In vitro evaluation of third generation PAMAM dendrimer conjugates

Article abstract of DOI:10.3390/molecules22101661

The present study compares the use of high generation G3 and low generation G0 Polyamidoamine (PAMAM) dendrimers as drug carriers of naproxen (NAP), a poorly water soluble drug. Naproxen was conjugated to G3 in different ratios and to G0 in a 1:1 ratio via a diethylene glycol linker. A lauroyl chain (L), a lipophilic permeability enhancer, was attached to G3 and G0 prodrugs. The G3 and G0 conjugates were more hydrophilic than naproxen as evaluated by the measurement of partitioning between 1-octanol and a phosphate buffer at pH 7.4 and pH 1.2. The unmodified surface PAMAM-NAP conjugates showed significant solubility enhancements of NAP at pH 1.2; however, with the number of NAP conjugated to G3, this was limited to 10 molecules. The lactate dehydrogenase (LDH) assay indicated that the G3 dendrimer conjugates had a concentration dependent toxicity towards Caco-2 cells. Attaching naproxen to the surface of the dendrimer increased the IC50 of the resulting prodrugs towards Caco-2 cells. The lauroyl G3 conjugates showed the highest toxicity amongst the PAMAM dendrimer conjugates investigated and were significantly more toxic than the lauroyl-G0-naproxen conjugates. The permeability of naproxen across monolayers of Caco-2 cells was significantly increased by its conjugation to either G3 or G0 PAMAM dendrimers. Lauroyl-G0 conjugates displayed considerably lower cytotoxicity than G3 conjugates and may be preferable for use as a drug carrier for low soluble drugs such as naproxen.

Full text of DOI:10.3390/molecules22101661

molecules
Article
In Vitro Evaluation of Third Generation PAMAM
Dendrimer Conjugates
*
Mohammad Najlah 1, ID , Sally Freeman 2, Mouhamad Khoder 3, David Attwood 2 and
Antony D’Emanuele 4
1
Faculty of Medical Science, Anglia Ruskin University, Bishops Hall Lane, Chelmsford CM1 1SQ, UK
Division of Pharmacy and Optometry, School of Health Sciences, Manchester Academic Health Sciences
Centre, University of Manchester, Manchester M13 9PL, UK; sally.freeman@manchester.ac.uk (S.F.);
David.Attwood@manchester.ac.uk (D.A.)
School of Life Sciences, Pharmacy and Chemistry, Kingston University London,
Kingston upon Thames KT1 2EE, UK; m.khoder@kingston.ac.uk
2
3
4
Leicester School of Pharmacy, De Montfort University, Leicester LE1 9BH, UK;
antony.demanuele@dmu.ac.uk
*
Correspondence: mohammad.najlah@anglia.ac.uk or m_najlah@yahoo.co.uk; Tel.: +44-845-196-4682
Received: 26 September 2017; Accepted: 30 September 2017; Published: 4 October 2017
Abstract: The present study compares the use of high generation G3 and low generation G0
Polyamidoamine (PAMAM) dendrimers as drug carriers of naproxen (NAP), a poorly water soluble
drug. Naproxen was conjugated to G3 in different ratios and to G0 in a 1:1 ratio via a diethylene glycol
linker. A lauroyl chain (L), a lipophilic permeability enhancer, was attached to G3 and G0 prodrugs.
The G3 and G0 conjugates were more hydrophilic than naproxen as evaluated by the measurement
of partitioning between 1-octanol and a phosphate buffer at pH 7.4 and pH 1.2. The unmodified
surface PAMAM-NAP conjugates showed significant solubility enhancements of NAP at pH 1.2;
however, with the number of NAP conjugated to G3, this was limited to 10 molecules. The lactate
dehydrogenase (LDH) assay indicated that the G3 dendrimer conjugates had a concentration
dependent toxicity towards Caco-2 cells. Attaching naproxen to the surface of the dendrimer increased
the IC50 of the resulting prodrugs towards Caco-2 cells. The lauroyl G3 conjugates showed the
highest toxicity amongst the PAMAM dendrimer conjugates investigated and were significantly more
toxic than the lauroyl-G0-naproxen conjugates. The permeability of naproxen across monolayers of
Caco-2 cells was significantly increased by its conjugation to either G3 or G0 PAMAM dendrimers.
Lauroyl-G0 conjugates displayed considerably lower cytotoxicity than G3 conjugates and may be
preferable for use as a drug carrier for low soluble drugs such as naproxen.
Keywords: PAMAM dendrimers; transepithelial transport; Caco-2 cells; dendrimer prodrugs;
oral drug delivery
1. Introduction
Dendrimers represent highly branched macromolecules that have a well-defined structure with
precisely controlled size and shape as well as terminal group functionality [
of dendrimers in several pharmaceutical applications including controlled drug delivery has been
extensively investigated [ ]. The high degree of branching allows drug encapsulation and the
formation of dendrimer-drug conjugates [ ], in addition, the ability to modify terminal functional
1]. The potential use
2–4
5
groups allows for the surface engineering of dendrimers for different applications such as the
enhancement of drug solubility [6–8] and permeability [9].
In dendrimer-drug conjugates, the drug is combined through a covalent bond either directly or
via a linker/spacer to the dendrimer. Our previous work showed that the direct conjugation of a drug
Molecules 2017, 22, 1661; doi:10.3390/molecules22101661
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1661
2 of 12
to PAMAM dendrimer (via an amide bond) resulted in a stable conjugate that would not be suitable
for use as a drug delivery system [10]. In contrast, using diethylene glycol as a linker/spacer between
the drug and dendrimer resulted in ester prodrugs that showed high chemical stability at pH 1.2,
7.4 and 8.5 (37 ◦C), but that readily released the drug in human plasma (in vitro). Such conjugates
have potential as carriers for low solubility drugs, such as naproxen. More recently, surface modified
G0-naproxen conjugates were found to increase significantly the permeability of naproxen across
epithelial cells [11].
In the present study, naproxen (Figure 1) is conjugated to both G0 and G3 PAMAM dendrimers,
via a diethylene glycol linker. The biological properties (in vitro) of the conjugates are compared
including their cytotoxicity (LDH assay) and transport across Caco-2 monolayers. The influence of
attaching a surface modifier (lauroyl chain) on transport properties is also evaluated and the respective
merits of using G0 and G3 as oral drug carriers are considered.
Figure 1. Structure of (a) G3 PAMAM dendrimer with an ethylenediamine core, (b) G0 PAMAM
dendrimer with an ethylenediamine core and (c) Naproxen.

Molecules 2017, 22, 1661
3 of 12
2. Results and Discussion
2.1. Synthesis and Characterization of G3 PAMAM Dendrimer-Naproxen Conjugates
The synthesis and characterization of G0 dendrimer conjugates were previously reported [10,11]
in a study in which diethylene glycol was used as a linker between naproxen, a poorly water-soluble
drug, and G3 PAMAM dendrimer (Figure 1). Naproxen-diethylene glycol (NAP-deg) was attached
to the primary amines of G3 dendrimer using 4-nitrophenyl chloroformate activation. NAP-deg was
initially reacted with 4-nitrophenyl chloroformate to obtain NAP-deg-(4-nitrophenyl carbonate) which
1
was purified and characterized by H-NMR and 13C-NMR spectroscopy [10]. G3 dendrimer and
NAP-deg-(4-nitrophenyl carbonate) were reacted in appropriate molar ratios to form a carbamate bond
between the diethylene glycol linker and the G3 dendrimer. Lauroyl alcohol was attached to a primary
amine of the G3 dendrimer-naproxen conjugates through a carbamate bond using 4-nitrophenyl
chloroformate activation (Figure 2a).
Figure 2.
(a) Illustrative scheme for the synthesis of G3 PAMAM conjugates and (b) G3 PAMAM
dendrimer-naproxen conjugates (letters are added to aid 1H-NMR assignments).
G3-(deg-NAP)x and LyG3-(deg-NAP)x were purified by size exclusion chromatography,
solubilization in water (NAP-deg and lauroyl alcohol are poorly soluble in water) and dialysis
(MW cutoff = 3500 Da) for 48 h.

Molecules 2017, 22, 1661
4 of 12
1H-NMR spectroscopy of the conjugates showed the presence of NAP-deg and/or lauroyl chain
and G3 PAMAM dendrimer in the product (Figures 2b, 3 and 4). The formation of a carbamate bond
between the diethylene glycol linker and/or lauroyl chain and the amine surface of G3 dendrimer
was confirmed by the appearance of a peak(s) at 159.2 ppm on the 13C-NMR spectrum, and were
assigned with the aid of DEPT-135 (Figure 5). No traces of the free drug, drug linker or lauroyl chain
were detected using RP-HPLC of the conjugates (data not shown), which confirmed that naproxen
or/and lauroyl chain is covalently (rather than electrostatically) attached to the dendrimer. The drug
and lauroyl chain average payloads in the conjugates were estimated using the relative intensities of
the peaks in the 1H-NMR spectrum originating from attached ligands compared to those of dendrimer
(Table 1). It is worth noting that size exclusion was used to produce conjugates with the narrowest
possible polydispersity in terms of size and molecular weight.
Table 1. Conjugation ratios and concentrations PAMAM dendrimer conjugates used in the
transport studies.
Equivalent
Concentration of
Naproxen (µM)
Conjugation Ratio
G3:Naproxen †
Conjugation Ratio
G3:Lauroyl Chain †
Average
wt/mol (g/mol)
Conjugate
Concentration (µM)
G3
n/a
1:5.2
1:10.5
1:4.8
1:5.4
n/a
1:1
n/a
n/a
n/a
1:6.4
1:11.6
n/a
n/a
1:1
6909
8636
10,363
9916
11,196
517
n/a
19.2
9.5
n/a
99.8
99.8
99.8
n/a
G3-(deg-NAP)5
G3-(deg-NAP)10
L6G3-(deg-NAP)5
L12G3-(deg-NAP)5
G0
20.8
n/a
n/a
100.0
100.0
n/a
G0-deg-NAP
L-G0-deg-NAP
861
100.0
100.0
1:1
1073
†
Determined by 1H-NMR spectroscopy.
1
Figure 3. H-NMR (d4-MeOD) spectra of (a) G3-(deg-NAP)10 and (b) G3-(deg-NAP)5.

Molecules 2017, 22, 1661
5 of 12
1
Figure 4. H-NMR (d4-MeOD) spectra of (a) L12G3-(deg-NAP)5 and (b) L6G3-(deg-NAP)5.
Figure 5. 13C-NMR and DEPT-135 (d4-MeOD) spectra of G3-(deg-NAP)10.
2.2. Determination of Partition Coefficient and Solubility
Solubility is one of the physicochemical parameters that influence drug permeability, and therefore
its pharmacokinetic profile and therapeutic activity. In order to increase the solubility of lipophilic
naproxen, it was attached to the surface groups of highly water soluble PAMAM dendrimer through
a degradable linkage [10]. Since determination of solubility would require the synthesis of large
amounts of conjugate, a maximum of 50 mg conjugate per mL was used to evaluate the enhanced
solubility of NAP after conjugation at pH 1.2 (i.e., naproxen (pKa 4.2) is not ionised at this pH).
The apparent partition coefficients (Kapp(o/w)) of naproxen and conjugates between 1-octanol and
phosphate buffer (pH 7.4) were determined as an indicator reflecting the change in the lipophilicity
of naproxen after attaching to G0 or G3 PAMAM dendrimers. The conjugation of naproxen to

Molecules 2017, 22, 1661
6 of 12
both G0 and G3 dendrimers resulted in a significant decrease in the lipophilicity of naproxen as
indicated by the apparent partition coefficients of dendrimer naproxen prodrugs (Table 2). For G0
conjugates, the surface modified conjugate L-G0-deg-NAP showed higher lipophilicity compared to
the unmodified surface conjugates G0-deg-NAP (but lipophilicity is still significantly (p < 0.05) lower
than that of naproxen) as a result of the presence of lipophilic chains (lauroyl chains) on the surface
of dendrimer (Table 2). In the case of G3 conjugates, surface modified conjugates (i.e., those having
attached lauroyl chains) showed no significant difference (p > 0.05) in lipophilicity compared to the
unmodified surface conjugates. Apart from Naproxen (pKa 4.2) which is ionised at pH 7.4, there was
no significant difference in log Kapp(o/w) values of the conjugates at both pH 7.4 and pH 1.2.
Correspondingly, the covalent conjugation of naproxen to both generations (G0 and G3) resulted
in significant enhancements (p < 0.05) in its solubility. The highest solubility of conjugated naproxen is
shown for the conjugates that are not surface modified (p < 0.05, Table 3). However, attaching more
than five naproxen molecules and 12 lauroyl chains to G3 dendrimers resulted in products that were
insoluble in water (data not shown).
◦
Table 2. The log Kapp(o/w) (pH 7.4 and pH 1.2) values of naproxen and its conjugates at 37 C.
Compound
pH 7.4
pH 1.2
NAP
G0-deg-NAP
0.11 ± 0.01
−1.07 ± 0.09
−0.19 ± 0.07
−0.36 ± 0.12
−0.40 ± 0.13
−0.40 ± 0.09
−0.18 ± 0.04
2.8 ± 0.7
−1.17 ± 0.1
−0.18 ± 0.05
−0.35 ± 0.19
−0.37 ± 0.17
−0.32 ± 0.11
−0.17 ± 0.09
L-G0-deg-NAP
G3-(deg-NAP)5
G3-(deg-NAP)10
L6G3-(deg-NAP)5
L12G3-(deg-NAP)5
◦
Table 3. The solubility (pH 1.2) naproxen and its conjugates at 37 C.
Compound
Solubility mg/mL
Solubility Eq. con. NAP * (mM)
NAP
G0-deg-NAP
0.06
>50
0.26
>58
L-G0-deg-NAP
G3-(deg-NAP)5
G3-(deg-NAP)10
L6G3-(deg-NAP)5
L12G3-(deg-NAP)5
35 ± 12
32 ± 11
>50
>28
>50
>48.2
29 ± 17
14 ± 8.2
18 ± 12
8 ± 5.7
* Equivalent concentration of naproxen (mM).
2.3. The Effect of PAMAM Dendrimer Prodrugs on Caco-2 Cell Viability
The influence of PAMAM dendrimer prodrugs on the viability of Caco-2 cells is reported using
the LDH assay. LDH, a cytosolic enzyme, has been found to leak out of cells upon damage to cell
membranes [12]. G3 PAMAM dendrimer and conjugates showed a concentration-dependent toxicity
(Figure 6 and Table 4) with approximately 20% cytotoxicity at concentrations of 100 µM after 180 min
incubation. Attaching 5 molecules of naproxen + linker to the surface of G3 PAMAM dendrimer
had no significant impact on the cytotoxicity of the resulting prodrug (G3-(deg-NAP)5). However,
a significant decrease in cytotoxicity was found after attaching 10 molecules of naproxen + linker to
give G3-(deg-NAP)10. The reduced cytotoxicity might result from reduction and shielding of the
cationic charges on the dendrimer surface. Similar findings have been previously reported [13–15].

Molecules 2017, 22, 1661
7 of 12
Table 4. The effect of G3-naproxen conjugates (LyG3-(deg-NAP)x) and L-G0-deg-NAP on the viability
of Caco-2 cells as determined by IC50 (mean ± S.D., n = 4).
Compound
IC50 (µM)
G3
247 ± 35
225 ± 32
357 ± 57
189 ± 28
76 ± 15
G3-(deg-NAP)5
G3-(deg-NAP)10
L6G3-(deg-NAP)5
L12G3-(deg-NAP)5
L-G0-deg-NAP
3181 ± 455
Figure 6. The effect of G3 PAMAM dendrimer, G3-naproxen conjugates (LyG3-(deg-NAP)x),
and L-G0-deg-NAP on the viability of Caco-2 cells (LDH assay) (mean ± S.D., n = 4).
As shown in Table 4 (IC50 results) and Figure 6, the presence of 6 lauroyl chains increased
(p < 0.05) conjugate cytotoxicity (L6G3-(deg-NAP)5) and an even more pronounced increase (p < 0.05) in
cytotoxicity was found when 12 lauroyl chains were attached (L12G3-(deg-NAP)5), which was excluded
from transport studies due to its high cytotoxicity. These results may be explained by the intrinsic
cytotoxicity of the lauroyl chain [11,13,14,16]. In contrast, previous studies from our group reported that
the cytotoxicity of G3 PAMAM can be decreased by modifying it’s surface with 6 lauroyl chains, but was
significantly increased when the number of attached chains was increased to 9. The reason for these
differences may be that in the earlier work, a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay was used rather than the LDH assay. A recent study by our group has found that
the MTT assay can yield anomalous data in the assessment of high generation dendrimer cytotoxicity
in Caco-2 cells (unpublished data). A comparison between the IC50 of LG0-deg-NAP and that of G3
PAMAM dendrimer conjugates shows that the G0 prodrug is significantly less toxic (p < 0.05) than G3
and its conjugates at the same concentrations (Table 4 and Figure 6). This is in agreement with previous
observations which showed that the cytotoxicity of PAMAM dendrimers was due to concentration,
generation, and charge dependency [13,17].

Molecules 2017, 22, 1661
8 of 12
2.4. Transport of Naproxen, G0 PAMAM Dendrimer and Conjugates across Caco-2 Monolayers
The transport of naproxen, G3 conjugates and L-G0-deg-NAP across Caco-2 monolayers was
investigated in both A B and B A directions at non-toxic concentrations (as determined by the
LDH assay). The concentration of conjugates used in the study were all equivalent to 100
→
→
µM
naproxen (Table 1). Approximately 8% of the conjugate was hydrolyzed during permeation through
the cells to release naproxen, which was detected in the receiver compartment. Therefore, in order
to compare transport data for the range of conjugates, permeability was expressed as the percentage
of equivalent naproxen transported across Caco-2 monolayers after 180 min (Figure 7) rather than
apparent permeability coefficient.
Figure 7. The A-B (ꢀ) and B-A (ꢁ) transport of naproxen across Caco-2 cell monolayers for naproxen
and conjugates (each equivalent to 100 µM naproxen) after 3 h of incubation (mean ± S.D., n = 4).
The transport of G3-(deg-NAP)5 and G3-(deg-NAP)10 across Caco-2 monolayers, especially
in the A
→
B direction, was similar and both were significantly higher (p < 0.05) than that of
naproxen itself. No significant difference (p > 0.05) can be observed between the transport of G3
conjugates (G3-(deg-NAP)5 and G3-(deg-NAP)10) and G0-deg-NAP. These results suggest that the
PAMAM dendrimer functions as a drug carrier that is able to cross cellular barriers and enhance the
permeability of the drug regardless of the generation. Moreover, these findings are in agreement with
previous observations that low generation PAMAM dendrimers (G0 and G1) exhibit significantly less
cytotoxicity and higher permeability than higher generations (G2, G3 and G4) [17].

Molecules 2017, 22, 1661
9 of 12
A comparison between the transport of naproxen-diethylene glycol (NAP-deg) and naproxen
confirms that the enhanced permeability of the conjugate arises from the attachment of the drug to
the PAMAM dendrimer. The high permeability of PAMAM dendrimer conjugates may be explained
by the presence of positively charged amine surface groups. Knipp et al. [18] reported that positively
charged molecules permeate at a higher rate across Caco-2 monolayers compared to neutral or anionic
molecules because of the favourable electrostatic interaction with the negatively charged epithelial
surfaces. Similarly, Jevprasesphant et al. [13] have found that the permeability of cationic dendrimers
(G2, G3, and G4) was higher than that of anionic dendrimers (G2.5 and G3.5).
To enhance the permeability of PAMAM dendrimer conjugates, lauroyl alcohol, a permeability
enhancer [19], was used as a surface modifier of G0 and G3 PAMAM dendrimers. As expected,
the resulting surface modified G0 and G3 PAMAM dendrimers (L-G0-deg-NAP and L6G3-(deg-NAP)5)
showed further enhancement (p < 0.05) in the transport of naproxen across epithelial cells (Figure 7).
L6G3-(deg-NAP)5 showed the highest transport (p < 0.05) in both directions across Caco-2 cell
monolayers of all the PAMAM dendrimer conjugates evaluated. However, L-G0-deg-NAP showed
an appreciable enhancement of naproxen transport (Figure 7) but is much less cytotoxic (p < 0.05)
compared to L6G3-(deg-NAP)5 (Table 4). In general, transepithelial transport of dendrimer conjugates
has been found to be due to both paracellular and transcellular pathways [16].
3. Materials and Methods
3.1. Materials
Third generation PAMAM dendrimers (G3) with ethylenediamine cores were purchased
from Dendritech Inc. (Midland, MI, USA). Trifluoroacetic acid (TFA), triethylamine (TEA),
(S)-(+)-6-methoxy-α-methyl-2-naphthaleneacetic acid (naproxen), 4-nitrophenyl chloroformate,
1-dodecanol (lauroyl alcohol), ethylenediamine tetraacetic acid (EDTA), lactate dehydrogenase assay
kits, trypan blue, Sephadex 25, and Sephadex LH-20 were purchased from Sigma-Aldrich Co. Ltd.
(Poole, Dorset, UK). Cell culture materials were from Gibco BRL Life Technologies (Paisley, Scotland).
Polycarbonate cell culture inserts (Transwell® 12 mm diameter) and cluster plates (96 well) were
purchased from Corning Costar UK (High Wycombe, Bucks, UK). Naproxen-diethylene glycol
(NAP-deg), NAP-deg-(4-nitrophenyl carbonate), lauroyl (4-nitrophenyl carbonate), G0-diethylene
glycol-naproxen (G0-deg-NAP) and lauroyl-G0-diethylene glycol-naproxen (L-G0-deg-NAP) were
synthesized and characterized as previously described [10,11]. G3-naproxen conjugates were
characterized using 13C and 1H-NMR spectroscopy (300 MHz, Bruker Avance 300, Bruker, Coventry,
UK). 13C-NMR spectra were assigned with the aid of DEPT-135. HPLC analyses were carried out
using a Hewlett-Packard Series II 1090 (Hewlett-Packard, Waldbronn, Germany) instrument equipped
with a Luna 5
µ
m, C18 column (250
×
4.6 mm, Phenomenex, Cheshire, UK). The solvent system
for characterization of the conjugates was methanol:ACN:H3PO4 (0.05% w/v) (10:32:58) for 2 min
then (48:32:20) for the remaining elution time. The flow rate was 1.2 mL/min and UV detection was
at
λ = 230 nm. HPLC solvent systems for the apparent partition coefficients and transport studies:
methanol:aq H3PO4 (0.05% w/v) (80:20) using phenanthrene as an internal standard. The flow rate was
1.2 mL/min and UV detection was at λ = 230 nm.
3.2. Synthesis of G3-(Diethylene glycol-naproxen)x (G3-(deg-NAPx))
In order to yield the target ratio NAP:G3 in anhydrous dimethylformamide (DMF) (1 mL),
10% excess of the required amount of NAP-deg-(4-nitrophenyl carbonate) was added dropwise over
2 hours (h) to a stirred solution of G3 dendrimer in DMF (2 mL). The reaction mixture was stirred
for 5 days. DMF was evaporated under vacuum. The residue was dissolved in 3 mL water and
filtered. The filtrate was purified by size exclusion chromatography, using Sephadex 25, eluting with
methanol:water (1:5). G3-(deg-NAP)5 and G3-(deg-NAP)10 were synthesized and characterized by
1H-NMR and 13C-NMR spectroscopy.

Molecules 2017, 22, 1661
10 of 12
3.3. Synthesis of (Lauroyl)y-G3-(diethylene glycol-naproxen)x (LyG3-(deg-NAP)x)
In order to yield the target ratio L:G3 in 1 mL DMF, 10% excess of the required amount of lauroyl
(4-nitrophenyl carbonate) was added dropwise over 2 h to a stirred solution of G3-(deg-NAP)5 in DMF
(2 mL). The reaction mixture was stirred for 5 days. DMF was evaporated under vacuum and the
residue was purified by size exclusion chromatography using Sephadex LH 20 with methanol:water
(5:1 v/v). The resulting product was dissolved in water and filtered. The filtrate was concentrated
under vacuum and purified again by size exclusion chromatography using Sephadex 25, eluting with
methanol:water (1:5 v/v). L6G3-(deg-NAP)5, L12G3-(deg-NAP)5, were synthesized and characterized
by 1H-NMR and 13C-NMR spectroscopy (Bruker Avance 300, Bruker, Coventry, UK).
3.4. Determination of Partition Coefficients and Soluiblities
The apparent partition coefficients (Kapp(o/w)) of G0 and G3 dendrimer conjugates between
1-octanol and phosphate buffer (pH 7.4) and pH 1.2 (0.06 M hydrochloric acid buffer) were determined
at 37 ◦C. Before use, the 1-octanol was saturated with phosphate buffer for 24 h by stirring vigorously.
A known amount of conjugate in phosphate buffer (pH 7.4, 5 mL) was shaken for 72 h with 1-octanol
(5 mL) to achieve equilibrium, and the phases were separated by centrifugation at 10,000 revolution per
minute (rpm) for 5 minutes (min). All experiments were performed in triplicate. The concentrations
of the compounds in the buffer phase before and after partitioning were determined by HPLC
(Hewlett-Packard, Waldbronn, Germany).
The solubility of G0 and G3 dendrimer conjugates in pH 1.2 (0.06 M hydrochloric acid buffer) were
determined at 37 ◦C. All synthesized amount of conjugate (50 mg) added to the buffer (pH 1.2, 1 mL)
was shaken for 72 h to achieve saturation. The unsolubilised conjugate was then filtered (Millipore,
Feltham, UK, 0.45 µm). All experiments were performed in triplicate. The concentrations of the
compounds in the buffer were determined by HPLC.
3.5. Lactate Dehydrogenase Leakage (LDH) Assay
Human intestinal adenocarcinoma cells (Caco-2) (passage 110–120) were seeded at 10,000 cells/well
◦
in 96-well plates and maintained at 37 C in an atmosphere of 5% CO2 and 95% relative humidity
in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% foetal bovine serum,
1% non-essential amino acids, 50 IU/mL penicillin. and 50 mg/mL streptomycin. After 24 h the
medium was removed and the cells were washed in phosphate buffered saline and 200 µL Hanks
Balanced Salt Solution (HBSS) containing G3 PAMAM dendrimer, conjugates or G0 PAMAM dendrimer
at different concentrations (concentrations were calculated using the average molecular weight (Table 1)
for each conjugate as a whole macromolecule). Caco-2 cell monolayers were also treated with blank
◦
HBSS and 1% Triton X-100 as low and high controls, respectively. After 3 h of incubation at 37 C,
100 µL/well supernatant was removed carefully and transferred into corresponding wells of an optically
clear 96-well flat bottom microplate. LDH leakage in the compartment was quantified using an LDH
assay kit.
The percentage was calculated using the following equation:
Cytotoxicity (%) = ((exp. − low control)/(high control − low control)) × 100
where exp. is the experimental value.
3.6. Transport Studies of G3 PAMAM Dendrimer and Conjugates
Caco-2 cells (passage 83–92 for G0 conjugates and 115–122 for G3 conjugates) were seeded onto
polycarbonate 12-well Transwell filters (pore size 3.0
µ
m, at a density of 1.2
×
105 cells/cm2. Cells were
◦
grown at 37 C in an atmosphere of 5% CO2 and 95% relative humidity in Dulbecco’s Modified
Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum, 1% non-essential amino acids,
50 IU/mL penicillin and 50 mg/mL streptomycin. The medium was changed on alternate days for

Molecules 2017, 22, 1661
11 of 12
21–22 days. The integrity of cells was assessed by measuring the transepithelial electrical resistance
(TEER) using a voltohmmeter (EVOM, World Precision Instruments, Sarasota, FL, USA) before and
after the experiments. Prior to transport experiments, cells were equilibrated with HBSS for 20 min
◦
at 37 C, and the TEER was determined. The TEER value, corrected for the blank filter resistance,
was in the range of 800–950 Ω·cm2. Only confluent monolayers were used for the transepithelial
transport studies. Transport of compounds was determined in both apical-to-basolateral (A
→
B)
and basolateral-to-apical (B
N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid (HEPES) and was placed in the donor and
receiver compartments. Naproxen, G0 or G3 dendrimer conjugates (each equivalent to 100 M of
naproxen, Table 1) were placed in the donor compartment, and cells were incubated in a humidified
atmosphere at 37 ◦C. TEER was measured every 30 min during the experiment, samples (50
L) were
→
A) directions. The transport medium (TM) was HBSS with 25 mM
µ
µ
removed from the receiver compartment at time zero, and after 60, 120 and 180 min, and from the
donor compartment after 180 min. In order to calculate the percentage of naproxen released from the
conjugate during transport studies, an additional sample was taken from the receiver after 180 min,
and TFA was added to release all conjugated naproxen. The samples were analyzed by HPLC.
3.7. Statistical Analysis
All experiments were performed at least three times using at least three different batches and
the results are presented as the mean
±
SD. A one-way analysis of variance (ANOVA) statistical test
was performed to allow comparison of more than two groups and Student’s t-test was conducted to
compare two sets of results. The differences were considered to be statistically significant if the p-value
was ≤ 0.05.
4. Conclusions
The synthesis, characterization, cytotoxicity and transport studies of G3 PAMAM dendrimer-
naproxen conjugates using diethylene glycol as spacer/linker are reported. Partition coefficient results
indicate that G3-naproxen conjugates were significantly more hydrophilic than the parent drug.
A comparison was made between the cytotoxicity and transport profiles of G3 dendrimer conjugates
and that of G0 dendrimer conjugates. Cytotoxicity studies showed that G0 dendrimer conjugates were
only slightly toxic towards Caco-2 monolayers, and their toxicity was significantly lower than that of G3
dendrimer conjugates. Conjugation of naproxen to G0 and G3 PAMAM dendrimer significantly increased
its permeability in both directions across Caco-2 monolayer. A more pronounced increase of naproxen
transport was observed when lauroyl chains were used as surface modifiers. No significant difference
was shown between the transport profiles of G3 and G0 PAMAM conjugates. However, surface modified
G3 dendrimer conjugates showed a higher permeability and significantly higher cytotoxicity than that
of surface modified G0 dendrimer conjugates. Future investigations may focus more on the impact of
conjugation on the density of surface charge for dendrimer conjugates and the aggregation behaviour of
these conjugates in aqueous solutions.
Overall, our results suggest that G0 PAMAM dendrimers demonstrate potential as nanocarriers
for the enhancement of oral bioavailability of naproxen as a model for low aqueous solubility drugs.
Acknowledgments: Thank you to Jeff Penny for the Caco-2 facilities.
Author Contributions: M.N. contributed in the initial planning, experimental design, experimental work and
data analysis, wrote the first draft and led the submission. S.F. and D.A. contributed in the experimental design
and data interpretation and edited the drafts. M.K. involved in the experimental work (the solubility part),
reviewed calculations and edited the manuscript. A.D. contributed in the initial planning, experimental design
and data analysis and edited the final draft.
Conflicts of Interest: The authors declare no conflicts of interest.

Molecules 2017, 22, 1661
12 of 12
References
1.
2.
3.
Tomalia, D.A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. A new class
of polymers: Starburst-dendritic macromolecules. Polym. J. 1985, 17, 117–132. [CrossRef]
D’Emanuele, A.; Attwood, D.; Abu-Rmaileh, R. Dendrimers. In Encyclopedia of Pharmaceutical Technology;
Swarbrick, J.B.J.C., Ed.; Marcel Dekker: New York, NY, USA, 2003; pp. 1–21.
Kesharwani, P.; Gothwal, A.; Iyer, A.K.; Jain, K.; Chourasia, M.K.; Gupta, U. Dendrimer nanohybrid carrier
systems: An expanding horizon for targeted drug and gene delivery. Drug Discov. Today 2017, in press.
[CrossRef] [PubMed]
4.
5.
6.
7.
Natfji, A.A.; Osborn, H.M.I.; Greco, F. Feasibility of polymer-drug conjugates for non-cancer applications.
Curr. Opin. Colloid Interface Sci. 2017, in press. [CrossRef]
D’Emanuele, A.; Attwood, D. Dendrimer-drug interactions. Adv. Drug Deliv. Rev. 2005, 15, 2147–2162.
[CrossRef] [PubMed]
Milhem, O.M.; Myles, C.; McKeown, N.B.; Attwood, D.; D’Emanuele, A. Polyamidoamine starburst
dendrimers as solubility enhancers. Int. J. Pharm. 2000, 197, 239–241. [CrossRef]
Yiyun, C.; Tongwen, X. Dendrimers as potential drug carriers. Part I. Solubilization of non-steroidal
anti-inflammatory drugs in the presence of polyamidoamine dendrimers. Eur. J. Med. Chem. 2005, 40,
1188–1192. [CrossRef] [PubMed]
8.
9.
Nasr, M.; Najlah, M.; D’Emanuele, A.; Elhissi, A. Pamam dendrimers as aerosol drug nanocarriers for
pulmonary delivery via nebulization. Int. J. Pharm. 2014, 461, 242–250. [CrossRef] [PubMed]
D’Emanuele, A.; Jevprasesphant, R.; Penny, J.; Attwood, D. The use of a dendrimer-propranolol prodrug to
bypass efflux transporters and enhance oral bioavailability. J. Control. Release 2004, 95, 447–453. [CrossRef]
[PubMed]
10. Najlah, M.; Freeman, S.; Attwood, D.; D’Emanuele, A. Synthesis, characterization and stability of dendrimer
prodrugs. Int. J. Pharm. 2006, 308, 175–182. [CrossRef] [PubMed]
11. Najlah, M.; Freeman, S.; Attwood, D.; D’Emanuele, A. In vitro evaluation of dendrimer prodrugs for oral
drug delivery. Int. J. Pharm. 2006, 336, 183–190. [CrossRef] [PubMed]
12. Decker, T.; Lohmann-Matthes, M.L. A quick and simple method for the quantitation of lactate dehydrogenase
release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J. Immunol. Methods
1998, 115, 61–69. [CrossRef]
13. Jevprasesphant, R.; Penny, J.; Attwood, D.; McKeown, N.B.; D’Emanuele, A. Engineering of dendrimer
surfaces to enhance transepithelial transport and reduce cytotoxicity. Pharm. Res. 2003, 20, 1543–1550.
[CrossRef] [PubMed]
14. Jevprasesphant, R.; Penny, J.; Jalal, R.; Attwood, D.; McKeown, N.B.; D’Emanuele, A. The influence of surface
modification on the cytotoxicity of pamam dendrimers. Int. J. Pharm. 2003, 252, 263–266. [CrossRef]
15. Teow, H.M.; Zhou, Z.; Najlah, M.; Yusof, S.R.; Abbott, N.J.; D’Emanuele, A. Delivery of paclitaxel across
cellular barriers using a dendrimer-based nanocarrier. Int. J. Pharm. 2013, 441, 701–711. [CrossRef] [PubMed]
16. Najlah, M.; D’Emanuele, A. Crossing cellular barriers using dendrimer nanotechnologies. Curr. Opin. Pharmacol.
2006, 6, 522–527. [CrossRef] [PubMed]
17. El-Sayed, M.; Ginski, M.; Rhodes, C.; Ghandehari, H. Transepithelial transport of poly(amidoamine)
dendrimers across Caco-2 cell monolayers. J. Control. Release 2002, 81, 355–365. [CrossRef]
18. Knipp, G.T.; Ho, N.F.H.; Barsuhn, C.L.; Borchardt, R.R. Paracellular diffusion in Caco-2 cell monolayers:
Effect of perturbation on the transport of hydrophilic compounds that vary in charge and size. J. Pharm. Sci.
1997, 86, 1105–1110. [CrossRef] [PubMed]
19. Aungst, B.J. Intestinal permeation enhancers. J. Pharm. Sci. 2000, 89, 429–442. [CrossRef]
Sample Availability: Samples of the compounds not available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).