A

Pttidcctc©

Ka

1

a(os(oA1O

2T

0d

Available online at www.sciencedirect.com

International Journal of Pharmaceutics 350 (2008) 113–121

Permeability of surface-modified polyamidoamine (PAMAM)dendrimers across Caco-2 cell monolayers

Dipak S. Pisal a, Venkata K. Yellepeddi a, Ajay Kumar a, Radhey S. Kaushik b,Michael B. Hildreth c, Xiangming Guan a, Srinath Palakurthi a,∗

a Department of Pharmaceutical Sciences, 1 Administration Lane, South Dakota State University, Brookings, SD 57007, United Statesb Department of Biology & Microbiology, 1 Administration Lane, South Dakota State University, Brookings, SD 57007, United States

c Department of Veterinary Sciences, 1 Administration Lane, South Dakota State University, Brookings, SD 57007, United States

Received 28 March 2007; received in revised form 17 August 2007; accepted 21 August 2007Available online 26 August 2007

bstract

Aim of this study was to prepare polyamine-conjugated PAMAM dendrimers and study their permeability across Caco-2 cell monolayers.olyamines, namely, arginine and ornithine were conjugated to the amine terminals of the G4 PAMAM dendrimers by Fmoc synthesis. The apical-

o-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (Papp) for the PAMAM dendrimers increased by conjugatinghe dendrimers with both of the polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration ofncubation. The correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with both the PAMAMendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelialells. Cytotoxicity of the polyamine-conjugated dendrimers was evaluated in Caco-2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-

onjugated dendrimers were slightly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations onhe possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer–polyamineonjugates as drug carriers to increase the oral absorption of drugs.

2007 Elsevier B.V. All rights reserved.

e (TE

spmebd1sf

eywords: PAMAM dendrimers; Polyamines; transepithelial electrical resistancssay

. Introduction

Polyamines (ornithine, putrescine, spermidine and spermine)re ubiquitous and essential for cell growth and differentiationPegg, 1988; MacRae et al., 1998). Intracellular concentrationsf the polyamines are maintained via their biosynthesis and tran-cellular uptake (Schiper et al., 2003). Ornithine decarboxylaseODC) is a key enzyme that regulates the decarboxylation ofrnithine to putrescine in polyamine biosynthesis (Pegg, 1988;

uvinen et al., 1992; Harold and Margaret, 1996; O’Brien et al.,997; Mafune et al., 1999; Liu et al., 2000; Schiper et al., 2003).nce putrescine is formed, the activity of the other polyamine

∗ Corresponding author at: College of Pharmacy, 1 Administration Lane, Box202 C, South Dakota State University, Brookings, SD 57007, United States.el.: +1 605 688 6072; fax: +1 605 688 6232.

E-mail address: Srinath.palakurthi@sdstate.edu (S. Palakurthi).

tJbr

tist

378-5173/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.ijpharm.2007.08.033

ER); Caco-2 cells; Transepithelial transport; methylthiazoletetrazolium (MTT)

ynthetic enzymes (spermidine synthase and spermine synthase)rovide for the sequential formation of spermidine and sper-ine. The gastrointestinal mucosa has rapid turnover rate which

quates to high rates of cell growth and differentiation. It haseen reported that polyamines are involved in the growth andifferentiation of gastrointestinal cells (Heby, 1981; Johnson,988; Seiler et al., 1996). In the stomach luminal polyaminestimulate repair of gastric mucosal injury (Luk et al., 1988),or example, putrescine injection was shown to stimulate oxyn-ic mucosal growth (Majumdar and Johnson, 1987; Wang andohnson, 1990). The polyamines from food sources are found toe absorbed by passive diffusion process through paracellularoute (Devens et al., 2000).

Efforts to design novel formulations to increase the absorp-

ion of poorly soluble and poorly absorbable drugs have beenncreasing recently. Dr. Kimura’s research group has usedodium laurate (C12) with taurine or l-glutamine to enhancehe absorption of poorly absorbable drug (e.g., rebamipide)

1 al of

faMnraSl2at2

lcoigdTeGts(fSadimEcmtlofl(d(dnwba(

pGettspsa

ms

2

2

wg[(dd4wTppNyAN2(Fsr

2d

wsiAmeeo2twflvaewdaAs

14 D.S. Pisal et al. / International Journ

rom colon and rectum without much toxicity (Messing etl., 1999; Aungst, 2000; Yata et al., 2001; Endo et al., 2002;iyake et al., 2003). However, the absorption enhancement was

ot efficient enough for oral administration. Subsequently, thisesearch group has explored other absorption enhancers suchs polyamines, spermidine and spermine (Miyake et al., 2003).everal absorption enhancers such as cationic amino acids, poly--arginine (Miyake et al., 2006), poly-l-lysine (Ohtake et al.,002) cationic gelatin (Ahnet et al., 2004), and cationic lipidsnd liposomes have also shown significant increase in absorp-ion but with considerable toxicity (Li et al., 2005; Seki et al.,005).

Polyamidoamine (PAMAM) dendrimers are macromolecu-ar hydrophilic polymers. They are synthesized from an initiatorore (ammonia or ethylene diamine) around which the branchesf the dendrimer originate. The dendritic structure is character-zed by ‘layers’ between each focal point (or cascade) calledenerations (Boas and Heggard, 2004). The core is sometimesenoted generation zero (G0), as no cascade points are present.he number of surface groups of PAMAM dendrimers increasesxponentially from 4 to 64 as the generation size increases from0 to G4. Because of their highly branched structure with easy

ailorability of surface groups with targeting moieties, and pos-ibility of entrapment of guest molecule in the dendrimer coredendritic box), they have been widely investigated as carriersor drug delivery and targeting (Janasen et al., 1994; Roy, 1997;akthivel et al., 1999; Wiwattanapatapee et al., 2000; Ma etl., 2005). The permeability of different generations of the den-rimers across the epithelial cell monolayers was reported to ben the order of G4 � G0 ≈ G1 > G3 > G2, and the rate of per-

eability was concentration dependent (Florence et al., 2000;l-Sayed et al., 2001). In addition to size, concentration andharge also influence the dendrimer transport presumably byodulating the intercellular tight junctions. It is suggested that

he possible mechanism of dendrimer transport across epithe-ial cells is adsorptive mediated endocytosis, in addition tother active transport mechanisms including receptor-mediated,uid-phase endocytosis, as well as carrier mediated processesFlorence, 1997). Recent studies by D’Emanuele et al. (2004)emonstrate that conjugation of small molecular weight drugsthat are known P-glycoprotein substrates) to PAMAM den-rimers increases their transport indicating that dendrimers areot substrates for P-glycoprotein. Use of palmitoyl carnitine,hich was found to decrease the lipid order of intestinal brushorder membrane vesicles, along with G2-NH2 dendrimers waslso shown to increase the Caco-2 cell monolayer permeabilityD’Emanuele et al., 2004).

Polyamines are assumed to be transported into the cell byolyamine transporter protein (PAT) system (Cullis et al., 1999;ardner et al., 2004). Based on their intestinal permeation

nhancement effect, and their absorption by carrier mediatedransport, we hypothesized that conjugation of the polyamineso the PAMAM dendrimers increases their permeability pre-

umably because of dendrimer uptake by a combination ofaracellular pathway as well as by the PAT system. As a firsttep to realize this objective, we have prepared ornithine andrginine-conjugated PAMAM (G4) dendrimers and their per-

Orbm

Pharmaceutics 350 (2008) 113–121

eabilities across Caco-2 cell monolayer and cytotoxicity weretudied.

. Materials and methods

.1. Materials

PAMAM dendrimers (both G4-NH2 and 3.5 G-COOH)ere obtained from Dendritic Nanotechnologies Inc., Michi-an, USA. Fluoresceine isothiocyanate dye (FITC), 3-4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromideMTT), piperidine, N,N-dimethylformamide (DMF), N,N-iisopropylethylamine (DIPEA), diethyl ether, D2O andihydrobenzoic acid (DHB), tetramethyl silane (TMS) and-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)ere purchased from Sigma–Aldrich (St. Louis, MO, USA).rifluoroacetic acid (TFA), phosphate buffered saline (PBS,H 7.4), acetone and acetonitrile (both HPLC grade) wereurchased from Fisher Scientitifc (Pittsburgh, PA, USA).-Hydroxybenzotriazole (HOBt), and 2-(1H-benzotriazole-1-l)-1,1,3,3-tetramethyluronium (HBTU) were purchased fromnaspec (San Jose, CA, USA). Fmoc-L-Arg(pbf)-OH was fromovabiochem (San Diego, CA, USA). For permeability studies,4-well Transwell® were purchased from Corning costar Inc.New York, USA). Cell culture materials were purchased fromisher Scientific Ltd. (Chicago, IL, USA). Hank’s balanced saltolution (HBSS) with calcium and magnesium (without phenoled) was purchased from Cellgro (Herndon, VA).

.2. Synthesis of the arginine- and ornithine-conjugatedendrimers

Arginine- and ornithine-conjugated PAMAM dendrimersere prepared using Fmoc synthesis (a schematic of the synthe-

is is shown in Fig. 1). Briefly, to 1.0 mmol of G4-NH2 dendrimern 3.0 mL of DMF, 70 mmol each of HOBt, HBTU, Fmoc-L-rg(pbf)-OH and 70 mmol of DIPEA were added. The reactionixture was allowed to stir for 4.0 h at room temperature (Choi

t al., 2004). The product was precipitated in about 5 mL ofthyl ether and washed with excess ether. The Fmoc groupsf Fmoc-Arg(pbf)-coupled dendrimer were removed by adding.0 mL of 30% piperidine in DMF (v/v). After 1.0 h of depro-ection reaction, the mixture was precipitated in ethyl ether andashed with excess ethyl ether. A 3.0 mL of the reagent, tri-uoroacetic acid/triisopropylsilane/deionized H2O (95:2.5:2.5,/v/v) was used for deprotection of pbf groups of arginine (6 ht room temperature) and the final product was precipitated inthyl ether and washed with excess ethyl ether. The productas then solubilized in deionized water and dialyzed againsteionized water at 4 ◦C overnight and freeze-dried to obtainwhite powder of dendrimer-arginine conjugate (PAMAM-RG). Dendrimer-ornithine conjugate (PAMAM-ORN) was

ynthesized the same way as above using Fmoc-ornithine(Boc)-

H and the deprotection of the BOC group was achieved by

eacting with 90% TFA for 1 h at room temperature. The yield foroth dendrimer–ornithine and dendrimer–arginine was alwaysore than 95%.

D.S. Pisal et al. / International Journal of Pharmaceutics 350 (2008) 113–121 115

Fig. 1. A schematic of the conjugation of ornithine to the amine terminals of the G4 PAMAM dendrimers. Similar protocol was followed for the synthesis ofarginine-conjugated PAMAM dendrimers using Fmoc-l-arginine-(pbf)-OH (Note: though G4 dendrimer has 64 surface amine groups, only a few amine groups wered

2

TdSTdpo

2

p(d

sdsdctslt5ArP

epicted in this figure for convenience).

.3. NMR and mass spectral analysis

1H NMR spectra (chemical shift in ppm with respect toMS set at zero) of arginine- and ornithine-conjugated PAMAMendrimers were recorded on a Bruker AMX-400 (400 MHz)pectrometer using D2O as the solvent (with 0.03% (v/v)MS). To confirm the molecular weight of surface-modifiedendrimers, mass spectral analysis of the dendrimers waserformed on a Bruker MALDI TOF (matrix assisted laser des-rption/ionization time-of-flight) using DHB as the matrix.

.4. Florescence labeling of dendrimers

Fluorescently (FITC)-labeled PAMAM dendrimers were pre-ared according to the methodology reported in the literatureEl-Sayed et al., 2001; Jevaprasesphant et al., 2003). PAMAMendrimer solution was diluted in PBS, pH 7.4. A 5 mg/mL

wfp5

olution of FITC in acetone was prepared and added to theendrimer solution at a molar ratio of 1:1. The mixture wastirred overnight at room temperature. Fluorescently labeledendrimer solution was purified by Sephacryl S-300 columnhromatography with acetonitrile:Tris buffer (70:30) as the elu-ion buffer. The elution fractions corresponding to the dendrimerize were collected, dialyzed against deionized water at 4 ◦C,yophilized and stored at 4 ◦C for further studies. Stability ofhe FITC-labeled dendrimers was studied at 4 and 37 ◦C (for

days) by determining the free FITC in the samples usinggilent 1100 high-pressure liquid chromatography with fluo-

escent detector (Agilent Technologies, Santa Clara, CA, USA).hosphate buffered saline (PBS pH 7.4):acetonitrile (80:20)

as used as the mobile phase. No second peak representing

ree FITC (retention time of 5.5 min) was found in the sam-les indicating that the labeled dendrimers were stable fordays.

1 al of

2

RDflufce

2t

TwhwticoCte1AI(

2c

Taao3tbtSwimtaatd5tw2(

bo51tat

P

wmc

2

MCwsM1t2m5aasanawt

2

tp

3

3

tdratd

16 D.S. Pisal et al. / International Journ

.5. Cell culture

Caco-2 cells (passage number 37) were a kind gift from Dr.adhey Kaushik, Department of Biology & Microbiology, Southakota State University. The cells were grown at 37 ◦C in T-75asks in an atmosphere of 5% CO2 and 95% relative humiditysing RPMI 1640 Medium (pH 7.4) supplemented with 10%etal bovine serum, 1% penicillin–streptomycin solution. Theells were passaged by using 0.25% trypsin containing 0.20%thylenediamine tetraacetic acid (EDTA).

.6. Effect on monolayer integrity by measuringransepithelial electrical resistance (TEER)

Caco-2 cells were seeded onto the polystyrene 24-wellranswell® filters at a density of 1 × 105 cells/cm2. The cellsere grown in an atmosphere of 5% CO2 and 95% relativeumidity using RPMI 1640 Medium (pH 7.4) supplementedith 10% fetal bovine serum, 1% penicillin–streptomycin solu-

ion. The growth medium was changed every 2 days. Followingncubation for 21 days, mean TEER value across the Caco-2ell monolayers was found to be 1000 ± 58 � cm2. The effectf PAMAM dendrimers and surface-modified dendrimers onaco-2 cell monolayers was determined by measuring the

ransepithelial electrical resistance across Caco-2 cell monolay-rs in presence of the dendrimers at donor concentrations ofand 10 �g/mL upon both apical and basolateral incubation.EVOMXTM epithelial tissue voltammeter (World Precision

nstruments, Sarasota, FL) was used to measure TEER valuesKitchens et al., 2006).

.7. Permeability of dendrimers across polarized Caco-2ell monolayers

Caco-2 cells were seeded onto the polystyrene 24-wellranswell® filters of 3.0 �m mean pore size, 0.33 cm2 surfacerea (Corning Costar Inc., NY) at a density of 1 × 105 cells/cm2,nd the growth medium was changed every 2 days. Polarizationf the cells was monitored by measuring the TEER once everydays. The TEER of the blank Transwell® filters was found

o be 178 ± 31 �. A TEER value of 1000 ± 58 � (including theackground filter resistance) was observed after 21–28 days andhese cell monolayers were used for the transport studies (El-ayed et al., 2001, 2002; Kitchens et al., 2006). The cells wereashed with HBSS pH 7.4 buffer (with 10 mM HEPES) and

ncubated with HBSS pH 7.4 buffer for 1 h prior to the experi-ent. For A–B transport study, 0.6 mL of the buffer was added

o the basal side and a 0.1 mL of test solution was added to thepical side. For B–A transport study, 0.6 mL of the buffer wasdded to the apical side and a 0.1 mL of test solution was addedo the basal side. Following addition of the test solution (den-rimers at 1 and 10 �g/mL), the plates were incubated at 37 ◦C,% CO2, 95% humidity, and shaking at 50 rpm while main-

aining sink conditions. Two hundred microlitres of samplesere removed from the donor compartment at 30, 90, 150 and10 min and analyzed for fluorescence using SpectraMax M2Molecular devices, Sunnyvale, CA). The calibration curves for

iddd

Pharmaceutics 350 (2008) 113–121

oth PAMAM-arginine (Y = 15.598X + 224.69) and PAMAM-rnithine (Y = 85.935X − 140.57) were linear between 0.1 and�g/mL. Minimum detectable concentration was found to beng/mL. The volume of the receptor compartment was main-

ained constant at 0.6 mL by replacing it with fresh HBSS. Thepparent permeability coefficient (Papp) was calculated usinghe equation:

app = ∂Q

AC0∂t

here ∂Q/∂t is the permeability rate, A the surface area of theembrane filter and C0 is the initial concentration in the donor

ompartment (Irvine et al., 1998; Kitchens et al., 2005).

.8. In vitro cytotoxicity

In vitro cytotoxicity of the dendrimers was evaluated byTT assay (Sgouras and Duncan, 1990; Fischer et al., 2003).aco-2 cells were seeded at a density of 4 × 104 cells perell in a 96-well plate. The cells were grown in an atmo-

phere of 5% CO2 and 95% relative humidity using RPMI 1640edium (pH 7.4) supplemented with 10% fetal bovine serum,

% penicillin–streptomycin solution. Following 24 h incuba-ion period, different concentrations (0.1, 1, 10, 50, 100 and00 �g/mL) of the native PAMAM dendrimers and surface-odified dendrimers were added. After a 4 h incubation period,

0 �L of 1:10 diluted MTT stock solution (5 mg/mL) was addednd the cells were incubated for 4 h. The medium was removednd 150 �L of dimethyl sulfoxide (DMSO) was added to dis-olve the MTT crystals and the optical density was read usingSpectraMax M2 microplate reader (Molecular devices, Sun-

yvale, CA) with 590 nm as excitation wavelength and 650 nms the background. The viability of cells exposed to dendrimersas expressed as a percentage of the viability of cells grown in

he absence of dendrimers.

.9. Statistical analysis

The statistical analysis was carried out using the Student’s t-est in GraphPad InStat 3.05 version software (San Diego, CA).< 0.05 was considered significant.

. Results

.1. Polyamine-conjugation of the dendrimers

Initially, ornithine and arginine were coupled to the amineerminals of the PAMAM dendrimers by EDC (1-ethyl-3-[3-imethylaminopropyl] carbodiimide hydrochloride) couplingeaction (Malik et al., 2000). However, only a few ornithine andrginine molecules (seven molecules each) were found attachedo the G4 PAMAM dendrimer. Increasing the molar ratio ofendrimer:arginine (and ornithine) did not yield any increase

n the number of molecules attached to the dendrimer, insteadimer formation (such as dendrimer–ornithine–dendrimer,endrimer–arginine–dendrimer) was observed. To avoidimerization and to increase the degree of conjugation, Fmoc

D.S. Pisal et al. / International Journal of

Fig. 2. (a) 1H NMR (400 MHz) spectra of (A) PAMAM, (B) PAMAM-ornithineand (C) PAMAM-arginine. (b) MALDI-TOF spectra of (A) standard PAMAMG4 dendrimer, (B) ornithine coupled dendrimer showing 62 molecules cou-pm

srtdiaouddssdaa2HPH(a

H(

3

icPBwTcttmt(at

3m

ddavdHdodpah((fBsoa(Bspass

monolayers at each time duration (not the mean P )

led to PAMAM dendrimer and (C) arginine coupled dendrimer showing 55olecules coupled to PAMAM dendrimer.

ynthesis protocol was used. To start with, different molaratios of the dendrimers and the polyamines were usedo optimize the conjugation reaction. When 1:35 ratio ofendrimer:Fmoc–polyamine (molar ratio) was used, approx-mately 28 molecules of ornithine and 26 molecules of therginine were found coupled to the PAMAM dendrimers. Basedn these preliminary results, a higher molar ratio of 1:70 wassed to get all the 64 amine groups of the G4 PAMAM den-rimer conjugated with the polyamines. The surface-modifiedendrimers were characterized by 1H NMR and MALDIpectroscopic analysis (Fig. 2a and b). The spectral analysishowed that out of 64 surface amine groups of G4 PAMAMendrimer, 62 molecules of ornithine and 55 molecules ofrginine were attached. The 1H NMR data of the dendrimersre as follows—PAMAM Dendrimer: 2.44, NCH2 CH2 CO;.63, CONHCH2 CH2 N and NCH2CH2N; 2.72, CON-CH2CH2N; 2.83 NCH2CH2CO; 3.25, ONHCH2CH2N.

AMAM-ornithine: 1.73, HCCH2CH2CH2NH (ORN); 1.91,

CCH2CH2CH2NH (ORN); 3.41, HCCH2CH2CH2NH

ORN); 3.92, HCCH2CH2CH2NH (ORN). PAMAM-rginine: 1.68, HCCH2CH2CH2NH (ARG); 1.93,

wod

Pharmaceutics 350 (2008) 113–121 117

CCH2CH2CH2NH (ARG); 3.25, HCCH2CH2CH2NHARG); 4.01, HCCH2CH2CH2NH (ARG).

.2. Transepithelial electrical resistance (TEER)

Transepithelial electrical resistance was measured at timentervals of 30, 90, 150 and 210 min in presence of differentoncentrations (1 and 10 �g/mL) of the dendrimers (PAMAM,AMAM-arginine and PAMAM-ornithine) from both AB andA directions. The TEER value with Caco-2 cell monolayeras 1000 ± 58 � (without background resistance subtracted).he decline in TEER for each type of the dendrimer wasoncentration and time dependent in both AB and BA direc-ions as shown in Fig. 3a–d. Among the dendrimers tested,he decrease in TEER by arginine-conjugated dendrimer was

ost prominent and showed significant decrease as comparedo PAMAM dendrimers (p < 0.05). The basolateral incubationBA) also showed faster and more pronounced decline in TEERs compared to apical incubation (AB) under similar condi-ions.

.3. Permeability of dendrimers through Caco-2 cellonolayers

PAMAM dendrimers, ornithine- and arginine-conjugatedendrimers were labeled with FITC. Attachment of FITC to theendrimers was verified by 1H NMR and spectrofluorimetry. Anverage molar ratio of 1:0.5 dendrimer:FITC was obtained. Initro stability tests were performed to ensure that FITC-labeledendrimer was stable during the period of experimentation.PLC analysis revealed that no free FITC was found in theendrimer-FITC samples incubated at 4 and 37 ◦C (PBS pH 7.4)ver a period of 5 days. At 1 �g/mL as well as 10 �g/mL, den-rimers showed incubation time and concentration dependentermeability profile as shown in the Fig. 4a–f. Both ornithine-nd arginine-conjugated dendrimers showed significantlyigher AB permeability than the native PAMAM dendrimersp < 0.05). For instance, mean apparent permeability coefficientPapp) of PAMAM dendrimers was (0.34 ± 0.01) × 10−6 cm/sor A–B direction and (0.61 ± 0.07) × 10−6 cm/s for the–A direction after incubation for 90 min with 10 �g/mL

olution. Under the similar conditions, mean Papp of PAMAM-rnithine and PAMAM-arginine was (0.46 ± 0.07) × 10−6

nd (0.63 ± 0.13) × 10−6 cm/s for AB direction, and0.86 ± 0.03) × 10−6 and (0.98 ± 0.05) × 10−6 cm/s forA direction, respectively. Because all the dendrimers havehown some degree of toxicity when incubated over 4 h,ermeabilities were not determined after 210 min. At both 1nd 10 �g/mL concentrations, permeability coefficients of theurface-modified dendrimers were significantly higher than theimple PAMAM dendrimers (p < 0.05).

Permeabilities of the dendrimers across Caco-2 cell

app

ere calculated by determining the amount permeatednly during that particular time period. Papp of all theendrimers increased with time. For instance, Papp of PAMAM-

118 D.S. Pisal et al. / International Journal of Pharmaceutics 350 (2008) 113–121

Fig. 3. (a) and (b) Effect of polyamine-conjugated dendrimers (1 �g/mL) on transepithelial electrical resistance (TEER) across Caco-2 cell monolayers uponi MAMP on trai

Oa1wi(mtdjda

3

PFpCwnP8iPatb

d(

4

daapc2ddiwIpageac

t

ncubation on (a) apical side and (b) basolateral side (n = 4 and p < 0.05). (PAAMAM). (c) and (d) Effect of polyamine-conjugated dendrimers (10 �g/mL)ncubation on (c) apical side and (d) basolateral side (n = 4 and p < 0.05).

RN during 0–90 min was (0.46 ± 0.076) × 10−6 cm/s,nd it increased to (1.87 ± 0.011) × 10−6 cm/s during50–210 min (p < 0.01). In case of PAMAM-ARG, Papp

as (0.63 ± 0.13) × 10−6 cm/s during 0–90 min, whereas, itncreased to (2.28 ± 0.009) × 10−6 cm/s during 150–210 minp < 0.01). The increase in the rate of permeability across the cellonolayers, as well as a decrease in TEER with time indicate

hat the increase in permeability with the surface-modifiedendrimers may be due to disruption of intercellular tightunctions as one of the mechanisms. However, effect of theseendrimers on the tight junctional proteins such as occludinnd actin has to be investigated to confirm the mechanism.

.4. In vitro cytotoxicity studies

The toxicity of the native PAMAM, PAMAM-arginine andAMAM-ornithine to Caco-2 cells was studied by MTT assay.ig. 5 shows the concentration of different dendrimers and theercent cell viability after 4 h incubation of dendrimers withaco-2 cells at 37 ◦C. No change in cell viability was observedith any of the dendrimers tested up to 2 h of incubation (dataot shown). At 0.1 �g/mL concentration, cell viability withAMAM, PAMAM-ORN and PAMAM-ARG were 87.4 ± 1.7,2.7 ± 8.5 and 70.1 ± 4.5%, respectively. As the concentrationncreased to 200 �g/mL, percent cell viability with PAMAM,

AMAM-ORN and PAMAM-ARG were 61.2 ± 4.2, 55.9 ± 4.7nd 45.4 ± 3.7%, respectively. Interestingly, at all the concen-rations tested, PAMAM-ARG was significantly more toxic thanoth PAMAM, PAMAM-ORN (p < 0.01). Toxicity of the den-

o7cn

-ORN: ornithine-conjugated PAMAM; PAMAM-ARG: arginine-conjugatednsepithelial electrical resistance (TEER) across Caco-2 cell monolayers upon

rimers increased with increasing in the duration of incubationdata not shown).

. Discussion

Dendrimers have been investigated as drug carriers for oralelivery as they have been shown to cross the epithelial layerst sufficient rates (Florence et al., 2000; Wiwattanapatapee etl., 2000). Anionic PAMAM dendrimers exhibited rapid trans-ort rates across everted rat intestine as compared to positivelyharged dendrimers (El-Sayed et al., 2002; Kitchens et al.,006). The difference in transepithelial transport between theseendrimers was attributed to the difference in interaction of theendrimers with the negatively charged cell membrane. Pos-tively charged dendrimers were shown to strongly associateith the tissue compared to the negatively charged dendrimers.

n this study, PAMAM-NH2 dendrimers were conjugated witholyamines (ornithine and arginine) and their permeabilitycross Caco-2 cell monolayers, and cytotoxicity were investi-ated. Though dendrimer–arginine conjugates were reported tonhance the gene transfection efficiency in 293 cells (Choi etl., 2004), their potential to increase permeability across Caco-2ell monolayers has not been investigated.

The extent of dendrimer–polyamine conjugation may be con-rolled by adjusting the molar ratio of the Fmoc arginine/Fmoc

rnithine and the PAMAM dendrimer. At a molar ratio of0, most of the surface amino groups of the dendrimer wereonjugated with the polyamine molecules (55 molecules of argi-ine/62 molecules of ornithine). The TEER studies indicate that

D.S. Pisal et al. / International Journal of Pharmaceutics 350 (2008) 113–121 119

Fig. 4. (a–c) Permeability of polyamine-conjugated dendrimers across polarized Caco-2 cell monolayer in AB (apical-to-basolateral) and BA (basolateral-to-apical)directions at a concentration of 1 �g/mL after incubation for different time intervals: (a) 90 min; (b) 150 min; (c) 210 min. Results are reported as mean ± S.E. (S.E.M.)(n = 4 and p < 0.05). (PAMAM-ORN: ornithine-conjugated PAMAM; PAMAM-ARG: arginine-conjugated PAMAM). (d–f) Permeability of polyamine-conjugateddendrimers across polarized Caco-2 cell monolayer in AB (apical-to-basolateral) and BA (basolateral-to-apical) directions at a concentration of 10 �g/mL afteri esulto .

tdrTtms

cPocd

sdctdcbSi

ncubation for different time intervals: (d) 90 min; (e) 150 min; (f) 210 min. Rrnithine-conjugated PAMAM; PAMAM-ARG: arginine-conjugated PAMAM)

he native PAMAM dendrimers and the surface-modified den-rimers (polyamine-conjugated dendrimers) have significantlyeduced the TEER values as compared to the control (p < 0.05).he reduction in TEER was dependent on dendrimer concen-

ration and duration of incubation. The reduction in TEER wasore pronounced on the apical side as compared to the basal

ide.At all the time intervals (90, 150 and 210 min), and the con-

entrations (1 and 10 �g/mL) tested, Papp of arginine-conjugated

AMAM dendrimers was significantly higher than the PAMAM-rnithine and native PAMAM dendrimers (p < 0.05). Theorrelation between monolayer permeability (Papp) and theecrease in TEER with both the PAMAM dendrimers and the

tmpi

s are reported as mean ± S.E. (S.E.M.) (n = 4 and p < 0.05). (PAMAM-ORN:

urface-modified dendrimers allows us to speculate that theseendrimers might have been transported across the epithelialells via paracellular route. The higher BA permeabilities ofhe dendrimers over AB permeability may be attributed toifferences in tight junctional structure, for example, Caco-2ell monolayers possess tight junctional proteins at the apicalut not the basolateral membrane (Noach et al., 1993; El-ayed et al., 2002). The mechanism by which the polyamines

mprove the permeation of the dendrimers remain to be inves-

igated, it is possible that the acidic molecules such as acidic

ucopolysaccharides on the surface of the membrane and phos-hatidyl serine, and/or polyamine transport system (PAT) arenvolved.

120 D.S. Pisal et al. / International Journal of

Fc(

pta2neOtSaP

ecidasttipl

5

theaicHbpmpe

A

IeS

R

A

A

A

B

C

C

D

D

E

E

E

F

F

F

F

G

H

H

HI

ig. 5. Effect of polyamine-conjugated dendrimers (0.1–200 �g/mL) on Caco-2ell viability after 4 h of incubation at 37 ◦C. Results are reported as mean ± S.E.S.E.M.) (n = 4 and p < 0.05).

Many of the cell penetrating peptides such as TAT-derivedeptide, protein transduction domains (PTD) or membraneranslocalization signals (MTS) contain positively chargedminoacid residues arginine and lysine (Futaki, 2002; Henry,003; Tung and Weissleder, 2003; Choi et al., 2004). It isot known if they enter the cell by endocytosis or other non-ndocytic pathways. Arginine is a precursor for ornithine.rnithine and other polyamines (such as putrescine) were shown

o be transported into the cell by PAT system (Shao et al., 1996;atriano et al., 2001). Though it is mere a speculation, ornithinend arginine-conjugated dendrimers uptake may also involveAT system.

Dendrimers were reported to exhibit concentration and gen-ration dependent toxicity in vitro. However, both acute andhronic toxicity evaluation in mice did not show any behav-oral changes, weight loss or immunogenicity with G3 and G5endrimers (Roberts et al., 1996). In the present study, PAMAM-rginine was found to be more toxic than PAMAM-ORN andimple PAMAM dendrimer. It is reasonable to attribute theoxicity of PAMAM-arginine to its high permeability. Thoughhe study was conducted only with G4 PAMAM dendrimers,t will be interesting to see the effect of generation of theolyamine-conjugated dendrimers on their toxicity and mono-ayer permeability.

. Conclusion

The concept of conjugation of polyamines to the dendrimerso increase the flux across Caco-2 cell monolayers is novel andas not been reported. PAMAM dendrimer permeability wasnhanced by conjugating with polyamines such as ornithine andrginine. The enhanced permeability and concomitant reductionn TEER with these modified dendrimers indicate the para-ellular pathway as one of the mechanisms of their transport.owever, it is well known that the polyamines are transportedy polyamine transporter system in a metabolism dependent

athway. Further studies are needed to understand the involve-ent of polyamine transporter protein (PAT) in the transport of

olyamine-conjugated dendrimers across Caco-2 cell monolay-rs.

J

Pharmaceutics 350 (2008) 113–121

cknowledgements

This work was partly supported by a grant from Nationalnstitutes of Health (NIH R15 CA121980-01) to SP, and the Gov-rnor’s Individual Research Seed Grant, SD and SDSU Researchupport Fund, South Dakota State University, SD.

eferences

hnet, C.H., Chae, S.Y., Bae, Y.H., Kim, S.W., 2004. Synthesis of biodegradablemulti-block copolymers of poly(l-lysine) and poly(ethylene glycol) as anon-viral gene carrier. J. Control. Release 97, 567–574.

ungst, B.J., 2000. Intestinal permeation enhancers. J. Pharm. Sci. 89, 429–442.

uvinen, M., Parsinen, A., Anderson, L.C., Holtta, E., 1992. Ornithine decar-boxylation activity is critical for cell transformation. Nature 360, 355–358.

oas, U., Heggard, P.M.H., 2004. Dendrimers in drug research. Chem. Soc. Rev.33, 43–63.

hoi, J.S., Nam, K., Park, K.J.Y., Kim, J.B., Lee, J.K., Park, J.S., 2004. Enhancedtransfection efficiency of PAMAM dendrimer by surface modification withl-arginine. J. Control. Release 94, 445–456.

ullis, P.M., Green, R.E., Merson-Davies, L., Travis, N., 1999. Probing themechanism of transport and compartmentalization of polyamines in mam-malian cells. Chem. Biol. 6, 717–729.

’Emanuele, A., Jevapresesphant, R., Penny, J., Deadwood, D., 2004. The useof dendrimer-propranalol prodrug to bypass efflux transporters and enhanceoral bioavailability. J. Control. Release 95, 447–453.

evens, B.H., Weeks, R.S., Burns, M.R., Carlson, C.L., Brawer, M.K., 2000.Polyamine depletion therapy in prostate cancer. Prostate Cancer. Prost. Dis.3, 275–279.

l-Sayed, M., Kiani, M.F., Naimark, M.D., Hikal, A.H., Ghandehari, H., 2001.Extravasation of poly (amidoamine) (PAMAM) dendrimers across microvas-cular network endothelium. Pharm. Res. 18, 23–28.

l-Sayed, M., Ginski, M., Rhodes, C., Ghandehari, H., 2002. Transepithelialtransport of poly(amidoamine) dendrimers across Caco-2 cell monolayers.J. Control. Release 81, 355–365.

ndo, Y., Hanada, K., Miyake, M., Ogawara, K., Higaki, K., Kimura, T., 2002.Mechanisms of cytoprotective effect of amino acids on local toxicity causedby sodium laurate, a drug absorption enhancer, in intestinal epithelium. J.Pharm. Sci. 91, 730–743.

ischer, D., Li, Y., Ahlemeyer, B., Krieglstein, J., Kassel, T., 2003. In vitrocytotoxicity testing of polycations: influence of polymer structure on cellviability and hemolysis. Biomaterials 24, 1121–1131.

lorence, A.T., 1997. The oral absorption of micro- and nanoparticles: neitherexceptional nor unusual. Pharm. Res. 14, 259–266.

lorence, A.T., Sakthivel, T., Toth, I., 2000. Oral uptake and translocation of apolylysine dendrimer with a lipid surface. J. Control. Release 65, 253–259.

utaki, S., 2002. Arginine-rich peptides: potential for intracellular delivery ofmacromolecules and the mystery of translocation mechanisms. Int. J. Pharm.245, 1–7.

ardner, R.A., Delcros, J.G., Konate, F., Breitbeil III, F., Martin, B., Sigman,M., Huang, M., Phanstiel IV, O., 2004. N1-Substituent effects in the selectivedelivery of polyamine conjugates into cells containing active polyaminetransporters. J. Med. Chem. 47, 6055–6069.

arold, B.B., Margaret, A.P., 1996. Circular dichromism assay for decarboxyla-tion of optically pure aminoacids: application to ornithine. Anal. Biochem.238, 191–194.

eby, O., 1981. Role of polyamines in the control of cell proliferation anddifferentiation. Differentiation 19, 1–20.

enry, C.M., 2003. Breaching barriers. Chem. Eng. News 81, 35–43.rvine, J.D., Takahashi, L., Lockhart, K., Cheong, J., Tolan, J.W., Selick, H.E.,

Grove, J.R., 1998. MDCK (Madin-Darby Canine Kidney) cells: a tool formembrane permeability screening. J. Pharm. Sci. 88, 28–33.

anasen, J.F.G.A., de Brabander-van den Berg, E.M.M., Meijer, E.W., 1994.Encapsulation of guest molecules into a dendritic box. Science 266,1226–1229.

al of

J

J

K

K

L

L

L

M

M

M

M

M

M

M

M

N

O

O

P

R

R

S

S

S

S

S

S

S

T

W

W

D.S. Pisal et al. / International Journ

evaprasesphant, R., Penny, J., Attwood, D., McKeown, N.B., D’Emanuele, A.,2003. Engineering of dendrimer surfaces to enhance transepithelial transportand reduce cytotoxicity. Pharm. Res. 20, 1543–1550.

ohnson, L.R., 1988. Regulation of gastrointestinal mucosal growth. Physiol.Rev. 68, 456–502.

itchens, K.M., El-Sayed, M.E.H., Ghandehari, H., 2005. Transepithelial andendothelial transport of poly(amidoamine) dendrimers. Adv. Drug Del. Rev.57, 2163–2176.

itchens, K.M., Kolhatkar, R.B., Swaan, P.W., Eddington, N.D., Ghandehari,H., 2006. Transport of poly(amidoamine) dendrimers across Caco-2 cellmonolayers: influence of size, charge and fluorescent labeling. Pharm. Res.23, 2818–2826.

i, W., Ishida, T., Okada, Y., Oku, N., Kiwada, H., 2005. Increasing gene expres-sion by cationic liposomes (TFL-3) in lung metastases following intravenousinjection. Biol. Pharm. Bull. 28, 701–706.

iu, X., Wang, L., Lin, Y., Teng, Q., Zhao, C., Hu, H., Chi, W., 2000. Ornithinedecarboxylase activity and its gene expression are increased in benign hyper-plastic prostate. Prostate 43, 83–87.

uk, G.D., Marton, L.J., Baylin, S.B., 1988. Ornithine decarboxylase is impor-tant in intestinal mucosal maturation and recovery from injury in rats. Science210, 195–198.

a, Z., Li, J., He, F., Wilson, A., Pitt, B., Li, S., 2005. Cationic lipids enhancesiRNA-mediated interferon response in mice. Biochem. Biophys. Res. Com-mun. 330, 755–759.

acRae, M., Kramer, D.L., Coffino, P., 1998. Developmental effects ofpolyamine depletion in caenorhabditis elegans. Biochem. J. 333, 309–315.

afune, K., Tanaka, Y., Mimori, K., Mori, M., Takubo, K., Makuochi, M., 1999.Increased expression of ornithine decarboxylase messenger RNA in humanesophageal carcinoma. Clin. Cancer Res. 5, 4073–4078.

ajumdar, A.P., Johnson, L.R., 1987. Effect of putrescine on oxyntic gland andcolonic mucosal growth in rats. Life Sci. 41, 961–966.

alik, N., Wiwattanapatapee, R., Klopsch, R., Lorenz, K., Frey, H., Weener,J.W., Meijer, E.W., Paulus, W., Duncan, R., 2000. Dendrimers: relationshipbetween structure and biocompatibility in vitro and preliminary studies onthe biodistribution of 125I-labeled polyamidoamine dendrimers in vivo. J.Control. Release 65, 133–148.

essing, E.M., Love, R.R., Tutsch, K.D., Verma, A.K., Douglas, J., Pomplun,M., Simsiman, R., Wilding, G., 1999. Low-dose difluoromethylornithineand polyamine levels in human prostate tissue. J. Natl. Cancer Inst. 91,1416–1417.

iyake, M., Oka, Y., Minami, T., Toguchi, H., Odomi, M., Ogawara, K., Higaki,K., Kimura, T., 2003. Combinatorial use of sodium laurate with taurine orl-glutamine enhances colonic absorption of rebamipide, poorly absorbableanti-ulcer drug without any serious histopathological mucosal damages. J.

Pharm. Sci. 92, 911–921.

iyake, M., Minami, T., Hirota, M., Toguchi, H., Odomi, M., Ogawara, K.,Higaki, K., Kimura, T., 2006. Novel oral formulation safely improvingintestinal absorption of poorly absorbable drugs: utilization of polyaminesand bile acids. J. Control. Release 111, 27–34.

Y

Pharmaceutics 350 (2008) 113–121 121

oach, A.B.J., Kurosaki, Y., Blom-Roosemalen, M.C.M., Boer, A.G.D.,Breimer, D.D., 1993. Cell-polarity dependent effect of chelation on the para-cellular permeability of confluent Caco-2 cell monolayers. Int. J. Pharm. 90,229–237.

’Brien, T.G., Megosh, L.C., Gillard, G., Soler, A.P., 1997. Ornithine decar-boxylase over expression is a sufficient condition for tumor promotion inmouse skin. Cancer Res. 57, 2630–2637.

htake, K., Natsume, H., Ueda, H., Morimoto, Y., 2002. Analysis of transientand reversible effects of poly-l-arginine on the in vivo nasal absorption ofFITC-dextrans in rats. J. Control. Release 82, 263–275.

egg, A.E., 1988. Polyamine metabolism and its importance in neoplastic growthand as a target for chemotherapy. Cancer Res. 48, 759–774.

oberts, J.C., Bhalgat, M.K., Zera, R.T., 1996. Preliminary biological evaluationof polyamidoamine (PAMAM) starburst dendrimers. J. Biomed. Mater. Res.30, 53–65.

oy, R., 1997. Recent developments in the rational design of multivalent glyco-conjugates. Top. Curr. Chem. 187, 241–274.

akthivel, T., Toth, I., Florence, A.T., 1999. Distribution of lipidic 2.5 nmdiameter dendrimer carrier after oral administration. Int. J. Pharm. 183,51–55.

atriano, J., Isome, M., Casero Jr., R.A., Thomson, S.C., Blantz, R.C., 2001.Polyamine transport system mediates agmatine transport in mammaliancells. Am. J. Physiol. Cell Physiol. 281, 329–334.

chiper, R.G., Romijn, J.C., Cuijpers, V.M.J.I., Verhofstad, A.A.J., 2003.Polyamines and prostate cancer. Biochem. Soc. Trans. 31, 375–380.

eiler, N., Delcros, J.G., Moulinoux, J.P., 1996. Polyamine transport in mam-malian cells: an update. Int. J. Biochem. Cell Biol. 28, 843–861.

eki, T., Kanabayashi, H., Nagao, T., Chono, S., Tomita, M., Hayashi, M.,Tabata, Y., Morimoto, K., 2005. Effect of aminated gelatin on the nasalabsorption of insulin in rats. Biol. Pharm. Bull. 28, 510–514.

gouras, D., Duncan, R., 1990. Methods for the evaluation of biocompatibilityof soluble synthetic polymers which have potential for biomedical use. 1.Use of the tetrazolium-based colorimetric assay (MTT) as a preliminaryscreen for the evaluation of in vitro cytotoxicity. J. Mater. Sci. Mater. Med.1, 67–78.

hao, D., Xiao, L., Ha, H.C., Casero Jr., R.A., 1996. Isolation of a polyaminetransport deficient cell line from human non-small lung carcinoma line NCIH157. J. Cell Physiol. 166, 43–48.

ung, C.H., Weissleder, R., 2003. Arginine containing peptides as deliveryvectors. Adv. Drug Deliv. Rev. 55, 281–294.

ang, J.Y., Johnson, L.R., 1990. Luminal polyamines stimulate repair of gastricmucosal stress ulcers. Am. J. Physiol. 259, G584–G592.

iwattanapatapee, R., Carreno-Gomez, B., Malik, N., Duncan, R., 2000.

Anionic PAMAM dendrimers rapidly cross adult rat intestine in vivo: apotential oral delivery system? Pharm. Res. 17, 991–998.

ata, T., Endo, Y., Sone, M., Ogawara, K., Higaki, K., Kimura, T., 2001. Aminoacids protect epithelial cells from local toxicity by absorption enhancer,sodium laurate. J. Pharm. Sci. 90, 1456–1465.