Solubilization and anticancer-activity enhancement of Methotrexate by novel dendrimeric nanodevices synthesized in one-step reaction

Article abstract of DOI:10.1016/j.bioorg.2012.01.002

The one-step synthesis of nanodevices based on PAMAM framework for targeted cancer therapy is described. Four water-soluble nanodevices (named fractions F1 to F4) were rightly separated by size discrimination, and characterized. From biological assays of cell growth inhibition percentage, the anticancer activity of Methotrexate (chemotherapeutic drug) as part of a nanodevice, generally increases over cancer cell lines and notably, in case of human lymphocytes, the cell growth inhibition percentage decreases drastically (more than 80%), thus, the nanodevices exhibited a favorable discrimination between healthy and diseased cells. From the characterization it can be conclude that the synthesized nanodevices provide a dual scenario of drug transportation: encapsulation and conjugation.

Full text of DOI:10.1016/j.bioorg.2012.01.002

Bioorganic Chemistry 41-42 (2012) 13–21
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Solubilization and anticancer-activity enhancement of Methotrexate by novel
dendrimeric nanodevices synthesized in one-step reaction
a
a
b
a,
⇑
Delia Soto-Castro , Jorge A. Cruz-Morales , María Teresa Ramírez Apan , Patricia Guadarrama
a
Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, Apartado Postal 70-360, CU, Coyoacán, México DF 04510, Mexico
Instituto de Química, Universidad Nacional Autónoma de México, Apartado Postal 70-213, CU, Coyoacán, México DF 04510, Mexico
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 November 2011
Available online 27 January 2012
The one-step synthesis of nanodevices based on PAMAM framework for targeted cancer therapy is
described. Four water-soluble nanodevices (named fractions F1 to F4) were rightly separated by size dis-
crimination, and characterized. From biological assays of cell growth inhibition percentage, the antican-
cer activity of Methotrexate (chemotherapeutic drug) as part of a nanodevice, generally increases over
cancer cell lines and notably, in case of human lymphocytes, the cell growth inhibition percentage
decreases drastically (more than 80%), thus, the nanodevices exhibited a favorable discrimination
between healthy and diseased cells. From the characterization it can be conclude that the synthesized
nanodevices provide a dual scenario of drug transportation: encapsulation and conjugation.
Ó 2012 Elsevier Inc. All rights reserved.
Keywords:
Dendrimers
Nanodevices
Methotrexate
Conjugates
Encapsulation
1
. Introduction
Several polymers have been extensively studied as drug delivery
drug used in chemotherapy) and folic acid (targeting agent) cova-
lently linked, allowing therapeutic responses, not possible to get
with the free drug [16,17]. In order to reduce the cytotoxicity from
terminal amines, and prevent non-specific reactions at the periph-
ery of PAMAM, pegylation or acetylation reactions were carried out
[18]. The possible modification of the PAMAM surface, keeping the
nanoscale dimensions, high water solubility and biocompatibility,
place these macromolecules as excellent candidates in drug-
carrying processes.
In this paper is described the one-step synthesis of nanodevices
based on PAMAM framework for targeted cancer therapy, including
Methotrexate (MTX) as chemotherapeutic drug, and folic acid (FA)
as target agent. FA was chosen to preferentially target cancer cells
that have folate receptors expressed on their surfaces, and improve
the transit across the cell membranes. After the modification of the
PAMAM periphery with tris(hydroxymethyl) aminomethane (TRIS),
low stoichiometric ratios of MTX and FA were loaded in order to
maintain a proper hydrophobic/hydrophilic balance. From the char-
acterization it can be conclude that novel nanodevices with dual
behavior of drug transportation (encapsulation and conjugation)
in the same entity were obtained. The quantification of MTX and
FA molecules was determined by NMR and UV–vis spectroscopy.
In vitro assays of the new nanodevices on different cancer cell lines
show anticancer-activity enhancement of Methotrexate.
vehicles, especially for anticancer drugs [1,2]. The use of nanode-
vices, within the so called macromolecular therapy (MT), allows a
number of therapeutic, pharmacokinetic and pharmaceutical
advantages in comparison to conventional low-molecular weight
therapeutics with free drugs [3–8].
Among the macromolecules likely to be used in MT, monodis-
perse dendrimers are prime candidates, with a great versatility
due to their unique architecture and functionality at nanoscale
[
9,10]. There are generally two different scenarios of transport/
delivery of drugs where dendrimers have been tested; the encap-
sulation by non-covalent interactions [11,12] and the formation
of conjugates [13] (drug molecules covalently attached to a macro-
molecule). Both scenarios lead to increased solubility of the drugs
and a lessened cytotoxicity; however, in case of encapsulation, the
control of the delivery rate is difficult to accomplish [14]. This can
be a disadvantage when prolonged periods of delivery-time are re-
quired, but it can turns into an advantage since high concentra-
tions of drug can be provided in short periods of time [15]. In
case of conjugates [4,5,7], the drug accumulation becomes more
selective and the circulation time in the body is larger, but it must
be taken into account that there is a limit of hydrophobic drug
loading, in order to avoid the formation of a molecular entity too
hydrophobic to be soluble in physiologic media.
2
. Materials and methods
The well-known PAMAM (polyamidoamine) dendrimers have
been modified as conjugates with Methotrexate (anti-metabolite
2.1. Chemistry
⇑
Corresponding author.
E-mail address: patriciagua@iim.unam.mx (P. Guadarrama).
Chemicals were obtained from commercial sources and used
without further purification. DMSO were dried over CaH (5% w/
2
0
045-2068/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2012.01.002

1
4
D. Soto-Castro et al. / Bioorganic Chemistry 41-42 (2012) 13–21
v) overnight, filter and freshly distilled at reduced pressure prior to
use. NMR spectra were recorded on a Bruker Avance 400.
FT-IR spectra were measured on a Nicolet 6700 spectrometer. MS
concentrated to obtain the dendrimer 4 as amber oil. Yield of
two steps: 92%.
ꢀ1
FT-IR (cm ): 3305.1 (NH); 2951.7, 2823.5 (CAH aliph.); 1729.9,
1
(
FAB) mass spectra were measured Jeol JSM AX102A spectrometer.
1643.1(C@O). NMR (CDCl
J = 6.50 Hz), 2.43 (t, 32H, ACCH
28H, ACCH NA, AN(CH NA), 2.74 y 2.80 (2t, 56H, ANCH
J = 6.70 Hz y J = 6.61 Hz), 3.22 a 3.30 (m ancho, 24H, ACON-
3
) H (d(ppm)): 2.36 (t, 24H, ACCH
COOA, J = 6.60 Hz), 2.50-2-61 (m,
CA,
2
CONA,
UV–vis spectra were recorded using a UNICAM UV 300 UV/vis
Spectrometer Vision 32 software, in phosphates buffer solution
2
2
2
)
2
2
(
PBS) at pH 7.4.
1
3
HCH
2
CA), 3.62 (s, 48H, ACOOCH
3
).
3
C (d(ppm)): (CDCl );
0
31.2(ACCH CONA); 32.6 (ACCH COOA), 37.2(ACONHCH CA),
2
.2. Tetramethyl 3,3 ,3’’,3’’’-(ethane-1,2-
2
2
2
4
9.2 (ANCH
2
CA, ACCH
2
NA); 49.7 (ACCH
2
NA, AN(CH
2
)
2
NA); 51.7
diylbis(azanetriyl))tetrapropanoate (1)
(
3 2
COOCH ), 52.8 (ANCH CA), 171(ACCONHA), 173(ACCOOCA). ESI
(
+MS) calc. 2807.6; exp.2807.9.
2
g (33 mmol) of ethylenediamine (ETDA) are diluted in 30 mL
of methanol. The flask is sealed, purged and isolated from light.
Then 14.3 mL (158 mmol) of methyl acrylate are injected and the
reaction was allowed to elapse for 48 h. The reaction was moni-
tored by thin layer chromatography using ethyl acetate as eluent,
until the mark for ethylenediamine disappears. The reaction
mixture is concentrated under vacuum to obtain the product as
translucent oil in quantitative yield.
2
.6. PAMAM-TRIS (5)
A suspension of tris(hydroxymethyl) aminomethane (TRIS;
.03 g; 8.54 mmol) is prepared in 15 mL of methanol, and a solu-
1
tion of 1 g (0.35 mmol) of dendrimer 4 in 5 mL of methanol is
added, heating to boiling point for 7 days, until the FT-IR band
ꢀ
1
FT-IR (cm 1): 2946, 2821 (CAH aliph.) 1733 (C@O). NMR 1
d(ppm): (Acetone-d6, 2.05): 2.43 (t, 8H, ACCH COOA, J =
.98 Hz); 2.51 (s, 4H, AN(CH NA), 2.75 (t, 8H, ANCH CA,
J = 6.98 Hz), 3.61 (s, 12H, ACOOCH ). C (d(ppm): (Acetone-d6,
COOA), 51.67(ANCH CA), 52.54 (ACOOCH ),
NA), 174.2(ACCOOCA). FAB + m/z calc. 405.5;
ꢀ
around 1733 cm , associated to methyl ester carbonyl disappears.
The reaction mixture is concentrated to obtain amber oil that is
dissolved in the minimum amount of water and precipitated with
acetone. This procedure was repeated several times to get as a
slightly yellow solid product, extremely hygroscopic. Yield: 70%.
H
(
2
6
)
2 2
2
13
3
3
5
0.56); 34.28 (ACCH
4.11 (AN(CH
2
2
3
ꢀ1
FT-IR (cm ): 3287.4(NH, OH), 2938.8 (CAH aliph.) 1635.1
2 2
)
1
(
C@O). NMR (D
2
O) H (d(ppm)): 2.36–2.42 (m, 56H, ACCH
.54–2.70 (m, 36H, ACCH NA, AN(CH NA), 2.74–287 (m, 56H,
CA), 3.25–3.34 (m, 36H, , ACONHCH CA); 3.74 (s, 96,
O); 33.55 (ACCH COOA), 37.22
CA), 50.46 (AN(CH NA)), 51.79
), 62.42 (AHNC(CH OH) ), 175.75
2
COOA);
m/z exp.405.5.
.3. PAMAM-G1 (2)
g (4.9 mmol) of compound 1 dissolved in 20 mL of methanol
2
2
2 2
)
ANCH
2
2
2
1
3
ACCH
2
OH).
AOCNHCH
ACCH NA), 61.33 (AC(CH
ACCONHA).
C (d(ppm)): (D
2
2
2
(
(
(
2
CA), 49.52 (ANCH
2 2
)
2
2
2
OH)
3
2
3
and added dropwise to a solution of 22.3 g (371 mmol) of ethy-
lenediamine (ETDA) in 50 mL of methanol (the flask is purged
and isolated from light). The reaction proceeds by 7 days at room
temperature. The reaction mixture was concentrated and repeat-
edly washed with t-BuOH to remove ETDA until it is undetected
by GC.
2.7. Nanodevices
In a flask previously purged with Ar, 0.5 g (0.1187 mmol) of
PAMAM-TRIS and 17.7 mg (0.145 mmol) of 4-N,N-Dimethylamino-
pyridine (DMAP, 0.33 eq. respect to esterified COOH groups) were
dissolved in 15 mL of DMSO. Separately, in containers purged with
Ar, 0.1 g (0.22 mmol) of MTX and 0.1 g (0.22 mmol) of FA are dis-
solved in 10 ml of DMSO each. A solution of DCC (108.9 mg,
2.4. PAMAM-G1.5 (3)
2
.3 g (4.45 mmol) of tetra-amine product 2 was dissolved in
3
3
0 mL of methanol, the flask is purged, isolated from light and
.9 mL (42.7 mmol) of methyl acrylate are injected. The reaction
0
.528 mmol) was prepared separately in 3 mL of DMSO. The flask
with PAMAM-TRIS and DMAP remains with vigorous stirring and
slow (in a period of 3 h) and simultaneously are added MTX, AF
and DCC in solution. After the addition the reaction is left for 48 h
at room temperature, after which the mixture was concentrated un-
der reduced pressure and re-dissolved in 3 mL of MeOH to precipi-
tate unreacted DCU, FA and MTX, and remove by filtration. Viscous
oil (0.82 g) is obtained and re-dissolved in 1 mL of methanol/3 drops
of water, to be passed through a Sephadex-LH20 column, eluting
with MeOH. The separation by size discrimination allows four water
soluble fractions of low polydispersity and removes the remaining
DCU, FA and MTX (see Scheme 1).
was left for 48 h, after which the reaction mixture was concen-
trated to obtain the dendrimer 3 as amber oil. Two steps yield: 95%.
ꢀ1
FT-IR (cm ): 3304 (NH); 2951, 2823 (CAH aliph.); 1729, 1644
1
(
C@O); NMR (Acetone-d6) H (d(ppm)): 2.46 (t, 24H, ACCH
ACCH CONA, J = 6.83 Hz); 2.56 (t, 8H, ACCH NA, J = 6.53 Hz), 2.76
t, 20H, ANCH CA, AN(CH NA, J = 6.83 Hz), 2.88 (t, 8H,
ANCH CA, J = 6.83 Hz), 3.24 (t, 8H, ACONHCH CA, J = 6.43 Hz),
.64 (s, 24H, ACOOCH (d(ppm)): (Acetone-d6); 33
ACCH CONA, ACCH COOA), 38 (ACONHCH CA), 49 (ANCH CA,
ACOOCH ), 52 (ANCH CA), 53 (ACCH NA), 54 (AN(CH NA),
72(ACCONHA), 173(ACCOOCA). ESI (+MS) m/z calc. (1206.4);
m/z exp. 1206.0.
2
COOA,
2
2
(
2
2 2
)
2
2
13
3
(
3
).
C
2
2
2
2
3
2
2
2 2
)
1
2.8. Biological assays
2.5. PAMAM-G2.5 (4)
2.8.1. Cell lines culture and culture medium
The dendrimers were screened in vitro against human cancer cell
lines: HCT-15 (human colorectal adenocarcinoma), MCF-7 (human
mammary adenocarcinoma), K562 (human chronic myelogenous
leukemia), U251 (human glyoblastoma), PC-3 (human prostatic
adenocarcinoma), SKLU-1 (human lung adenocarcinoma), cell lines
were supplied by National Cancer Institute (USA). Besides human
lymphocytes MT2 cell lines, the human tumor cytotoxicity was also
determined by using the proteinbinding dye sulforhodamine B
(SRB) in microculture assay to measure cell growth as described in
0
.5 g (2.9 mmol) of dendrimer 3 (G = 1.5) were dissolved in
4
(
0 mL of methanol and added slowly to a solution of 48 g
798.6 mmol) of ETDA. After 9 days at room temperature, the mix-
ture is concentrated and washed repeatedly with t-BuOH to re-
move the remaining ETDA. Then 3.3 g (2.3 mmol) of eight-amine
product was dissolved in 60 ml of methanol, the flask is purged,
isolated from light, and 4 ml (44.3 mmol) of methyl acrylate are in-
jected. The reaction was left for 48 h, after which the mixture is

D. Soto-Castro et al. / Bioorganic Chemistry 41-42 (2012) 13–21
15
H N
NH2
O
2
O
H N
O
O
HN
O
2
HN
NH2
O
O
H2N
NH2
N
N
N
O
N
O
O
O
NH
O
NH
O
O
O
1
H N
2
2
NH2
O
O
O
O
O
O
O
O
O
O
O
N
O
N
N
O
N
O
O
O
O
HN
H
O
N
O
O
HN
O
N
O
O
O
O
N
O
O
N
O
N
HN
O
N
H
HN
O
N
O
O
N
N
NH
H
O
O
N
H
N
O
NH
N
O
HN
NH
O
N
O
O
O
O
N
O
H
N
N
O
O
NH
O
O
O
O
O
N
N
N
N
H
NH
O
O
O
N
O
N
O
O
O
O
O
O
O
O
O
O
3
O
O
4
OH
OH
OH
HO HO
HO
HO
HO
H
N
HO OH
OH
OH HO
O
O
HO
HO
H
O
N
N
O
N
O
HO
HO
HN
HN
N
OH
N
OH
OH
O
O
O
HO
O
O
N
H
H
N
N
HN
O
N
OH
OH
O
O
N
HN
HO
HO
HO
O
O
H
N
O
N
OH
N
O
NH
O
O
O
N
NH
H
N
N
OH
HO
HO
H
O
N
OH
O
O
HO
O
NH
N
NH OH
OH
OH
OH
NH
O
N
O
N
HO
HO
N
O
O
OH
OH
OH
HO
H
OH
OH
OH
HO
HO OH
5
(PAMAM-TRIS)
Scheme 1. Synthesis of PAMAM G2.5 and its functionalization with TRIS at the periphery.
the protocols established by the NCI [19]. The cell lines were cul-
tured in RPMI-1640 medium supplemented with 10% fetal bovine
dye sulforhodamine B (SRB) in microculture assay to measure cell
growth as described in the reference of Monks et al. 1991 [19]. The
cells were removed from the tissue culture flasks by treatment
with trypsin, and diluted with fresh media. From these cell suspen-
serum, 2 mM
0,000 g/ml streptomycin sulfate and 25
Gibco) and 1% non-essential amino acids (Gibco). They were main-
tained at 37 °C in humidified atmosphere with 5% CO . The viability
L
-glutamine, 10,000 units/ml penicillin G sodium,
1
l
l
g/ml amphotericin B
(
sion, 100
into 96 well microtiter plates (Costar) and the material was
incubated at 37 °C for 24 h in a 5% CO atmosphere. Subsequently,
100 L of a solution of the dendrimers obtained by diluting the
stocks were added to each well. The cultures were exposed for
48 h to the compound at concentrations 50 M. After the incuba-
tion period, cells were fixed to the plastic substratum by the addi-
tion of 50 L of cold 50 % aqueous trichloroacetic acid. The plates
lL containing 5000–10,000 cell per well, were pipetted
2
of the cells used in the experiments exceeded 95% as determined
with trypan blue.
2
l
2.8.2. Cytotoxicity assay
l
Cytotoxicity after treatment of the tumors cells and normal cell
with the test compounds was determined using the protein-binding
l

1
6
D. Soto-Castro et al. / Bioorganic Chemistry 41-42 (2012) 13–21
Fig. 1. (a) FT-IR spectra showing the evolution of the functionalization of PAMAM G2.5 with TRIS in MeOH; (b) NMR 1H spectrum of PAMAM-TRIS.
OH
OH
OH
OH
OH
O
OH
H
N
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
H
N
NH2
O
N
N
NH2
N
OH
H N
N
NH
O
HO
N
OH
OH
OH
NH
OH
NH
N
OH
OH
H2N
N
O
O
O
O
N
N
NH
N
N
N
N
NH
N
H
O
O
N
OH
NH
O
O
NH
N
OH
OH
N
N
O
O
OH
OH
NH
O
O
NH
N
N
NH
OH
OH
OH
O
OHH N
N
NH
O
O
HN
O
O
HN
O
O
OH
OH
O
N
OH
NH
OH
HO
HO
OH
NH
OH
O
O
O
O
N
N
O
OH
O
OH
O
OH
OH
MTX
OH
OH OH
N
O
FA
H
OH
OH
OH
OH OH
OH
OH
Separation by sephadex column
CFA[mol FA/ mgconjugate]x10-8
CMTX[mol MTX/ mgconjugateo]x10-8
F1
F2
F3
F4
FA-PAMAM-MTX
PAMAM-MTX
FA-PAMAM-MTX
FA-PAMAM-MTX
CFA = 4.56
CFA = 6.26
CMTX =5.94
CMTX =40.906
FA
C = 129.54
CMTX =1.98
CMTX=25
306mg
31 mg
7
1 mg
1
75mg
Fig. 2. Synthesis of nanodevices and proportional concentration of FA/MTX determined by UV–vis.
were incubated at 4 °C for 1 h, washed with tap H
The trichloroacetic-acidfixed cells were stained by the addition of
2
O, and air-dried.
bound dye was solubilized by the addition of 10 mM unbuffered
Tris base (100 L). The plates were placed on a shaked for 5 min,
l
0
1
.4% SRB. Free SRB solution was then removed by washing with
% aqueous acetic-acid. The plates were then air-dried, and the
and the absorption was determined at 515 nm using an ELISA
plates reader (BioTex Instruments).

D. Soto-Castro et al. / Bioorganic Chemistry 41-42 (2012) 13–21
17
3
. Results and discussion
3.1. Formation of nanodevices
The synthesis of PAMAM G2.5 was carried out according to ear-
lier reports [20], however, the periphery modification by TRIS
groups was significantly improved (Scheme 1). Avoiding the labo-
To avoid the saturation of the periphery with hydrophobic
molecules of FA and MTX, a stoichiometry of 2 MTX: 2 FA: 1
PAMAM-TRIS was used.
rious conditions reported before [21,22] (anhydrous K
DMSO at 50 °C for 72 h), PAMAM-TRIS was obtained in refluxed
MeOH, maintaining an excess (50%) of TRIS.
2
CO
3
and
The one-step reaction (Fig. 2) was carried out in anhydrous con-
ditions, by slow and simultaneous addition of MTX, FA and DCC
(coupling agent), previously dissolved in DMSO, under vigorous
stirring (see experimental section). Once concentrated, the viscous
oil obtained was re-dissolved in methanol–water mixture and sep-
arated in a column packed with Sephadex LH20, eluting with
MeOH.
Four water-soluble fractions were rightly separated by size dis-
crimination, and characterized by FT-IR, NMR and UV–vis. During
the separation, free FA, MTX and DCU (dicyclohexylurea) were
eliminated.
Even though the reaction time was extended from 3 days to
7
days, this new protocol offers several advantages like easy mon-
itoring of the reaction by the disappearance of the carbonyl band in
ꢀ
1
FT-IR (ꢁ1720 cm ) from methyl ester; an easier work up of the
reaction, removing MeOH instead of DMSO, and a significant in-
crease in yield (from 54% to 70%). PAMAM G2.5 functionalized with
TRIS (PAMAM-TRIS) was fully characterized by FT-IR and NMR
(Fig. 1).
From the NMR spectrum, according to the integration of the sin-
From Fig. 2, all average molar concentration ratios of MTX and
FA were determined by a quantitative analysis of UV–vis spectra,
as will be shown below. By reacting MTX and FA simultaneously,
the largest fraction by weight (F3 (306 mg)) contains only MTX,
and the remaining fractions contain a higher proportion of FA,
since MTX was practically consumed in F3. The different reactivity
of MTX and FA can be rationalized by pKa values for both mole-
cules. Considering the coupling mechanism with DCC, the major
glet in 3.74 ppm corresponding to the methylenes of TRIS, there
was complete substitution of the periphery. The surface modifica-
tion with TRIS is beneficial from both, biological and chemical
viewpoints. In terms of biological applications, provides a com-
pletely water-soluble material, able to prevent non-specific inter-
actions with cell walls, decreasing thus the cytotoxicity. On the
other hand, the hydroxyl groups facilitate the formation of conju-
gates since they are suitable for anchoring targeting agents like
FA and drug molecules of MTX by ester links.
acidity of MTX (pKa
1
= 3.8; pKa
2
= 4.8) makes this molecule more
= 4.7; pKa = 6.8), which explains
reactive, compared to FA (pKa
1
2
Fig. 3. FT-IR spectra of PAMAM-TRIS, FA and F3.

1
8
D. Soto-Castro et al. / Bioorganic Chemistry 41-42 (2012) 13–21
NH2
N
N
H2N
N
N
3
1.6
N
O
MTX
NH
HO
O
OH
OH
OH
OH
O
O
OH
OH
OH
OH
O
OH
HO
OH
OH
OH
1H de PAMF3 en D2O
S. Delia/Dra. Guadarram
Bruker Avance 400mHz
GCV IIm UNAM
O
O
R
OH
O
N
N
O
OH
OH
OH OH
OH
NH
O
N
NH
O
O
O
O
OH
NH
N
OH
N
NH
OH
OH
N
NH
O
O
O
NH
N
R
2
.8
1
5
4
2
6
2
.0
2.1
7
8
1
.1
3
1
.0
1.01.0
Dendrímero
8
.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
Fig. 4. NMR 1H spectrum of F3 in D
O.
2
1
the formation of a major fraction containing only MTX. It is worth
mentioning that, although the reaction was carried out in DMSO,
pKa data can be extrapolated because, regardless of the dielectric
constant of the solvent, the tendency remains.
Fractions F1 and F2 are viscous oils, while F3 and F4 are yellow
solids. By their appearance is confirmed that fractions F1 and F2
have a very low load of MTX and FA, thus maintaining the physical
properties of the dendrimer PAMAM-TRIS, while fractions F3 and
F4, with a much heavier load of MTX and FA, retain the physical
properties of these molecules (yellow solids).
From the H NMR spectrum of fraction F3 (Fig. 4), two well de-
fined regions are observed: an aromatic region where three signals
corresponding to MTX are located in 8.4 ppm (singlet, pteridine
ring), 6.65 ppm and 7.6 ppm, related to protons of the aromatic
ring. The other region contains overlapping signals from dendrimer
PAMAM-TRIS and drug molecule. As will be corroborated below by
UV–vis, F3 contains only MTX molecules linked to PAMAM-TRIS.
Regarding the second most abundant fraction; F4, according to
1
NMR ( H, Fig. 5) both molecules, FA and MTX are present and dis-
tinguished by the signals corresponding to the respective aromatic
rings, a doublet in 6.64 ppm assigned to two of the protons of FA,
and the doublet in 6.75 ppm assigned to the counterpart in MTX.
The ratio between signals allows for a 5 to 1 stoichiometry of mol-
ecules of FA and MTX, respectively, which is confirmed by UV–vis.
Thus, by NMR was possible to discriminate fractions with
different contents of FA and MTX, and these fractions exhibit very
different behaviors in assays in vitro, as shown below.
As mentioned before, some of the fractions correspond to
nanodevices with a dual behavior of carrier (encapsulation and
conjugation) in the same entity. From the NMR spectra, signals
around 3.9 ppm are assigned to the methylenes of TRIS already
esterified, which accounts for molecules of MTX/FA covalently an-
chored, forming conjugates (as example, see NMR spectrum from
F1; Supporting information). On the other hand, signals between
2.2 and 3.8 ppm, corresponding to PAMAM-TRIS, exhibit a splitting
due to two different chemical environments assignable to ‘‘free’’
dendrimer, and dendrimer encapsulating FA/MTX molecules via
non-covalent interactions (see spectra from Figs. 4 and 5). Accord-
The four fractions obtained, clearly are vehicles to enhance the
solubility of both MTX and FA, and can be considered as nanode-
vices for drug-carriage.
3.2. Characterization of nanodevices
FT-IR spectra of all fractions are similar. Characteristic bands in
ꢀ
1
2
100–3500 cm due to carboxylic acids from FA/MTX were ob-
ꢀ1
served. A band around 3270 cm , corresponding to terminal
hydroxyl groups from PAMAM-TRIS is also present. A broad band
ꢀ1
in 1565 cm
is assigned to carbonyls from amide groups
(
PAMAM-TRIS/FA/MTX conjugates). Illustratively, spectra of the
PAMAM-TRIS, FA and fraction F3 (with only MTX) are shown in
Fig. 3.
The IR spectra of fractions F1 and F2 show two bands in
ꢀ1
ꢀ1
3
324 cm and 2926 cm , presumably due to encapsulated DCU,
therefore, these fractions were not considered for further biological
assays.

D. Soto-Castro et al. / Bioorganic Chemistry 41-42 (2012) 13–21
19
H2N
N
N
N
NH2
N
H2N
N
N
N
N
N
H
N
O
O
HO
HO
H
N
O
NH
O
OH
OH
OH
OH OH
O
O
OH
OH
OH
N
H N
MTX
1
H de PAMF2 en D2O
OH
OH
OH
AF
O
O
S. Delia/Dra. Guadarrama
Bruker Avance 400MHz
GCV IIM UNAM
6.1
OH
O
OH
OH
O
O
N
O
O
O
OH OH
OH
NH
O
OH
OH
NH
O
N
4.9
O
O
OH
O
N
NH
OH
OH
OH
NH
N
N
NH
H
O
N
O
NH
O
N
4
3
.0
N
3
11
12
1.0
1
8
8
.6
8.4
8.2
8.0
7.8
7.6
7.4
7.2
7.0
6.8
6.6
6.4
1
0.4
2
7
6 6.4
15
9
.2
5
14
5
2
.9
3.1
3.2
13
10
4.4
4.2
4.0
3.8
3.6
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
Fig. 5. NMR 1H spectrum of F4 in D
2
O.
ing to integral ratio of the signals in 8.50 and 3.66 ppm (corre-
sponding to the proton of pteridine ring of MTX/FA, and methylene
protons of TRIS, respectively), the MTX:PAMAM-TRIS ratio is 42:1
for fraction F3 and 103:1 for fraction F4 (containing both, MTX
and FA). Such a high charge of MTX/FA per dendrimer can only
be explained by the formation of supramolecular structures medi-
ated by non-covalent interactions, which are still completely solu-
ble in aqueous media. Mass spectra for these fractions were
attempted by ESI-MS, but the fragmentation patterns led to mis-
leading conclusions. Future work includes mass determinations
by MALDI-TOF.
It is important to mention that the nanodevices are stable in
aqueous solutions, in dark and dryness conditions, at room tem-
perature for several months. When the concentrations of FA and
MTX are determined after these periods of time, they remain the
same.
3
3
.3. Biological section
.3.1. Pamam tris
Since non-cytotoxicity is a necessary attribute for any biomate-
From UV–vis spectra of nanodevices (measured in phosphates
buffer solution (PBS) at pH 7.4; Fig. 6), with the exception of F3,
all fractions contain both FA and MTX, anchored/encapsulated to
PAMAM-TRIS.
To determine the average molar concentrations of MTX in frac-
tion F3, the Lambert–Beer law together with the calibration curve
of MTX, measured at 304 nm (Fig. 7), was used.
It is important to mention that the absorption maxima of FA and
MTX overlap, thus is not possible to quantify directly the concen-
tration of each analyte; therefore, in case of F1, F2 and F4 contain-
ing FA and MTX, it was considered the additive character of the
Lambert–Beer‘s law for binary mixtures and the concentrations
were determined as detailed in support information, the results
are summarized in Table 1.
rial, the harmlessness of dendrimer PAMAM-TRIS was evaluated by
assays on human lymphocytic cells (MT2) [23]. The results, ob-
tained in triplicate by Monks protocol (see Ref. [19]), as well as
the standard error, are shown in Table 2. A sample of polyglycidol
was included as reference of innocuity [24].
Although the main interest for the biological study was the ver-
ification of non-cytotoxycity of the new materials in normal cell
lines, additional assays on human cancer cell lines were carried
out for forthcoming studies.
In Table 2, the + sign denotes cell growth compared with the
control, which means that the compounds are non cytotoxic; and
the values without + sign correspond to cell growth inhibition
percentage.
From these results it can be observed that dendrimer PAMAM-
TRIS, exhibits cytotoxycity towards two cancer cell lines only, and
is harmless towards normal cells. Even though polyglycidol (refer-
Thus, in case of fraction F4 is corroborated the 5:1 ratio of
FA:MTX by UV–vis, as was observed above by NMR.

2
0
D. Soto-Castro et al. / Bioorganic Chemistry 41-42 (2012) 13–21
F1
AF
1
0
0
.0
.5
.0
F2
1
.0
.5
MTX
MTX
0
0.0
200
300
400
500
2
00
300
400
λ (nm)
500
λ (nm)
2
1
1
0
0
.0
.5
.0
.5
.0
MTX
F3
MTX
F4
1.0
0
.5
.0
0
2
00
300
400
500
200
300
400
500
600
λ(nm)
λ (nm)
Fig. 6. UV–vis spectra of fractions F1 to F4 in phosphate buffer solution (pH 7.4). F3 and MTX (red) spectra are shown together.
2
2
1
1
0
0
.5
.0
.5
.0
.5
.0
1.8
A304 = 32265.0
C
± 216.63
1
1
1
1
0
0
.6
.4
.2
.0
.8
.6
2
r
= 0.9997
0.00002
0.00003
0.00004
0.00005
0.00006
C [
M ]
200
300
400
λ (nm)
500
600
Fig. 7. Absorption spectra of MTX in PBS buffer at pH 7.4 and calibration curve at 304 y 370 nm in a concentration range of 1.8 ꢂ 10ꢀ5 to 5.5 ꢂ 10ꢀ5 M.
ence compound) showed cytotoxycity in some cancer cell lines, it
was also non cytotoxic towards normal cells (lymphocytes).
Table 1
Average concentrations of FA and MTX in fractions F1 to F4.
3
.3.2. Nanodevices
In order to evaluate the cytotoxic behavior of MTX embedded in
CFA ꢂ 104
M
CMTX ꢂ 104
M
CFA ꢂ 10 M/mg
8
CMTX ꢂ 10 M/mg
8
F1
F2
F3
F4
0.3633
0.2919
–
0.3444
0.1265
1.15
6.264
4.561
–
5.938
1.977
40.91
25.95
dendrimeric nanodevices, assays were conducted to determine the
cell growth inhibition percentage in different cancer cell lines and
human lymphocytes cell line (MT2). The results of Table 3 shows
that the MTX activity not only is maintained but increases, except
2.33
0.47
129.54

D. Soto-Castro et al. / Bioorganic Chemistry 41-42 (2012) 13–21
21
Table 2
a
Cell growth inhibition percentage of human cancer cell lines and human lymphocytic cells .
Compound
MT2
U251
PC-3
K-563
HCT-15
MCF7
SKLU-1
PAMAM-TRIS
Polyglicidolb
+8.7 ± 2.7
+5.8 ± 0.6
+14.7 ± 2.1
1.6 ± 0.1
+3.89 ± 1.2
+5.4 ± 1.8
+17.1 ± 5.1
6.4 ± 0.08
+6.7 ± 5.9
4.8 ± 1.5
4.6 ± 3.7
5.3 ± 0.2
12.7 ± 1.6
5.5 ± 1.5
a
Cellular activity measured in presence of different compounds at 50 lM ± standard error. MT2: Human lymphocytes; Human cancer cell lines: U251 (human
glyoblastoma); PC-3 (human prostatic adenocarcinoma); K562 (human chronic myelogenous leukemia cells); HCT-15 (human colorectal adenocarcinoma); MCF-7 (human
mammary adenocarcinoma); SKLU-1 (human lung adenocarcinoma).
b
MW = 4591; polydispersity = 1.8.
Table 3
a
Cell growth inhibition percentage of human cancer cell lines and human lymphocytic cells .
Sample
MT-2
U251
PC-3
K562
HCT-15
MCF-7
SKLU-1
MTX
F3
F4
86.34
16.47 ± 5.1
+3.5 ± 3.8
61.8 ± 3.3
87.7 ± 6.0
88.8 ± 1.7
27.9 ± 5.6
18.4 ± 1.1
7.1 ± 0.1
44.0 ± 4.1
47.0 ± 4.2
41.7 ± 5.4
55.1 ± 2.4
94.4 ± 6.5
96.1 ± 1.8
50.6 ± 2.9
89.5 ± 10.6
87.9 ± 5.5
54.1 ± 1.9
67.2 ± 7.6
64.7 ± 3.8
a
Cell growth inhibition percentage measured in presence of different compounds at 50 lM ± standard error. MT2: Human lymphocytes; Human cancer cell lines: U251
(
(
human glyoblastoma); PC-3 (human prostatic adenocarcinoma); K562 (human chronic myelogenous leukemia cells); HCT-15 (human colorectal adenocarcinoma); MCF-7
human mammary adenocarcinoma); SKLU-1 (human lung adenocarcinoma); bMW = 4591; polydispersity = 1.8.
for cell line of prostatic cancer; PC-3. It is noteworthy that, in case
of human lymphocytes, the cell growth inhibition percentage de-
creases drastically (more than 80%) in presence of fraction F3 con-
taining only MTX, and fraction F4 containing FA and MTX,
promotes growth of this cell line, presumably due to the presence
of FA, which acts as a metabolite According to the results, the nan-
odevices, with or without FA, will discriminate between healthy
and diseased cells.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bioorg.2012.01.002.
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Acknowledgments
This research was carried out with the support of Grants 81924
from CONACyT, IN101109 from PAPIIT and 4316 from ICyTDF. The
authors kindly thank Gerardo Cedillo Valverde and Carmen
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