transepithelial and endothelial transport of poly (amidoamine) dendrimers

www.elsevier.com/locate/addr

Advanced Drug Delivery Revie

Transepithelial and endothelial transport of

poly (amidoamine) dendrimersB

Kelly M. Kitchens a,b, Mohamed E.H. El-Sayedc, Hamidreza Ghandehari a,b,d,*

aDepartment of Pharmaceutical Sciences, University of Maryland, Baltimore, Baltimore, Maryland, USAbCenter for Nanomedicine and Cellular Delivery, University of Maryland, Baltimore, Baltimore, Maryland, USA

cDepartment of Bioengineering, University of Washington, Seattle, Washington, USAdGreenebaum Cancer Center, University of Maryland, Baltimore, Baltimore, Maryland, USA

Received 29 June 2004; accepted 13 September 2005

Available online 11 November 2005

Abstract

This article summarizes our efforts to evaluate the potential of poly (amidoamine) (PAMAM) dendrimers as carriers for

oral drug delivery. Specifically, the permeability of a series of cationic PAMAM–NH2 (G0–G4) dendrimers across Caco-2

cell monolayers was evaluated as a function of dendrimer generation, concentration, and incubation time. The influence of

dendrimer surface charge on the integrity, paracellular permeability, and viability of Caco-2 cell monolayers was monitored

by measuring the transepithelial electrical resistance (TEER), 14C-mannitol permeability, and leakage of lactate

dehydrogenase (LDH) enzyme, respectively. Microvascular extravasation of PAMAM–NH2 dendrimers in relation to their

size, molecular weight, and molecular geometry is also discussed. Results of these studies show that transepithelial transport

and microvascular extravasation of PAMAM dendrimers are dependent on their structural features including molecular size,

molecular geometry, and surface chemistry. These results suggest that by optimizing the size and surface charge of PAMAM

dendrimers, it is possible to develop oral delivery systems based on these carriers for targeted drug delivery.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Oral drug delivery; Water-soluble polymers; Poly (amidoamine) dendrimers; PAMAM; Caco-2 cells; Endothelial barrier;

Microvascular extravasation; Intravital microscopy

0169-409X/$ - s

doi:10.1016/j.ad

Abbreviation

LDH, Lactate dB This review

* Correspondi

625, Baltimore,

E-mail addre

ws 57 (2005) 2163–2176

ee front matter D 2005 Elsevier B.V. All rights reserved.

dr.2005.09.013

s: AB, Apical-to-basolateral; BA, Basolateral-to-apical; FITC, Fluorescein isothiocyanate; G, Generation; GI, Gastrointestinal;

ehydrogenase; PAMAM, Poly (amidoamine); TEER, Transepithelial electrical resistance.

is part of the Advanced Drug Delivery Reviews theme issue on bDendrimers: a Versatile Targeting PlatformQ, Vol. 57/15, 2005.ng author. University of Maryland School of Pharmacy, Department of Pharmaceutical Sciences, 20 Penn Street, HSFII Room

MD 21201-1075, USA. Tel.: +1 410 706 8650; fax: +1 410 706 5017.

ss: [email protected] (H. Ghandehari).

K.M. Kitchens et al. / Advanced Drug Delivery Reviews 57 (2005) 2163–21762164

Contents

. . . . . 2164

. . . . . 2164

. . . . . 2164

. . . . . 2166

. . . . . 2169

. . . . . 2170

. . . . . 2172

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2. PAMAM dendrimers and their potential as carriers for oral drug delivery . . . . . . . . . . . . . .

2.1. Unique architecture of PAMAM dendrimers . . . . . . . . . . . . . . . . . . . . . . . . . .

2.2. Transepithelial transport of dendrimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.3. Effect of surface charge on PAMAM transepithelial transport . . . . . . . . . . . . . . . . .

2.4. Possible transport mechanisms of PAMAM dendrimers . . . . . . . . . . . . . . . . . . . .

3. Microvascular extravasation of PAMAM–NH2 dendrimers . . . . . . . . . . . . . . . . . . . . . .

4. Conclusions and future direction of this research . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . 2174
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2174

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2175

1. Introduction

The concept of targeted drug delivery systems

consisting of drug molecules and targeting moieties

attached to a polymeric carrier was first introduced by

Ringsdorf [1]. The main objective in mind for such

delivery systems was to successfully deliver therapeu-

tic drug molecules, which represent the bcargoQ to

specific target site(s) within the body. Water-soluble

polymers are commonly used as carriers in such

targeted delivery systems where conjugation of thera-

peutic drug molecules is achieved either by linking the

drug using pendant side chains along the polymer

backbone, allowing several drug molecules to be con-

jugated per polymer chain or by conjugation of the drug

to one or both terminal groups of the polymer chain [2].

Conjugation of drug molecules to a water-soluble

polymeric carrier results in changing several properties

of the drug such as its solubility, biodistribution, phar-

macokinetic profile, and intracellular trafficking [3].

This in turn can lead to improved therapeutic index and

decreased toxicity of water-soluble polymer-drug con-

jugates compared to free drug molecules [4–6]. Water-

soluble polymers that have been used in drug delivery

systems include synthetic polymers such as poly

(ethylene glycol) (PEG), N-(2-hydroxypropyl)metha-

crylamide (HPMA), and poly (amidoamine) (PAMAM)

dendrimers. Additionally, natural water-soluble poly-

mers including chitosan, dextran, and poly (amino

acids) have also been utilized as drug carriers [2].

Therapeutic water-soluble polymer-drug conjugates

are typically administered via the parenteral route

mostly by intravenous injections. This is due to the

limited absorption of such conjugates from the

gastrointestinal (GI) tract as a result of the large size

and molecular weight of the polymeric carriers.

However, development of orally active water-soluble

polymer-drug conjugates is of therapeutic interest as it

allows for preferential targeting of the whole system to

certain tissues or organs within the body without the

inconvenience of intravenous injection. Successful

development of therapeutically active polymer-drug

conjugates that can achieve appreciable oral perme-

ability requires a clear understanding of structure–

transport relationship of polymeric carriers. This can be

achieved by investigating the relationship between the

physicochemical properties of the polymer such as size,

molecular weight, molecular geometry, hydrophilicity,

and charge on one hand and their transepithelial

transport upon oral administration on the other. The

major barrier for oral delivery of macromolecules is the

epithelial barrier of the gut. Once absorbed, other

factors to consider are systemic distribution, extrava-

sation into the target tissue, subcellular trafficking and

elimination. The emphasis of this review will be on our

recent studies related to the transport of PAMAM

dendrimers across the intestinal barrier. Studies related

to the in vivo microvascular extravasation of the

dendrimers will be also discussed.

2. PAMAM dendrimers and their potential as

carriers for oral drug delivery

2.1. Unique architecture of PAMAM dendrimers

PAMAM dendrimers are the first complete den-

drimer family to be synthesized, characterized, and

Table 1

Structural features of PAMAM dendrimers

PAMAM

generation (G)

Surface

functionality

Number of

surface groupsaMolecular

weight (Da)a

G0 –NH2 4 517

G1 –NH2 8 1430

G2 –NH2 16 3256

G3 –NH2 32 6909

G4 –NH2 64 14,215

G2 –OH 16 3272

G3 –OH 32 6941

G4 –OH 64 14,279

G-0.5 –COOH 4 436

G0.5 –COOH 8 1269

G1.5 –COOH 16 2935

G2.5 –COOH 32 6267

G3.5 –COOH 64 12,931

G4.5 –COOH 128 26,258

The list refers to those reviewed in this manuscript.a Reported by the manufacturer (Dendritech, Inc., MI).

K.M. Kitchens et al. / Advanced Drug Delivery Reviews 57 (2005) 2163–2176 2165

commercialized [7]. Dendrimers are macromolecules

with defined mass, size, shape, topology and surface

chemistry (Table 1) [7–11]. Starburst dendrimers are

characterized by a unique tree-like branching archi-

tecture that starts from an initiator core around which

Initiator Core G0 G

Cationic Ne

Scheme 1. Schematic representation of PAMAM dendrimers. An increas

incremental increase in size, molecular weight and number of surface gr

terminated, neutral hydroxyl-terminated, or negatively charged carboxyl-te

the branches of the dendrimer originates (Scheme 1).

This method of dendrimer synthesis is known as the

divergent method [7,9,10]. A second method of

dendrimer synthesis is the convergent growth method,

in which the branching architecture originates from

the outer dendron surface and proceeds towards the

inner reactive dendron [7,9,10]. The surface groups of

PAMAM dendrimers increase exponentially and the

molecular mass of dendrimers increases almost by

double due to the ethylenediamine inner core multi-

plicity (nc=4) and branch cell multiplicity (nb=2)

[7,8]. Furthermore, the molecular size of PAMAM

dendrimers increases by approximately 1 nm with

each generation [8,9]. In addition to the precise

control in the size, shape and surface chemistry of

dendrimers, these nanostructures have been regarded

as mimics to globular proteins, micelles, and lip-

osomes [7,8,11].

PAMAM dendrimers have demonstrated potential

use as drug delivery systems due to the high degree of

branching, which allows the polymer-drug conjugate

to potentially have a high density of biological agents

in a compact system. A variety of molecules, such as

drugs and other therapeutic agents, can be loaded both

1 G2

utral Anionic

e in generation number (G0, G1, G2, etc.) produces a controlled

oups. PAMAM dendrimers can exist as positively charged amine-

rminated.


in the interior void space and on the surface functional

groups to control the rate of release of these agents in

the body [7,10]. Dendrimers are also capable of com-

plexing with oligonucleotides and have been proven to

enhance the cytosolic and nuclear uptake of nucleic

acids [10–14]. Recent research has examined the

potential of dendrimers [15–28] for oral drug delivery.

The major obstacle for oral delivery of macromolecular

carriers is their transport across the epithelial barrier of

the gut. Therefore, considerable research has focused

on the influence of structural properties of dendrimers

on their transport across this barrier.

2.2. Transepithelial transport of dendrimers

Several studies that have examined the oral uptake

and transport of dendrimers include the in vivo studies

of lipidic polylysine-based dendrimers [15,16]. The

biodistribution of a tritium labeled dendrimer of mean

diameter 2.5 nm was studied in Sprague–Dawley rats,

in which at least 20% of the dendrimer was recovered

from the stomach, small intestine and large intestine 6

h after oral administration [15]. These results indicate

rapid uptake of the dendrimers in the GI tract. After

24 h less than 1% of the administered dose was

recovered from the GI tract indicating clearance or

absorption of the dendrimers [15]. Further studies by

Florence et al. examined the influence of particle size

on absorption by comparing the uptake of dendrimer

and polystyrene latex nanoparticles of diameter 2.5

and 50–3000 nm, respectively [16]. The amount of

dendrimer recovered in the small intestine (15%) was

similar to that of 50 nm polystyrene particles (12%)

[16]. However, the cumulative uptake of dendrimer in

the liver, spleen, kidneys and blood was less than that

of the 50 nm polystyrene particles [16].

One would expect the smaller particles to have

greater uptake in the GI tract [17], thus Florence et al.

investigated the possibility of dendrimer aggregation

in the gastrointestinal fluid. Their studies found that

an increase in the concentration of alkyl chain-

terminated (C4–C12) polylysine dendrimers, as well

as increasing the generation from 5 to 6, led to the

formation of aggregates due to the hydrophobic

interactions between the dendrimer surface groups

[18]. Furthermore, dendrimers were found to form

aggregates in stomach fluid and were stable in

intestinal fluid [18]. These findings indicate that the

formation of dendrimer aggregates is influenced by

factors such as pH and dendrimer concentration. The

aggregation of dendrimers in stomach fluid may

explain the lower uptake of dendrimers with diameter

2.5 nm compared to the uptake of polystyrene

nanoparticles of 50 nm.

The first report in the literature demonstrating the

potential of PAMAM dendrimers as oral drug carriers

examined the rate of 125I-labelled PAMAM uptake and

transport in vitro using the everted rat intestinal sac

system [19]. Cationic PAMAM–NH2 dendrimers

showed greater tissue uptake than their serosal transfer

rates across everted rat intestinal sacs at each time

point [19]. In contrast anionic G2.5 and G3.5 showed

greater serosal transfer rates (3.4–4.4 AL/mg protein/h)

than their tissue uptake (0.6–0.7 AL/mg protein/h) in

the same model. Anionic G5.5 had greater tissue

uptake, 30–35% (2.48 AL/mg protein/h), than that of

G2.5 and G3.5 (15–20%), and the serosal transfer of

G5.5 (60–70% recovered in serosal fluid) was less than

that of G2.5 and G3.5 (80–85% recovered in serosal

fluid) [19]. This report demonstrated that there is a size

and charge window where PAMAM dendrimers can

potentially carry drugs across the GI epithelial barrier.

A variety of pre- and post-epithelial factors

influence the transport of compounds across isolated

intestinal tissues including everted sacs. To rule out

these factors and examine the influence of structural

properties of PAMAM dendrimers on their trans-

epithelial transport, we examined their transport

across epithelial cell culture monolayers. As a

preliminary screening tool, we studied the permeabil-

ity of fluorescently labeled cationic PAMAM–NH2

dendrimers across Madin–Darby Canine Kidney

(MDCK) cell monolayers [20]. The transport of

fluorescently labeled cationic PAMAM dendrimers

generations 0–4 (G0–G4) across MDCK cells was

studied as a function of generation number and

polymer concentration. The fluorescently labeled

dendrimers were fractionated by size exclusion

chromatography techniques to rule out the contribu-

tion of small molecular weight fragments to perme-

ability values [20]. The rank order of PAMAM–NH2

permeability was of G4NNG06G1NG3NG2 [20].

An increase in PAMAM–NH2 concentration resulted

in increased dendrimer permeability. These results

demonstrated that in addition to size, other factors

such as charge and concentration of the dendrimers


influence their transport possibly by modulating the

tight junctions or cytotoxicity. At this stage cytotox-

icity of the dendrimers on MDCK cells was not

evaluated. The next logical step was to systematically

evaluate the influence of size, charge, incubation time,

and dendrimer concentration on transepithelial trans-

port across Caco-2 cell monolayers, a well-established

cell culture model for studying intestinal transport.

The transport of fractionated FITC-labeled

PAMAM–NH2 (G0–G4) across Caco-2 cells as a

function of generation number, incubation time and

concentration was studied in both the apical to

basolateral (AB) and basolateral to apical (BA)

directions [21]. Cytotoxicity of the probes was also

studied as a function of dendrimer generation,

concentration, and incubation time, using the lactate

(A)

1.03

1.81

0.26

1.361.18

0.10

2.382.02

0

1

2

3

4

5

6

7

8

G0 G1 G2 G3 G4

Per

mea

bili

ty x

106

(cm

/sec

)

* *

(B

0

1

2

3

4

5

6

7

8

Per

mea

bili

ty x

106

(cm

/sec

)

(C)

3.51

2.19

1.77

4.16

6.04 7.

0

1

2

3

4

5

6

7

8

G0 G1

Per

mea

bili

ty x

106

(cm

/sec

)

Fig. 1. AB (5) and BA (n) Caco-2 permeability of PAMAM–NH2 dendr

times of: (A) 90 min; (B) 150 min; and (C) 210 min. AB and BA perme

Results are reported as meanF standard error of the mean (SEM). From R

dehydrogenase (LDH) assay. The smaller fluores-

cently labeled PAMAM–NH2 dendrimers, G0–G2,

had similar AB permeability values at a donor

concentration of 1.0 mM (Fig. 1), despite their drastic

difference in molecular weights (Table 1) [21]. In

addition, these smaller dendrimers exhibited the

highest permeability while exerting no toxicity at this

concentration towards Caco-2 cell monolayers. LDH

studies demonstrated that an increase in generation

number, concentration or incubation time results in an

increase in cytotoxicity [21]. G3 dendrimers were

cytotoxic at all concentrations upon incubation with

Caco-2 cells for 210 min, whereas G4 exhibited

cytotoxicity at all concentrations and incubation time

points [21]. G2 dendrimers showed cytotoxicity at the

higher concentration of 10.0 mM and incubation time

)

0.610.27

1.581.681.18

4.84

3.48

1.81

G0 G1 G2 G3 G4**

41

G2 G3 G4** **

imers (G0–G4) at a donor concentration of 1.0 mM and incubation

ability values are not reported (**) at toxic incubation time points.

ef. [21] with copyright permission.


of 210 min. The BA permeability of each dendrimer

was generally higher than the corresponding AB

permeability [21]. This could probably be attributed

to the difference in tight junction characteristics at the

apical and basolateral surfaces. The occludin protein

responsible for fusing adjacent enterocytes is present

at the apical surface but not at the basolateral surface

[29–31]. Permeability of PAMAM–NH2 dendrimers

also increased with the increase in incubation time

(Fig. 1). The AB and BA permeability profiles of each

dendrimer exhibited similar generation- and incuba-

tion time-dependent permeability at 1.0 and 10.0 mM

donor concentration [21]. However, the permeability

values for a given generation at 10.0 mM were higher

than that of 1.0 mM [21].

(A)

1.263.08

6.198.13

1.96 2.58

6.28

11.38

0

5

10

15

20

25

30

35

Per

mea

bili

ty x

106

(cm

/sec

)

* * * *

Control G0 G

Control G0 G1 G2 G3 G4

(C)

1.26

7.06

1.96

16.32

10.66

0

10

20

30

40

50

60

70

Per

mea

bili

ty x

106

(cm

/sec

)

Fig. 2. AB (5) and BA (n) Caco-2 permeability of 14C-mannitol at an incu

(C) 10.0 mM solution of PAMAM–NH2 dendrimers (G0–G4). AB and B

Results are reported as meanFSEM. From Ref. [21] with copyright perm

The effect of PAMAM–NH2 dendrimers on the

paracellular pathway was investigated through TEER

measurements and the permeability of a known

paracellular permeability marker, 14C-mannitol.

TEER values decreased with the increase in

PAMAM–NH2 generation number, donor concentra-

tion and incubation time [21]. This data correlates

with the PAMAM–NH2 permeability profiles, in

which prolonged interaction of the dendrimers with

the epithelial surface, and increased surface charge

density due to increased generation number and/or

concentration, can result in modulation of the tight

junctions, which would lead to the decline in TEER

values. PAMAM–NH2 dendrimers were capable of

enhancing the paracellular permeability across Caco-2

(B)

2.93 4.684.071.26

33.41

15.14

7.51

1.96

0

10

20

30

40

50

60

70

Control G0 G1 G2 G3 G4

1 G2 G3 G4

Per

mea

bili

ty x

106

(cm

/sec

)

* * * *

26.35

** * * * *

bation time of 210 min in the presence of: (A) 0.1 mM; (B) 1.0 mM;

A permeability values are not reported (**) at toxic concentrations.

ission.


cell monolayers as evident by increased 14C-mannitol

permeability in a generation-, concentration- and

incubation time-dependant fashion (Fig. 2) [21]. The

decrease in TEER values and the increase in 14C-

mannitol permeability suggest that PAMAM–NH2

dendrimers enhance net paracellular transport by

loosening tight junctions of Caco-2 cells. These

results collectively show that PAMAM–NH2 G0–G4

exhibit an appreciable permeability across Caco-2 cell

monolayers relative to their large size and molecular

weights at nontoxic polymer concentrations. This is in

contrast to the transport properties of hydrophilic,

linear and neutral polymeric carriers such as poly

(ethylene glycol) (PEG) where permeability is dras-

tically reduced above a molecular weight of 600 Da

[32].

2.3. Effect of surface charge on PAMAM

transepithelial transport

Terminal surface groups of PAMAM dendrimers

may be charged or neutral at physiological pH

(Scheme 1). As described above, initial studies

suggest that both positively and negatively charged

dendrimers permeate across epithelial barriers to an

appreciable extent relative to their size [19–21]. We

then studied the influence of surface charge of

PAMAM dendrimers on the transport of 14C-mannitol

across Caco-2 cells and evaluated their cytotoxicity.

PAMAM dendrimers with neutral surface groups

(PAMAM–OH) did not significantly influence TEER

or 14C-mannitol permeability across Caco-2 mono-

layers while those with negatively charged surface

groups (PAMAM–COOH) had a generation-depen-

dent effect on TEER and 14C-mannitol permeability

[22]. G-0.5, G0.5 and G1.5 caused no decline in

TEER values, whereas G2.5 and G3.5 caused a

significant decline in TEER compared to control

values [22]. G4.5 also caused a decline in TEER

values with an increase in incubation time and donor

concentration; however, this was due to cytotoxicity

as evidenced by lactate dehydrogenase (LDH) assay

[22]. The permeability data for 14C-mannitol in the

presence of anionic dendrimers (Fig. 3) supports the

observed decline in TEER: G-0.5, G0.5 and G1.5

caused no change in 14C-mannitol permeability

compared to control values; G2.5 and G3.5 increased

AB 14C-mannitol permeability up to 6-fold; 14C-

mannitol permeability in the presence of G4.5 was

toxic to Caco-2 monolayers at a donor concentration

of 10.0 mM and incubation time of 210 min [22].

Cytotoxicity of PAMAM–NH2, and PAMAM–COOH

dendrimers generally increased in a generation-,

incubation time-, and concentration-dependent man-

ner based on LDH results [21,22]. G-0.5 dendrimers

were non-toxic to Caco-2 cells except at a donor

concentration of 10.0 mM following incubation for

210 min, while G0.5–G4.5 dendrimers were generally

non-toxic to Caco-2 cells after 90 min, except at donor

concentration 10.0 mM for G3.5 and G4.5 [22].

Overall, anionic dendrimers were less toxic to Caco-2

cells than cationic polymers. PAMAM–OH den-

drimers (G2–G4) were not toxic to Caco-2 cells at

any of the evaluated concentrations or incubation time

points [22].

Ethylenediaminetetraacetic acid (EDTA) is a well-

known anionic permeation enhancer, which appears to

modulate the integrity of the tight junctions by

chelation of extracellular Ca2+ and Mg2+ ions that

are required to maintain the integrity of the tight

junctions [29]. Thus, it is likely that PAMAM–COOH

dendrimers similarly modulate the tight junctions by

chelation of the extracellular Ca2+ and Mg2+ ions. The

observed decline in TEER and increase in 14C-

mannitol permeability within a specific size/molecular

weight window (G2.5 and G3.5) can potentially be

due to the fact that a certain surface charge density is

required to bind extracellular divalent ions to effec-

tively open the tight junctions and enhance para-

cellular permeability [29]. The observations in this

study examining the transport of 14C-mannitol across

Caco-2 cell monolayers in the presence of anionic

dendrimers were consistent with previous studies of

PAMAM–COOH transport across rat intestinal

everted sacs [19], in which a specific size window

of PAMAM–COOH dendrimers demonstrate en-

hanced transport. Collectively, these results suggest

that the permeability of PAMAM dendrimers, or

paracellular markers in the presence of PAMAM

dendrimers, across Caco-2 cell monolayers is depen-

dent on terminal surface charge. Positively charged

PAMAM dendrimers increased 14C-mannitol transport

as a function of increase in generation number,

concentration and incubation time, whereas negatively

charged dendrimers caused the highest 14C-mannitol

transport within a specific dendrimer size window.

(A)

1.90

4.00

3.53

1.140.911.011.120.92

1.41

2.68

3.393.00

1.00

2.02

1.501.21

0

1

2

3

4

5

6

Control EDTA G -0.5 G 0.5 G 1.5 G 2.5 G 3.5 G 4.5 Control EDTA G -0.5 G 0.5 G 1.5 G 2.5 G 3.5 G 4.5

Control EDTA G -0.5 G 0.5 G 1.5 G 2.5 G 3.5 G 4.5

Per

mea

bili

ty x

106

(cm

/sec

)

(B)

2.93

0.92 0.76

1.81

0.910.94

5.38 5.50

1.211.76 1.52 1.16

2.44

4.08

4.80

6.55

0

1

2

3

4

5

6

7

8

9

10

Per

mea

bili

ty x

106 (

cm/s

ec)

(C)

0.92

5.36

2.903.42

1.071.851.79

4.81

8.81

1.21

26.99

3.44

0

5

10

15

20

25

30

35

Per

mea

bili

ty x

10

6 (c

m/s

ec)

****

Fig. 3. AB (5) and BA (n) Caco-2 permeability of 14C-mannitol at an incubation time of 90 min in the presence of: (A) 0.1 mM; (B) 1.0 mM;

(C) 10.0 mM solution of EDTA and PAMAM-COOH dendrimers (G-0.5–G4.5). AB and BA permeability values are not reported (**) at toxic

concentrations. Results are reported as meanFSEM. From Ref. [22] with copyright permission.


2.4. Possible transport mechanisms of PAMAM

dendrimers

Several mechanistic studies are possible to elucidate

the transport mechanisms utilized by macromolecules.

One characteristic mechanism is endocytosis (Scheme

2), which is an energy-dependent process [33]. It has

been suggested that the rate of drug permeability and

absorption in active processes such as endocytosis and

carrier-mediated transport (Scheme 2) decreases at

lower temperatures [33]. The transport mechanisms of

PAMAM–NH2 generation 2 (G2–NH2) were investi-

gated in Caco-2 cell monolayers since G2–NH2 had

appreciable permeability and a large number of surface

amine groups that can be used to bind drug molecules

[21]. The AB permeability of G2–NH2 (10.0 mM)

across Caco-2 cell monolayers was investigated at 37

and 4 8C to investigate the contribution of adsorptive-

mediated endocytosis for G2–NH2 transport. G2–NH2

permeability increased with increased incubation times

of 90 to 150 min at 37 8C, while its permeability was

significantly lower at 4 8C than observed at 37 8C for

both incubation times (Fig. 4A) [23]. Other studies also

observed decreased permeability of surface modified

PAMAM dendrimers at 4 8C compared to permeability

at 378C [27]. This data suggests the contribution of

adsorptive-mediated endocytosis to G2–NH2 transport

across Caco-2 cell monolayers. It is important to note

that this observation is only suggestive since reduced

permeability at lower temperatures may also suggest

A B C D

Apical Side

(luminal/mucosal side)

Basolateral Side

(blood/serosal side)

Paracellular PassiveDiffusion

EndocytosisCarrier-mediated

Scheme 2. Schematic representation of possible pathways for cellular uptake and transport. (A) Paracellular pathway; (B) passive diffusion; (C)

carrier-mediated, which can either be facilitated (energy-independent) or active (energy-dependent); (D) endocytosis, which can either be

adsorptive-mediated, fluid-phase, or receptor-mediated. PAMAM dendrimers most likely are transported by routes (A) and (D).


the contribution of other active transport mechanisms,

including receptor-mediated and fluid-phase endocy-

tosis, as well as carrier mediated processes.

Since we observed the BA permeability of G2–NH2

was higher than the corresponding AB permeability

[21], we examined the contribution of an efflux

system, namely P-glycoprotein (P-gp), to PAMAM

transport. The AB and BA permeabilities of 14C-

paclitaxel (200 nM), a known P-gp substrate, were

measured in the absence and presence of G2–NH2

(10.0 mM) at incubation times of 90 and 150 min (Fig.

4B). The BA permeability of 14C-paclitaxel was

greater than the AB permeability, indicating a func-

tioning P-gp efflux system in the utilized Caco-2 cell

monolayers [23]. There was no significant difference

in AB and BA permeability of 14C-paclitaxel in the

presence of G2–NH2 [23]. Similarly, the BA perme-

ability of G2–NH2 was greater than the AB perme-

ability. The AB and BA permeability of G2–NH2 did

not change in the presence of 200 nM paclitaxel (Fig.

4C) [23], which suggests G2–NH2 is not a P-gp

substrate and is therefore not subject to the P-gp efflux

system. Recent studies by D’Emanuele et al. [28]

demonstrate that conjugation of small molecular

weight drugs that are substrates for P-gp to PAMAM

dendrimers increases their transport, also suggesting

that the dendrimers are not substrates for P-gp.

Application of palmitoyl carnitine to the intestinal

epithelium increases membrane fluidity, which results

in opening of the paracellular spaces as a result of

decreasing the lipid order of intestinal brush border

membrane vesicles [34]. The application of palmitoyl

carnitine (0.3 mM) caused statistically significant

increases in the AB permeability values of G2–NH2

and 14C-mannitol across Caco-2 cell monolayers at an

incubation time point of 150 min [23]. The similar

increase observed for G2–NH2 and14C-mannitol AB

permeability in the presence of palmitoyl carnitine

suggests G2–NH2 is in part transported via the

paracellular route (Fig. 4D). This observation was

further supported by the incubation time-dependent

decline in TEER values and the corresponding

increase in 14C-mannitol permeability across Caco-2

cells upon apical incubation with G2–NH2 [23].

These initial studies suggest that G2–NH2 is

transported across Caco-2 cell monolayers by a

combination of an energy-dependent process, such

as adsorptive-mediated endocytosis, and the para-

cellular pathway. Also, these studies indicate G2–NH2

is not a substrate for the P-gp efflux system. Recent

confocal studies in our laboratory (data not shown)

show the perinuclear distribution of PAMAM–NH2

and PAMAM–COOH dendrimers that is comparable

to the perinuclear distribution of the endocytosis

marker transferrin. Confocal studies also show the

disruption of cellular tight junctions after treatment

with PAMAM dendrimers, which indicates PAMAM

dendrimers influence tight junctions of Caco-2 cells.

However, further studies are necessary to evaluate the

contribution of other efflux systems that may contri-

(A)

0.16 0.11

4.21

2.37

0

1

2

3

4

5

6

90 min 150 min

Per

mea

bili

ty x

106

(cm

/sec

)

* *

(B)

8.20

2.01

10.32

3.22

0

2

4

6

8

10

12

AB BA

Per

mea

bili

ty x

106

(cm

/sec

)(C)

3.21

10.94

2.94

11.15

0

2

4

6

8

10

12

14

AB BA

Per

mea

bili

ty x

106

(cm

/sec

)

(D)

1.003.21

35.43

30.71

0

5

10

15

20

25

30

35

40

45

G2 Mannitol

Per

mea

bili

ty x

106

(cm

/sec

)

Fig. 4. (A) Permeability of G2–NH2 at 37 8C (5) and 4 8C (n). (B) AB and BA permeability of 14C-paclitaxel in absence (5) and presence (n)

of G2–NH2 (10.0 mM) at incubation time of 150 min. (C) AB and BA permeability of G2–NH2 (10.0 mM) in absence (5) and presence (n) of14C-paclitaxel (200 nM) at incubation time of 150 min. (D) Permeability of G2–NH2 and

14C-mannitol in absence (5) and presence (n) of 0.3

mM palmitoyl carnitine. Results are reported as meanFSEM. From Ref. [23] with copyright permission.


bute to PAMAM transport. Also, additional studies

have yet to be conducted to delineate the transport

mechanisms used by PAMAM–OH and PAMAM–

COOH dendrimers. The visualization of PAMAM

dendrimers in subcellular components can aid in the

elucidation of transport mechanisms used by PAMAM

dendrimers.

3. Microvascular extravasation of PAMAM–NH2

dendrimers

Extravasation is the process of movement of

different molecules from the blood circulation across

the endothelial lining of capillary walls into the

neighboring interstitial tissues [35]. Once absorbed

orally, drug molecules and drug delivery systems are

distributed and must extravasate from the systemic

circulation across the microvascular endothelium into

the interstitial tissue to reach the site of therapeutic

action [24]. We investigated the influence of size and

molecular weight of a series of PAMAM–NH2

dendrimers on their extravasation across the micro-

vascular endothelium. PAMAM–NH2 dendrimers

(G0–G4) and PEG (MW 6000 Da) were fluorescently

labeled with FITC and subsequently fractionated

using size exclusion chromatography following pro-

cedures described previously [21]. The elution vol-


umes of FITC-labeled PAMAM–NH2 dendrimers and

PEG were measured using size exclusion chromatog-

raphy to indicate the relative sizes of these polymers.

The elution volumes of the dendrimers were in the

order G0NG1NG2NG3NG4 following the expected

trend based on their size and molecular weight [24].

Based on the molecular weight (and not the hydro-

dynamic volume), the examined PEG was expected to

have an elution volume between that of G2–NH2

(MW 3256) and G3–NH2 (MW 6909). However,

PEG eluted earlier than G4–NH2 (MW 14,215), which

can be explained by examining the molecular struc-

tures of the PAMAM dendrimers compared to linear

PEG. PAMAM dendrimers have a compact spherical

shape in solution [8], while PEG molecules have a

random or coiled conformation in solution [36]. Thus,

PEG polymers have a larger hydrodynamic volume

than PAMAM–NH2 dendrimers of comparable mo-

lecular weight, and therefore elute earlier than the

PAMAM–NH2 dendrimers.

Intravital microscopy is a useful technique for the

qualitative and quantitative in vivo observation of

leukocyte–endothelial cell interactions [37,38], blood

flow rate within arterioles or post-capillary venules,

lymph vessel kinetics during inflammatory conditions,

microvascular permeability of macromolecules, and

measurement of the microvascular network architec-

ture and density [38]. The cremaster muscle of male

0

100

200

300

400

500

600

0 4000 80Molecular w

Ext

rava

sati

on

tim

e (s

ec)

Fig. 5. The relation between polymer extravasation time (s) (s) and polymer

[24] with copyright permission.

golden Syrian hamsters was used as an in vivo model

to measure the extravasation time (s) of fluorescentlylabeled polymers across the microvascular endotheli-

um using intravital microscopy techniques. Extrava-

sation time (s) was determined post-injection by the

time required of labeled polymer for the fluorescence

intensity in the interstitial tissue to reach 90% the

fluorescence intensity in the neighboring microvascu-

lature [24]. Extravasation time (s) increased exponen-

tially with the increase in molecular weight and size of

the PAMAM dendrimers [24]. The order of extrava-

sation time for PAMAM–NH2 dendrimers was

G0bG1bG2bG3bG4, ranging from 143.9–422.7 s.

This size-dependent selectivity is due to the increased

exclusion of PAMAM–NH2 dendrimers from the

endothelial pores, 4–5 nm in radius, as the dendrimer

size was increased [24]. Furthermore, the extravasa-

tion time of linear PEG (MW 6000 Da) was greater

than that of all PAMAM–NH2 dendrimers. This data

correlates with the observed elution volume of PEG

compared to those of PAMAM–NH2 dendrimers (Fig.

5). Thus, these observations indicate PEG had longer

extravasation time (s) across the microvascular

endothelium because of its larger hydrodynamic

volume and neutral charge compared to the cationic

and spherical PAMAM–NH2 dendrimers.

Based on the reported molecular sizes of

PAMAM–NH2 dendrimers, ranging from 1.5–4.5

00 12000 16000eight (Da)

molecular weight; (!): PAMAM dendrimers, (E): PEG. From Ref.


nm [8], it seems that dendrimers would cross the

microvascular endothelium through the endothelial

pores of diameter 4–5 nm [24]. Thus, the increase in

molecular size of the studied probes results in an

increase in their degree of exclusion from the

endothelial pores, which explains the increase in

extravasation time (s) with an increase in polymer

molecular size. Additionally, the microvascular endo-

thelium is lined with the glycocalyx layer, which is

composed of negatively charged sulfated glycosami-

noglycan [39]. The observed extravasation of

PAMAM–NH2 dendrimers and PEG molecules may

be a function of the electrostatic interactions between

the polymers and the negatively charged endothelium.

PAMAM–NH2 dendrimers are positively charged at

physiological pH, whereas PEG molecules are neutral.

As a result, the electrostatic interaction between

PAMAM–NH2 dendrimers and the negatively charged

glycocalyx lining was more favorable compared to the

neutral PEG molecules, which led to the faster

extravasation time observed for PAMAM–NH2 den-

drimers compared to PEG.

There are some limitations to the use of the cremaster

muscle preparation as an in vivo model for extravasa-

tion experiments, including the contribution of uptake

of the polymers by different tissues in the body [24].

Another limitation is the possible interaction of the

polymers with plasma components [24], which would

result in the overestimation of extravasation time.

Therefore, appropriate in vitro studies of PAMAM

extravasation across endothelial cell monolayers is

necessary to further elucidate the physiological factors

that influence their microvascular permeability. Over-

all, this study demonstrated that an increase in

molecular weight and size of polymers results in

increased extravasation time across the microvascular

endothelium. Molecular geometry and surface charge

also influence the microvascular extravasation of

water-soluble polymers across the endothelial barrier,

in which compact, spherical, positively charged

PAMAM dendrimers have shorter extravasation times

than random-coiled, neutral PEG chains.

4. Conclusions and future direction of this research

Studies to date in our laboratory [20–24] and others

[19,25–28] demonstrate the potential of PAMAM

dendrimers as carriers of bioactive agents for oral

delivery. Clearly factors such as dendrimer surface

charge, surface modification, size, concentration, and

incubation time play a role. There is a window of

opportunity where these factors can be optimized for

specific oral drug delivery needs. However the

mechanism(s) of transport of these dendrimers must

be further elucidated. Although the influence of

PAMAM surface charge has been investigated, a

systematic correlation between the effect of size and

charge of these polymers on their transport mecha-

nism(s) across the intestinal epithelial barrier has not

been evaluated to date. To what extent these den-

drimers are transported paracellularly versus across

the cells, how they enhance paracellular transport,

mechanism(s) of endocytosis, subcellular trafficking

and localization, as well as interaction with efflux

pumps and receptors are still unknown. In addition,

limited data is available about the relationships

between their structural features on one hand and GI

stability on the other. Basic mechanistic studies will

add to our current knowledge of polymer interaction

with intestinal epithelial barriers. By studying the

transport mechanism(s) of PAMAM dendrimers and

structural features that influence their transport while

reducing their toxicity and maximize stability, it

would be possible to develop novel polymeric

systems that enhance oral bioavailability of poorly

bioavailable drugs as well as target drugs to specific

sites in the body. This will be a substantial improve-

ment over current polymeric drug delivery systems

that are primarily administered via the parenteral

route.

Acknowledgements

The research described in this article was supported

by Guilford Pharmaceuticals, Inc., the American

Association of Colleges of Pharmacy, and the Univer-

sity of Maryland School of Pharmacy. Financial

support for Kelly Kitchens was made possible by a

National Research Service Award Predoctoral Fellow-

ship from the National Institutes of Health (GM67278-

01). We deeply appreciate the collaboration and advice

of a number of colleagues in our investigations related

to this research whose names are listed as coauthors in

Refs. [20–24].


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