nm. A calibrationcurve of NAP in methanol at different concentrations wasprepared and found linear over a concentration range of 0.25–8mg/mL with r2 value of 0.998. Encapsulated NAP amounts in thecomplexes were evaluated indirectly. After centrifugation ofdendrimers at 5000 rpm for 30 min, free NAP was precipitatedand separated from solubilized NAP in dendrimers. Two millilitreof methanol was added on the NAP to obtain drug solution. Afterrequired dilutions, non-encapsulated NAP amount was deter-mined via UV-Vis Spectrophotometer.

In vitro release studies

Dialysis method was used to obtain release profile of formulations(D’Souza & DeLuca, 2006). Release medium and time werechosen according to USP 35 Naproxen Tablet DissolutionMethod, using 0.1 M pH 7.4 phosphate buffer as the releasemedium. Medium volume was chosen as 20 mL to provide sinkconditions. Drug–dendrimer inclusion complex suspensions(1 mL in volume) were transferred into dialysis bags

(MWCO size 2 kDa). The dialysis bags were then placed in abeaker containing the release medium. The outer phase wasstirred continuously at 50 rpm. After a scheduled interval of timefor 2 h, 1 mL of sample was withdrawn from the outer phase, andthe outer phase was again replenished with 1 mL fresh medium.The absorbance of the samples was monitored at 231 nm using aspectrophotometer to quantitate the concentration of NAP (n¼ 3)(Kolhe et al., 2003).

Results

Spectral deconvolution of synthesized of dendrimers

In the synthesis of dendrimers, three functional groups werehighlighted. These are (i) esterifications of polyether core, (ii)hydrolysis of esters and (iii) aminolysis of esters. By examiningTable 1, these groups were easily monitored by spectroscopictechniques and elementary analysis. The characteristic n (NH2)and � (NH2) modes of primary amines (P0, P1) were observedin the regions 3400–3300 and 1650–1550 cm�1, respectively.The formation of ester (P0.5 e) has been verified by theappearance of very characteristic (C¼O) stretching vibrations inthe 1740–1720 cm�1 region. These results were supported with1H and 13C NMR spectral data. The 1H NMR spectra pattern haschanged significantly due to esterification of amines by appear-ance of several new signals and disappearance of broadunresolved amine protons. The 13C NMR data also tend tosupport ester formation by appearance of several new peaks,particularly for carboxyl (C¼O) chemical shifts at approximately173 ppm, typical for ester compound.

Ester hydrolysis pathway (formation of carboxylic acid den-drimer P0.5 a) could also be confirmed by IR and NMRspectroscopic data. The band corresponding to n(O–H) of theCOOH group is observed as a broad band at �3440 cm�1. Thismay be taken as evidence that the ester groups fully hydrolyzedto carboxylic acid groups. This was also confirmed by lowerfrequency shift of n(C¼O) characteristic mode from ester to acidfrom �1740 to �1650 cm�1. The 1H NMR spectra pattern haschanged significantly due to hydration of the ester groups tocorresponding carboxylic acid groups. This was confirmed by theappearance of a singlet in the 8–10 ppm region for the COOHproton chemical shift and by disappearance of singlet-methoxyproton chemical shift at 3.63 ppm. The formation of COOH canalso be supported by absent of 13C NMR signal for the methoxygroups from the ester and as well as appearance of a new band atlower chemical shift values for COOH groups. The formation ofamine from ester via aminolysis (P1) could be confirmed bysimilar strategic manner using vibrational and nuclear magneticresonance spectral data. Appreciate band assignments are pre-sented in Table 1.

GPC results show that first generation dendrimers (P0–P1)were relatively monodisperse. However, measured MWs weresmaller than expected value. This may be attributed to thereference molecules (polystyrene), which eluded much faster than

Table 3. Formulation codes of studied dendrimer formulations.

Surfacefunctionality

Generation numberof dendrimer

Concentration ofdendrimer (mg/mL)

Surface carboxylicacid number of dendrimer

Molecular weight ofdendrimers (g/mol)

Formulationcode

–COOH 2.5 0.5 32 4753 G2.5C0.5–COOH 2.5 1 32 4753 G2.5C1–COOH 3.5 0.5 64 9752 G3.5C0.5–COOH 3.5 1 64 9752 G3.5C1–COOH 3.5 5 64 9752 G3.5C5–COOH 4.5 0.5 128 19750 G4.5C0.5–COOH 4.5 1 128 19750 G4.5C1–COOH 4.5 5 128 19750 G4.5C5

DOI: 10.3109/02652048.2013.814727 Cytotoxicity and in vitro characterization of Jeffamine-cored PAMAM dendrimers 5

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our molecules. The dendrimers are expected to exhibit signifi-cantly larger chains stiffness than the calibration standardpolystyrene, because the steric repulsion of the voluminousdendritic side chains should stretch the polymeric backboneconsiderably. These chain stiffness leads to an increasedhydrodynamic volume, which causes the GPC molar mass tobecome much smaller than the true molar mass. These resultsare consistent with the literatures (Percec et al., 1997;Schluter, 1998).

Cytotoxicity assay

The cytotoxicity results of the ester-, amino- and carboxylic acid-terminated dendrimers were given in Figures 3 and 4. Amine-terminated dendrimers were found to be the most cytotoxicdendrimers among the studied ones (Figure 4). A concentration1 mg/mL of amine-terminated dendrimers resulted cell viabilityof 48%, and all the cells were lost when higher concentrationswere applied. According to the results, amine-terminated dendri-mer formulations were discarded from further studies.

As can be seen from Figure 4, generation 2.5 of ester-terminated dendrimers were found to be the least toxic generationon L929 cells. Highest toxicity was observed with generation 4.5.They caused a significant cytotoxicity against L929 cells, andeven at the lowest concentration the cell viability was below 50%.A concentration 1 mg/mL of generation 2.5 did not significantlydecrease the cell viability (100% viability), but unfortunately theencapsulation efficiencies and release profiles were lower thancarboxylic acid-terminated dendrimers.

In Figure 5, the cytotoxicity results of the carboxylic acid-terminated dendrimers were demonstrated. Obtained results showthat generation 3.5 and 4.5 of carboxylic acid-functionalizeddendrimers at concentrations of up to 5 mg/mL did not show anysignificant toxicity (p40.05) on the L929 cells (108.9% and97.8%, respectively, relative to the control group). Moreover,generation 2.5 did not affect the cell viability at 0.5 and 1 mg/mLconcentrations, but when the concentration was increasedto 5 mg/mL, approximately 35% cell death was observed.The similar amount of cell death was obtained with generation3.5 and 4.5 only at concentrations around 10 mg/mL.

Results obtained from cell culture studies indicate thatcarboxylic acid-terminated dendrimers were the optimum formu-lation regarding their cytotoxicity results, among the studieddendrimer types.

Particle size and zeta potential measurements

The mean diameter of NAP–dendrimer complexes were found tobe between 45 and 261 nm as seen in Table 4. Polydispersityindexes of formulations are between 0.08 and 0.16 (Table 4).NAP–PAMAM complexes showed increased particle size withincreasing generation number (p50.05). Zeta potential values arenegative, due to anionic carboxylic acid groups that exist on thesurface, and in the range of �3 to �20.8 mV (Table 4).

Differential scanning calorimetry analysis

As can be seen in Figure 5, NAP exhibited a sharp melting peak at153 �C. Between 105 �C and 150 �C, multiple peaks related tolyophilized dendrimer sample was observed. In the presence ofP4.5 a dendrimer, the melting point temperature of drug changedsignificantly with decreased heat of fusion. Disappearances ofsharp NAP melting, dendritic multiple peaks and appearance ofbroad endothermic peak could be attributed to successiveencapsulation of NAP with the PAMAM dendrimer.

Loading capacity

Encapsulation ability of different amounts of drug in dendrimer(PAMAM–COOH) was studied to estimate the ‘‘maximum’’amount of NAP molecules that can be incorporated in adendrimer molecule.

Preformulation studies were performed to determine maximumNAP amounts that can be encapsulated. Therefore, 1.106, 0.85,0.766, 1.67, 4.627, 0.808, 1.049 and 4.7 mg NAP were selected forG2.5C0.5, G2.5C1, G3.5C0.5, G3.5C1, G3.5C5, G4.5C0.5,G4.5C1 and G4.5C5, respectively. These amounts were usedthroughout in vitro characterization studies and also assumed as %100 loading was achieved for each formulation.

Designed systems show different loading capacities accordingto their concentration and generation number. Effects of

Figure 3. Cytotoxicity evaluation of amine- and ester-terminated dendrimers on L929 cells after 24 hours (n¼ 3, mean� standard deviation).

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generation number and dendrimer concentration on the encapsu-lation of NAP amounts were evaluated. Loading capacity refers toamount of NAP (mg) per amount of mg dendrimer. Additionally,encapsulated NAP amount was presented as percentage offormulation. The results showed that increased generationnumber and dendrimer concentration provide higher encapsulatedamount of NAP, but the relationship was found to be nonlinear asshown in Table 5.

In vitro release studies

In vitro release studies indicated that prepared inclusioncomplexes showed higher release than NAP base and NAP

Figure 5. DSC curves of (A) naproxen, (B) generation 4.5 Jeffamine-cored PAMAM and (C) G4.5C5 formulation.

Figure 4. Cytotoxicity evaluation of carboxylic acid-functionalized dendrimers on L929 cells after 24 hours (n¼ 3, mean� standard deviation).

Table 4. Particle size, polydispersity index and zeta potentialmeasurements of naproxen–PAMAM dendrimer complexes.

Formulation codeParticle

size (nm)Polydispersity

indexZeta potential

(mV)

G2.5C0.5 45.2� 21 0.15 �3.47� 0.7G2.5C1 56.4� 3.2 0.09 �10.4� 3.23G3.5C0.5 117.6� 2.4 0.12 �2.57� 1.3G3.5C1 129� 8.4 0.08 �3� 0.6G3.5C5 152� 1.9 0.11 �20.8� 1.08G4.5C0.5 181� 17 0.16 �3.1� 0.3G4.5C1 263� 10.8 0.14 �13.9� 0.73G4.5C5 291� 4.6 0.11 �14.2� 0.3

DOI: 10.3109/02652048.2013.814727 Cytotoxicity and in vitro characterization of Jeffamine-cored PAMAM dendrimers 7

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sodium (Figure 6). G2.5 dendrimers released 55% of their NAPamount regardless of the dendrimer concentration. Moreover, itwas found that, highest amount of NAP released from G3.5carboxylic acid-terminated PAMAM dendrimers at 1 mg/mLconcentration. Lower or higher dendrimer concentrations than1 mg/mL resulted in decreased release. As for G4.5, 95% ofcumulative release was achieved by using 5 mg/mL concentration.

Discussion

In this study, acceptable cytotoxicity of the dendrimer formula-tions was 5%, meaning that formulations which have more than5% cytotoxicity on the cells were evaluated as not-acceptable.Despite the fact that ester-functionalized dendrimers were lesscytotoxic than that of the amine-functionalized ones, they causedsignificant generation-dependent toxicity. When the ester-functionalized dendrimer formulations were considered, the onlygeneration and concentration that did not have any effect on cellviability was generation 2.5 and 1 mg/mL, but this formulationresulted in very low encapsulation efficiencies and less favourablein vitro characteristics (data not shown).

The main aim of this study was to choose a Jeffamine-coreddendrimer type and concentration that is safe regarding thecytotoxicity on L929 cells, while demonstrating suitable and

favourable in vitro characterization parameters including encap-sulation efficiency and release properties. Although Aydin et al.used Jeffamine-cored PAMAM dendrimers for gene delivery, theyonly investigated the cytotoxicity on HeLa cells, and the intendedadministration route was parenteral. A systematic cytotoxicitycomparison of PAMAM dendrimers intended for oral adminis-tration using L929 cell line was needed to decide nontoxicdendrimer type and concentration. It was shown earlier thatcationic dendrimers exert elevated cytotoxicity due to terminal–NH2 groups and high cationic charge (Malik et al., 2000;Kolhatkar et al., 2007). Epithelial cells are known to possessnegative charge and thus cationic dendrimers may show strongelectrostatic interactions and consequently disturb the cell mem-brane and thus result in high cellular toxicity (Perumal et al.,2008). But, surprisingly, Kissel’s group reported minimal cyto-toxicity of third-generation cationic PAMAM dendrimers (Fischeret al., 1999). Even though they found that the IC50 concentrationof the cationic PAMAM dendrimers was above 10 mg/mL(Fischer et al., 2003), our results were supportive to the previouslypublished studies, and indicated that amine-terminated dendri-mers were highly cytotoxic even at the lowest studied concentra-tion of 1 mg/mL (Kolhatkar et al., 2007; Goldberg et al., 2011;Sadekar & Ghandehari, 2012). Thus, they were discarded fromfurther studies.

Figure 6. In vitro release profiles of different formulations at pH 7.4 phosphate buffer solution. Cumulative released amount of free drug in releasemedium from NAP and naproxen sodium solutions at pH 7.4 phosphate buffer solution was given as insert.

Table 5. Encapsulation characterizations of formulations.

Formulationcode

Encapsulated NAPamount (mg)

Loading capacity(mg/mg)

Amount of NAP (mmol)per mmol dendrimer Percentage of NAP

G2.5C0.5 1.106� 0.02 2.2� 0.04 47.82� 0.87 68.75� 0.38G2.5C1 0.852� 0.03 0.85� 0.03 18.53� 0.657 46� 0.88G3.5C0.5 0.766� 0.006 1.532� 0.013 66.63� 0.57 60.53� 0.21G3.5C1 1.67� 0.09 1.67� 0.09 72.61� 4.14 62.5� 1.35G3.5C5 4.627� 0.007 0.934� 0.001 40.24� 0.068 48.06� 0.04G4.5C0.5 0.808� 0.01 1.616� 0.028 140.51� 2.4 61.77� 0.42G4.5C1 1.049� 0.005 1.049� 0.005 91.24� 0.44 51.2� 0.12G4.5C5 4.7� 0.1 0.94� 0.022 80.72� 1.9 48.45� 0.6

8 K. Ozturk et al. J Microencapsul, Early Online: 1–10

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Despite the fact that ester-functionalized dendrimers wereless cytotoxic than that of the amine-functionalized ones, theycaused significant generation-dependent toxicity. When the ester-functionalized dendrimer formulations were considered, the onlygeneration and concentration that did not have any effect on cellviability was generation 2.5 and 1 mg/mL, but this formulationresulted very low encapsulation efficiencies and less favourablein vitro characteristics (data not shown).

In general, cytotoxicity of the dendrimers increase with thegeneration number due to large number of surface groups resultedwith high cell penetration. On the other hand, in this case it wasfound that cytotoxicity of the carboxylic acid-terminated dendri-mers (Figure 4) decreased with increasing generation number. TheIC50 values of these formulations were 13.32 and 13.92 mg/mLfor generation 3.5 and 4.5, respectively. Although 10 mg/mLconcentration is below the IC50 values, at this concentration,generations 3.5 and 4.5 caused approximately 40% celldeath. Interestingly, while generation 2.5 of carboxylic acid-functionalized dendrimers did not demonstrate any significantcytotoxicity at 0.5 and 1 mg/mL, higher concentrations resulted inincreased toxicity than generation 3.5 and 4.5. This phenomenonof increased toxicity by decreased generation could be explainedby different hypotheses. Lee et al. reported that shielding theinternal core of the dendrimers by surface groups could causedecreased toxicity (Lee et al., 2003). Increased generation ofdendrimers means increased branching and higher surface densityof hydrophilic and biocompatible end groups like –COOH.A potentially toxic dendrimer core could be more accessible andexposed to the L929 cells when the lower generation of carboxylicacid-functionalized dendrimers was used. Moreover, it was foundthat cell type is also an important factor for cytotoxicityevaluation. Malik et al. found different cytotoxicity results indifferent cell lines by using same dendrimer formulations (Maliket al., 2000). It was also possible that the dendrimer formulationsnewly synthesized for this study could comprise impuritiesescaped from purification processes in lower generations.

Nevertheless, the obtained results indicate that amine-terminated PAMAM dendrimers were not suitable due to theirsignificantly high cellular toxicity and thus discarded fromstudies. Ester-terminated dendrimers were eliminated fromfurther studies because their non-cytotoxic concentrations andgenerations resulted in insufficient in vitro characterizationproperties when compared to their carboxylic-acid functionalizedcounterparts.

Prepared formulations were evaluated in terms of thermalbehaviours to show complexation. G4.5C5 formulation was usedas a selected formulation for DSC analysis. Results showed thattransition to amorphous structure from crystalline form of NAPoccurred in the dendrimer formulation. It can be concluded thatNAP is not presented on the surface of the dendrimers and thuscomplexation with dendrimers is achieved successfully.

In vitro characterization studies of anionic PAMAM dendri-mers showed that particle size increase of NAP–dendrimercomplexes was generation dependent. In the same generation,increasing of polymer concentration did not significantly changethe particle size. In addition, polydispersity index values indicatedthat monodisperse particle size distribution was obtained for allformulation. Zeta potential is also an important indicator forstability of colloidal systems. Colloidal stability of dendrimersuspension is important due to their monodispersity and homo-geneity. It is recommended that zeta potential should be between10 and 30 mV (negative or positive). Samples with low zetapotential tend to aggregate. The small size of dendrimerformulations can also cause stability problems. Hence, enhancingthe stability of this type of formulation still maintains itsimportance. It was clearly observed that augmentation of

dendrimer concentration at generation 3.5 and 4.5 provideshigher encapsulated amount of NAP. Although increased encap-sulation of NAP had been provided, because it was nonlinear,there is no augmentation at loading capacity and also at theamount of NAP (mmol) per mmol dendrimer. Regarding to in vitrorelease studies, it is critical for estimation of in vivo behaviour ofdrug delivery system. Oral delivery was the proposed route ofadministration of the dendrimer formulations developed in ourstudy, thus in vitro release studies were carried out according toUSP 35 Naproxen Sodium Tablet Dissolution monograph. All theformulations showed higher release from free NAP solution,while only three formulations, G4.5C5, G3.5C1 and G4.5C05,indicated higher release from free NAP sodium solution. Allformulations presented more than 50% release of naproxen at120 min except three of them which provided 80% release ofnaproxen at least.

Conclusion

This study is substantial due to the whole cytotoxicity analysis ofsynthesized PAMAMs with ester, amine and carboxylic acid endgroups. Surface charge and concentration dependence on cyto-toxicity of synthesized PAMAMs on L929 cell lines wereevaluated in point of oral drug delivery of NAP. Obtained resultsshowed that only carboxylic acid-terminated ones are appropriatefor administration and strategy of this research. Besides this, withrespect to the cytotoxicity results, appropriate formulations weredeveloped at the end of in vitro characterization stages. Theseformulations could be promising for oral administration.

Declaration of interest

We gratefully thank the Y|ld|z Technical University Project Office (2011-01-02-KAP04, 2011-01-02-KAP05, 2011-01-02-KAP06) and EULifelong Learning Programme (Webgentech Project number: 2010-1-TR1-LEO05-16728).

The authors report no conflicts of interest. The authors alone areresponsible for the content and writing of the article.

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