Development and characterization of triazine based dendrimers for delivery of antitumor agent

Article abstract of DOI:10.1166/jnn.2010.3003

In the present study we developed the novel kind of triazine dendrimers by utilizing differential reactivity of the cyanuric chloride (triazine trichloride) which overcome the limitations associated with the others classes of dendrimers like toxicity, low yield, high synthesis cost etc. Triazine dendrimers were synthesized by divergent method using triazine trichloride as core and diethanolamine as branching unit to avoid the use of protecting group and functional group interconversion up to third generation. These hydroxyl terminated dendrimers were characterized by FTIR, ¹HNMR, ¹³CNMR, ES mass spectroscopy, and by Elemental analysis. The yield of pure G3 dendrimers was 63%. This novel dendrimers increases the aqueous solubility of hydrophobic drug Paclitaxel up to 0.562 mg/ml as well as showed control release behavior. Hemolytic and toxicology studies of this dendrimer in mice showed no adverse toxicity to the kidneys and the liver up to 200 mg/kg dose (i.p). Triazine being a hydrophobic compound, the core of this dendrimer is hydrophobic and supposed to easily incorporate the hydrophobic guest while presence of hydroxyl group on periphery increases its water solubility and reduces its toxicity; and thus it is useful in various fields like gene delivery, MRI contrasting agents, vaccines or as solubilization tool. Copyright

Full text of DOI:10.1166/jnn.2010.3003

Copyright © 2010 American Scientific Publishers
All rights reserved
Printed in the United States of America
Journal of
Nanoscience and Nanotechnology
Vol. 10, 8395–8404, 2010
Development and Characterization of Triazine Based
Dendrimers for Delivery of Antitumor Agent
∗
Kuldeep K. Bansal, Deepak Kakde, Umesh Gupta, and Narendra K. Jain
Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences,
Dr. H. S. Gour University, Sagar, M.P. 470003, India
In the present study we developed the novel kind of triazine dendrimers by utilizing differential
reactivity of the cyanuric chloride (triazine trichloride) which overcome the limitations associated with
the others classes of dendrimers like toxicity, low yield, high synthesis cost etc. Triazine dendrimers
were synthesized by divergent method using triazine trichloride as core and diethanolamine as
branching unit to avoid the use of protecting group and functional group interconversion up to third
1
13
generation. These hydroxyl terminated dendrimers were characterized by FTIR, HNMR, CNMR,
ES mass spectroscopy, and by Elemental analysis. The yield of pure G3 dendrimers was 63%. This
novel dendrimers increases the aqueous solubility of hydrophobic drug Paclitaxel up to 0.562 mg/ml
as well as showed control release behavior. Hemolytic and toxicology studies of this dendrimer in
mice showed no adverse toxicity to the kidneys and the liver up to 200 mg/kg dose (i.p). Triazine
being a hydrophobic compound, the core of this dendrimer is hydrophobic and supposed to easily
incorporate the hydrophobic guest while presence of hydroxyl group on periphery increases its
water solubility and reduces its toxicity; and thus it is useful in various fields like gene delivery, MRI
Delivered by Publishing Technology to: Deakin University Library
contrasting agents, vaccines or as solubilization tool.
IP: 72.9.2.164 On: Mon, 04 Jan 2016 19:19:30
Keywords: Dendrimer sC, oP pa yc lri it ga xh et :l , AT mr ia ez ri ni ce a Tn r iSc hc l ioe r ni dt ei f ,i cD Pi e ut hb al ins oh l ea mr sine, Solubilization.
1
. INTRODUCTION
to achieve highly branched globular structures,12 which is
probably difficult and time consuming. These also provides
less hydrophobic interior compared to dendrimers having
Dendrimers are versatile, derivatisable, monodispersed,
highly branched, well-defined, compartmentalized poly-
mers with sizes and physicochemical properties resem-
bling those of biomolecules like proteins. The structure
of these materials has great impact on their physical and
chemical properties. Due to their unique behavior, den-
dritic molecules have made significant contributions in
many fields including but not limited to drug delivery,
protein mimicry, gene transfection, quantum dots and
13
aromatic core. However, in spite of their broad applica-
bility, toxicity associated due to the terminal –NH groups
1
2
(
cationic charge), limits these candidatures for their clin-
1
4
15
ical applications. Fréchet and Hawker developed less
toxic dendrimers using polyaryl ether, but they have poor
water solubility. Several research groups routinely synthe-
sized new dendrimers or modified the previously estab-
2
3
4
5
1
6
lished class of dendrimers to overcome these limitations.
6
7
8
nano dots, sensors and detectors, image contrast agents,
Approaches to develop a simple and less toxic den-
drimers for drug delivery is an attractive and promising
area of research in dendrimer chemistry. Number of cores
and branching units has been earlier utilized for the devel-
opment of new dendrimers. Some important cores used
include diaminobutane, ethylenediamine, ammonia, amino
acid, bis(4-fluorophenyl) sulfone, carbosilane etc. however,
this list can never be completed.
9
10
catalysis and separations. Dendrimers bearing more than
one type of peripheral as well as branching groups can
be well prepared, but many times their synthesis, requires
1
1
numerous steps, which is sometimes challenging.
Because of the wide range of applicability of this macro-
molecule, efforts are continuing to improve the efficiency
of dendrimers in the drug delivery and to reduce its syn-
thesis cost. Although, polyamidoamine (PAMAM) and
polypropylene imine (PPI) dendrimers are most researched
category of dendrimers, however, this offers some limita-
tions. The synthesis up to higher generation is necessary
1
,3,5-Triazine derivatives have been known for a long
1
7
period of time. Triazine and its derivatives found
widespread applications in the pharmaceutical, textile, plas-
tic, and rubber industries, and have successfully been
used as pesticides, dyestuffs, optical bleaches, explosives,
∗
Author to whom correspondence should be addressed.
J. Nanosci. Nanotechnol. 2010, Vol. 10, No. 12
1533-4880/2010/10/8395/010
doi:10.1166/jnn.2010.3003
8395

Development and Characterization of Triazine Based Dendrimers for Delivery of Antitumor Agent
Bansal et al.
and surface active agents.18 The chemistry of this group
of compounds has been studied intensively and well
that are ionisable in a pharmaceutically useful range and
therefore manipulation in pH does not enhance its solu-
bility. Furthermore, common approaches to improve sol-
ubility like addition of charged complexing agents or by
producing alternate salts of the drug are not feasible in
case of paclitaxel. Moreover, the extensive clinical use
of this drug is somewhat hampered due to lack of appro-
priate delivery vehicles. This clearly establishes the need
for the development of alternate formulation of Paclitaxel
(PTX) having good aqueous solubility and at the same
time free from any side effects. Various approaches and
novel carriers like cosolvents, emulsions, micelles, lipo-
somes, nanoparticles, cyclodextrins, microspheres and pro-
drug are employed to resolve the problems associated with
the PTX delivery.
1
9
reviewed. Triazine trichloride is one of the versatile
derivatives of triazine family, first prepared in 18th cen-
tury. It is readily obtained though the cyclotrimeriza-
tion of cyanogen chloride using activated charcoal as the
2
8
2
0
catalyst. Because of its versatility it can be the promising
core used in dendritic synthesis.
Triazine trichloride as a “core” adding the new era in
dendrimer synthesis21 because the branching necessary for
dendrimer growth results from direct substitution onto the
core, rather than through functional groups appended to
the core as well as exhibits the aromatic nature and tem-
perature dependent substitution. Triazine trichloride has
become the molecule of choice for synthesis of novel
class of dendrimers as it possesses numerous advantages
including its ability to diversify the chemical function-
ality without using the protecting groups and the nucle-
ophilic aromatic substitution on its ring occurs sequentially
in temperature dependent manner thus no need of func-
tional groups manipulation required. The aromatic inte-
rior of these molecules makes them significantly more
hydrophobic likely to favor noncovalent sequestration of
hydrophobic guests.13 The synthetic versatility of this sys-
tem suggests that more “composition space” can be exam-
Dendrimers are also the carrier of choice to deliver PTX
safely because of its unique characteristics and therefore
many research groups worked extensively to establish the
2
9
ability of dendrimers for PTX delivery.
In the present study, we have synthesized and charac-
terized triazine trichloride “core” based novel dendrimers
having diethanolamine and triazine trichloride both, as
branching units in their structural framework for improved
delivery of Paclitaxel. In this sequence earlier Namazi and
3
0
Adeli synthesized dendritic triblock copolymer using tri-
azine trichloride and diethanolamine as A and B block.
2
2
ined with these molecules.
The differential reac tDi v ei tl iyv e or ef dt br i ya z Pi nu ebli tsr hi ci hn lgo rTi de ec h ins olog Ty ht eo :r De a ec at i ko inn cU o nn i dv iet ir os ni tsy iLn ibt rh ae r yr eported study were sim-
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manipulated in two ways, i.e., temperature and nucle-
ple, excluding protection and deprotection steps with
high yield.
Copyright: American Scientific Publishers
2
3
ophilicity (Scheme 1). Steffensen and Simanek demon-
strated differential reactivity of triazine trichloride with
various amines. Many types of triazine based dendrimers
2. EXPERIMENTAL DETAILS
2
4
have been reported in the literatures out of which
melamine dendrimers reported by Simanek group was
2.1. Materials
2
5
extensively explored for its drug delivery ability.
Triazine trichloride was purchased from Sigma Aldrich
Chemicals Pvt. Ltd. (India) and used as received. Ace-
tone, Tetrahydrofuran (THF) methanol was purchased
from Qualigens Fine Chemicals (India), 1-4 Dioxane
was purchased from Loba Chemie Pvt. Ltd. (India) and
diethanolamine and sodium bicarbonate was from Central
Drug House (P) Ltd. (India). All the reagents and solvents
for the synthesis and analysis were used as received. FTIR
Dreyer et al. synthesized triazine dendrimers, by uti-
lizing dialkylamine as linker molecule. However, number
of other molecules can be used as linker molecules like
2
6
diethanolamines, p-aminobenzylamine etc. for synthe-
2
7
sis. Further, Simanek and Hollink described synthesis of
functionally diverse macromolecules, using Boc-protected
NH(CH CH NH ꢀ as a linker by utilizing nucleophilic
2
2
2 2
aromatic substitution reactions.
−1
®
studies were carried out in the range of 600–4000 cm
using FTIR-470, Jasco (UK), instrument through KBr disc
Paclitaxel (Taxol ) is a promising anti-tumor agent with
poor aqueous solubility used widely in the treatment of
ovarian and breast cancer. Intravenous administration of
Paclitaxel formulation containing non-aqueous vehicle cre-
mophor EL causes allergic reactions and precipitation
on aqueous dilution. Paclitaxel lacks functional groups
1
13
and pellet method. H NMR and C NMR spectra were
recorded at 400 MHz in Brucker Avance II 400 (Germany).
The electrospray (ES) mass spectra were recorded on
Micromass Quattro II triple quadrupole mass spectrometer.
Cl
N
Cl
Cl
N
NH X
N
NHX
X'HN
N
NHX
2.2. Synthesis Method
2
X'HN
0
-4 °C
25 °C
NH X'
70 °C
N
N
N
N
N
N
N
N
NH X
NH X"
2
2
2
2.2.1. Generation 1
Cl
Cl
Cl
NHX"
Cyanuric chloride (4 g, 21.69 mmol) was dissolved in
50 mL of tetrahydrofuran (THF) containing sodium bicar-
Scheme 1. Temperature dependent nucleophilic aromatic substitution
of triazine trichloride where X, X , X denotes alkyl, halogens, or any
ꢀ
ꢀꢀ
substituting group.
bonate (NaHCO ; 65.07 mmol) and placed on ice bath.
3
8
396
J. Nanosci. Nanotechnol. 10, 8395–8404, 2010

Bansal et al.
Development and Characterization of Triazine Based Dendrimers for Delivery of Antitumor Agent
ꢁ
ꢁ
Diethanolamine (6.5 mL, 65.07 mmol) in excess was
added drop wise in the solution of cyanuric chloride at
at 0 C. The solution was stirred for 1.5 hr at 0 C and
for 1 hr at room temperature, and then refluxed for 6 hr,
filtered washed with acetone and dried under vacuum; a
white solid product was formed with 10.6 g yield.
ꢁ
ꢁ
0
C with stirring. Solution was stirred at 0 C for 1.5 hr;
for 12 hr at room temperature, and then refluxed for 28 hr
ꢁ
−1
−1
(ꢁC–H), 1777, 1716, 1647 cm
at 100 C. Product was washed with acetone and methanol
IR: 2779 cm
(ꢁC N), 1052 cm (ꢁC–O), 766 cm (ꢁC–Cl).
H NMR (In DMSO-d6): ꢂ 3.06–3.08 (60H) (Triazine-
N-CH ꢀ, ꢂ 3.70–3.72 (60H) (CH –O-Traizine).
−
1
−1
and dried in vacuum to get the G1 dendrimers which was
dark brown in color and honey like in consistency with
1
7
.8 g yield.
IR: 3364 cm (ꢁO–H), 2963 cm (ꢁC–H), 1738,
2
2
−1
−1
−
1
−1
1
621 cm (ꢁC N), 1064 cm (ꢁC–O).
2
.2.5. Generation 3
1
H NMR (In D O): ꢂ 3.21–3.25 (12H) (Triazine-N-
2
Generation 2.5 dendrimers (8 g, 1.41 mmol) was dis-
solved in the 100 mL of 1,4 dioxane containing NaHCO3
CH ꢀ, ꢂ 3.86–3.89 (12H) (CH –OH).
2
2
(
6
67.94 mmol). To this solution diethanolamine (6.56 mL,
7.94 mmol) was added in excess drop wise with stirring.
2
.2.2. Generation 1.5
Generation 1 dendrimers (7 g, 17.94 mmol) was dis-
solved in 40 mL of methanol containing NaHCO3
The solution was stirred at room temperature for 6 hr.
A clear solution was formed which was then refluxed for
28 hr at 100 C. After refluxing, the solution was cooled;
ꢁ
(
(
122.83 mmol) and kept in ice bath. Cyanuric chloride
19.81 g, 107.47 mmol) was dissolved in 200 mL of THF.
filtered and evaporated the solvent in vacuum to get the
product that was washed with acetone and methanol, puri-
fied by column chromatography to get the pure den-
drimers, which was light brown in color with 8 g yield.
The G1 dendrimers solution was added in solution of cya-
ꢁ
nuric chloride with stirring at 0 C. The solution was
ꢁ
stirred for 1.5 hr at 0 C and for 1 hr at room temperature,
− −1
1
and then refluxed for 6 hr, filtered, washed with acetone
and dried under vacuum; a white colored solid product was
formed with 12.4 g yield.
FTIR: 3413 cm (ꢁO–H), 2872 cm (ꢁC–H), 1611,
−
1
−1
1487 cm (ꢁC N), 1061 cm (ꢁC–O).
1
H NMR (In D O): ꢂ 3.53–3.56 (60H) (Triazine-N-
2
−
1
−1
IR: 2779 cm (ꢁC–H), 1716, 1621 cm (ꢁC N),
CH ꢀ, ꢂ 3.90–3.94 (60H) (CH –O-Traizine), ꢂ 3.42–3.44
2
2
−
1
−1
1
053 cm (ꢁC–O), 767 cm (ꢁC–Cl).
(192H) (Triazine-N-CH ꢀ, ꢂ 3.66–3.69 (192H) (CH –OH).
Library
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2
2
1
13
H NMR (In DMSO-d6): ꢂ 3.06I –P 3:. 07 82 . (91 . 22 H. 1) 6 (4T rOi a nz :i nMe -o n, 04 J aC n N2 M0 1R 6( 1I n9 :D1 9 O: 3) 0: 48 ppm (Triazine-N-CH ꢀ, 59 ppm
N-CH ꢀ, ꢂ 3.75–3.78 (12H) (CH –O- CT roi ap zy i rn i ge )h. t: American S c( Ci e Hn ti –f i Oc -PT ur ab i lzi si nh e e) ,r s5 6 ppm (CH –OH), 158 ppm (Triazine
2
2
2
2
2
2
Portion).
+
+
Calcd: [M] C345H600N156O126 mass = 8949ꢃ5, Found:
2
.2.3. Generation 2
ESI: [M+H] mass = 8950.
Generation 1.5 dendrimers (8 g, 6.25 mmol) was dis-
solved in 100 mL of 1,4 dioxane containing NaHCO3
2.3. Solubilization Studies
(75.10 mmol). To this solution diethanolamine (7.25 mL,
7
5.10 mmol) was added in excess, drop wise, with stir-
Solubilization of Paclitaxel using triazine dendrimers was
done according to the phase solubility studies performed
by Higuchi and Connors. Briefly, known amount of drug
(in excess) was added separately into 12 screw-capped
vials (USP Type I glass) (10 ml) containing different con-
ring. The solution was stirred at room temperature for 6 hr.
A clear solution was formed which was then refluxed for
2
filtered, and solvent was evaporated in vacuum to get the
product, which was washed with acetone and methanol
and dried in vacuum. The product was light brown color
powder with 7.2 g yield.
3
1
ꢁ
8 hr at 100 C. After refluxing the solution was cooled,
−
2
−2
−2
centrations (0ꢃ55 × 10 , 1ꢃ11 × 10 , 1ꢃ67 × 10 , and
−
2
2ꢃ23 × 10 mM) of triazine dendrimers (1.0, 2.0, 3.0 G)
in distilled water (4 vials for each generation). Separately
known amount of drug (in excess) was also added in a
vial containing only distilled water as control. Vials were
shaken for 48 hr in a metabolic shaker (Indian Equip-
ment Corporation, Mumbai, India) at room temperature
and allowed to stand for 24 hr to attain equilibrium. Solu-
tions were filtered using nylon membrane filter of pore
size 0.45 ꢄm (Pall Gelman Sciences, USA) and methanol
−
1
−1
IR: 3405 cm (ꢁO–H), 2876 cm (ꢁC–H), 1641,
−
1
−1
1
487 cm (ꢁC N), 1056 cm (ꢁC–O)
H NMR (In D Oꢀ: ꢂ 3.50–3.53 (12H) (Triazine-N-
1
2
CH ꢀ, ꢂ 3.99–4.02 (12H) (CH –O-Traizine), ꢂ 3.19–3.22
2
2
(48H) (Triazine-N-CH ꢀ, ꢂ 3.82–3.83 (48H) (CH –OH).
2
2
2
.2.4. Generation 2.5
(
5 ml×5 times) was passed through the same membrane
Generation 2 dendrimers (6 g, 2.85 mmol) was dissolved
in 50 mL of methanol containing NaHCO (68.5 mmol)
and placed on ice bath at 0 C. Cyanuric chloride (12.63 g,
6
filter to remove out insoluble Paclitaxel from filter and
each fraction of methanol was analyzed by HPLC, per-
3
2
3
ꢁ
®
formed on reverse phase Zorbax C18 column (25 cm ×
8.5 mmol) was dissolved in 100 mL of THF and to this
0ꢃ45 cm) at ꢅmax 227 nm (UV detector) after proper dilu-
solution, G2 dendrimers was added slowly with stirring
tion to determine indirectly the concentration of PTX in
J. Nanosci. Nanotechnol. 10, 8395–8404, 2010
8397

Development and Characterization of Triazine Based Dendrimers for Delivery of Antitumor Agent
Bansal et al.
dendrimers solution i.e., solubility. To establish the effect
of pH on solubilization ability of dendrimers, solvent of
different pH (4, 7.4 and 10) was used instead of distilled
water.
2.5.2. Acute In Vivo Toxicity Study
The BALB/c mice of uniform weight (20 ± 2 g) and 3–4
weeks old were cared under a protocol approved by the
university animal ethical committee of Dr. H. S. Gour Uni-
versity, Sagar (M.P). BALB/c mice were housed at con-
2
.4. In Vitro Studies
ꢁ
stant temperature (22± 2 C) and humidity (50–60%) and
2
.4.1. Drug Loading and Release
with 12 hr light/dark cycle, and fed a standard diet ad
libitum. The mice were assigned in 6 groups, each group
containing 6 animals. First five groups were administered
the 40, 60, 100, 200 and 500 mg/kg of dendrimers solution
Generally two approaches have been followed for loading
of drug within dendritic construct i.e., by inclusion com-
plex or by conjugation. In the present study we used inclu-
sion complex approach for loading of PTX because it is a
hydrophobic drug and hence can be physically entrapped
in the hydrophobic region of the triazine dendrimers. Drug
(
in saline) via I.P route, respectively. The last group served
as control and was administered only saline solution. After
8 hr blood was collected from each mice by retro orbital
4
plexus and analyzed for blood urea nitrogen (BUN) level
and for serum glutamine pyruvate transaminase (SGPT)
level in local pathology laboratory.
Data Analysis and Statistics: Statistical analysis was
performed using Student’s t-test with Prism 4 software
(
PTX) was loaded in dendrimers by reported procedure
33
with minor modifications. Briefly, known amount of drug
(
1
in excess) was added to the dendrimer solution (2ꢃ23 ×
−
2
0
mM in 10 ml of distilled water) and the mixture
was stirred for 72 hr at room temperature. This mixture
was then filtered with 0.45 ꢄm (Pall Gelman Sciences,
USA) membrane filter, and methanol (5 ml×5) was passed
through the same membrane filter to remove out unencap-
sulate Paclitaxel from filter and each fraction of methanol
was analyzed by HPLC to determine indirectly the amount
of drug encapsulated within dendrimers. The filtrate was
lyophilized and used for further studies.
(
GraphPad Software, San Diego, CA). For all statistical
analysis, the difference was considered significant when
P < 0ꢃ05.
3. RESULTS AND DISCUSSION
3
.1. Synthesis
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Drug release from known amounts of PTX loaded tri-
The chemoselectivity of triazine trichloride for different
amines are taken into account for the synthesis of this
IP: 72.9.2.164 On: Mon, 04 Jan 2016 19:19:30
azine dendrimers was determined using modified dissolu-
Copyright: American Scientific Publishers
34
tion method with slight modification. The release medium
used was the PBS (pH 7.4) having Tween 80 (0.1%). The
dialysis bag (MWCO 3000 Da) was filled with the solu-
tion of PTX loaded triazine dendrimers (50 mg in 1 ml of
novel triazine based dendrimers. Nucleophilic aromatic
substitution reaction took place when triazine trichloride
was allowed to react with diethanolamine with the release
of HCl. The proposed dendrimers synthesis has been
exclusively developed for the first time in our laboratory.
Since the secondary amine present on diethanolamine is
more nucleophilic as compared to hydroxyl group there-
fore the dendrimer growth proceeds in chemoselective
fashion (Scheme 2). One equiv of triazine trichloride was
allowed to react with 3 equiv of diethanolamine to get
generation 1 (G1) dendrimers which was dark brown in
color and honey like consistency. The substitution on tri-
azine ring takes place similarly as shown in Scheme 1.
To increase the generation, G1 dendrimers was allowed to
distilled water) and placed in 50 ml of release medium at
ꢁ
3
7 C with slow magnetic stirring under sink conditions.
Aliquots of 1 ml were withdrawn from the release medium
and replenished with the same volume of fresh medium
to maintain perfect sink condition. The aliquots were ana-
lyzed after proper dilution in HPLC at 227 nm to know the
amount of drug release from the system.
2
.5. Toxicity Studies
2
.5.1. Hemolysis Study
ꢁ
react with triazine trichloride at 0 C, and at room temper-
The RBC suspension was obtained by following reported
procedure for hemolytic studies. Briefly, the human
blood was collected in HiAnticlot blood collection vials
ature, giving generation 1.5 (G1.5) dendrimers which was
white powder, insoluble in water having chlorine group on
the periphery. Similarly diethanolamine, triazine trichlo-
ride and again diethanolamine were attached step-by-step
respectively to the half and full generation of dendrimers
to develop dendrimers up to third generation (G3) with
3
5
(Himedia Labs, India). The RBCs were separated out from
the blood by centrifugation and diluted with saline to make
the RBC suspension. Dendrimer solution (in saline) of
concentration 0.5, 1.0, 1.5, 2.0 and 5.0 mg/mL (0.5 mL)
was added to the RBC suspension (4.5 mL) and incubated
for 1 hr and 24 hr separately. After centrifugation, super-
natants were taken and diluted with an equal volume of
normal saline and absorbance was measured at 540 nm
63% yield. NaHCO was used in each synthesis step to
3
neutralize the released HCl. The full practical descriptions
of G1–G3 dendrimers was given in Table I.
In each reaction step, simply washing the reaction mix-
ture with acetone can remove both the excess triazine
trichloride and diethanolamine because both are soluble in
(ꢅmax of Hb).
8
398
J. Nanosci. Nanotechnol. 10, 8395–8404, 2010

Bansal et al.
Development and Characterization of Triazine Based Dendrimers for Delivery of Antitumor Agent
HO
OH
N
N
Cl
N
HO
OH
N
N
N
N
a′
HO
OH
+
N
N
N
H
Cl
Cl
OH
OH
Cyanuric chloride
Diethanolamine
G1 dendrimer
b′
HO
OH
HO
OH
HO
N
N
N
Cl
Cl
N
OH
N
N
N
N
N
N
N
HO
HO
N
N
N
O
O
Cl
N
O
N
O
N
N
Cl
OH
OH
N
N
N
N
HO
HO
N
N
N
N
Cl
Cl
N
N
N
N
N
N
N
N
O
N
O
N
OH
N
N
N
N
O
N
O
N
N
N
N
N
c′
OH
Cl
Cl
O
O
N
N
OH
HO
N
O
O
N
N
N
OH
Cl
N
N
N
N
OH
N
Cl
N
N
N
HO
HO
Cl
Cl
OH
HO
OH
OH
G2 dendrimer G1.5 dendrimer
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Copyright: American Scientific Publishers
b′
OH OH OHOH
HO HO OHHO OH HO
OH
HO HO
HO
N
HO
HO
N
OH
OH
Cl Cl
N
N
Cl
Cl
N
N
N
Cl
N
Cl
Cl
HO
HO
N
N
N
OH
OH
N
N
N
N
Cl
N
N
N
N
N
HO
HO
HO
HO
HO
HO
HO
HO
N
N
N
N
N
N
O
N
N
N
OH
OH
N
O
N
Cl
O
N
N
N
O
N
O
O
O
N
N
N
O
N
Cl
N
O
Cl
N
OH
O
N
Cl
Cl N
N
N
N
O
N
N
N
OH
OH
N
N
N
N
O
N
N
N
N
N
N
Cl
Cl
N
N
N
N
N
N
OH
Cl
N
N
N
N
N
N
O N
N
O
N
N
O
N
N
OH
O
O
N
N
N
N
N
N
N
NNN
N
HO
HO
N
N
OH
OH
Cl
N
O
O
N
Cl
Cl
O
O
N
N
N
N
N
O
N
O
OH
OH
Cl
N
HO
HO
N
N
N
N
N
N
N
O
N
O
N
Cl
Cl
N
O
OH
N
N
HO
N
N
N
N
O N
N
N
N
N
N
OH
OH
OH
Cl
Cl
N
N
O
N
HO
HO
N
N
N
N
N
N
O
N
O
N
N
N
O
N
N
O
N
N
N
N
N
N
N
O
N
c′
HO
HO
N
N
N
O
N
N N
O
N
N
N
N
O
N
OH
N
N
N
N
N
N
O
N
N
N
CI
HO
N
N
O
N
N
OH
OH
Cl
Cl
HO
HO
N
N
O
N
N
N
O
N
CI
N
N
OH
N
N
N
O
N
N
N N
O
N
N
OH
OH
OH
OH
OH
O
O
N
N
Cl
HO
HO
N
N
N
N
N
N
O
N
O
N
N
N
HO
HO
HO
HO
N
N
Cl
O
N
N
N
N
O
N
N
N
N
N
N
CI
Cl
N
N
N
N
N
O
N
N
N
O
N
Cl
Cl
N
O
O
N
O
O
NN
CI
Cl
N
N
HO
HO
N
N
N
O
N
N
N
OH
N
N
N
N
N
N
N
OH
N
N
N
O
O
N
Cl
N
HO
HO
N
OH
N
O
O
O
ClN
Cl
N
OH
N
N
N
N
Cl
N
N
N
N
N
HO
N
OH OH
OH OH
OH
N
Cl
N
N
N
N
HO
N
Cl
CI
OH
Cl Cl
N
N Cl
OH
HO HO
Cl Cl
HO
OH OH
HO OH
G2.5 dendrimer
G3 dendrimer
ꢀ
ꢁ
ꢁ
Scheme 2. Synthetic Strategy of Triazine Dendrimers. Reagent and conditions: (a ) THF, NaHCO , 0–2 C → 1.5 h, RT (room temperature) 25± 2 C
3
ꢁ
ꢀ
ꢁ
ꢁ
ꢀ
→
12 h, 100 ± 2 C → 28 h (b ) triazine trihloride, THF, Methanol, NaHCO , 0–2 C → 1.5 h, RT → 1 h, 60 ± 2 C → 6 h (c ) Diethanolamine,
3
ꢁ
Dioxane, NaHCO , RT → 6 h, 100± 2 C → 28 h.
3
acetone whereas dendrimers (half and full generations) are
insoluble in acetone thus minimizing the chances of for-
mation of side product in further steps. Full generation
dendrimers was washed with methanol to removes sodium
chloride formed during neutralization as salt was insoluble
in methanol. Additionally, G2 and G3 dendrimers were
purified by column chromatography using silica gel to
obtained product free from impurities.
J. Nanosci. Nanotechnol. 10, 8395–8404, 2010
8399

Development and Characterization of Triazine Based Dendrimers for Delivery of Antitumor Agent
Bansal et al.
Table I. Physical description of triazine dendrimers.
Dendrimer
generation
Molecular
formula
Appearance
color/form
Theoretical
molecular weight
Aqueous
solubility
Group at
periphery (number)
G1
G1.5
G2
G2.5
G3
C H N O
6
C H Cl N O
33 24 12 24 6
Dark brown/Semisolid
White/Solid
Brown/Solid
White/Solid
Light brown/Solid
390.78
1278.24
2101.92
5651.76
8949.50
Soluble
Insoluble
Soluble
Insoluble
Soluble
OH (6)
Cl (12)
OH (24)
Cl (48)
OH (96)
15
30
6
C H N O
81
144 36 30
C153H120Cl N108O30
48
C345H600N156O126
3
.2. Characterization
and G3 dendrimers were freely soluble in water and dark
brown to light brown in color. On the other hand G1.5 and
G2.5 dendrimers were insoluble in water and white in color.
TLC plates were run for identifying the consumption of
reactants and formation of products.
During synthesis the growth of dendrimer is preliminarily
detected by its water solubility, color and by TLC. G1, G2
In G1, G2 and G3 Dendrimers, hydroxyl groups were
present on periphery in contrast to chlorine groups on
the periphery of G1.5 and G2.5 dendrimers. This is the
basic difference in the structure of different generation
of dendrimers. Thus in FTIR spectroscopy G1, G2 and
Cl Cl
Cl
N
N
Cl
Cl
Cl
N
N
N
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
N
Cl
N
N
N
Cl
N
N
N
N
N
O
Cl
O
O
O
O
Cl
N
N
O
Cl
Cl
Cl N
Cl
N
N
N
N
N
Cl
Cl
N
Delivered by Publishing Technology to: Deak
N
in University Library
N
2
O
N
N
N
N
N
N
N
IP: 72.9.2.164 On: Mon, 04 Jan 2016 19:19:30
N
Cl
N
O
1
O
N
Cl
2
O
N
Copyright: American Scientifi
N
c P
ublishers
Cl
N
O
Cl
1
N
N
O
N
O
4
000
3000
2000
1000
500
N
N
N
N
Cl
Cl
Cl
Wavenumber [cm–1
N
]
N
O
N
N
N
N
Cl
O
N
N
N
N
N
O
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
N
N
O
N
N
N
N
N
N
Cl
Cl
Cl
N
O
N
Cl
N
O
O
N
N
Cl
O
N
N
N
N
O
N
N
N
N
Cl
Cl
O
N
N
N
N
Cl
N
N
N
O
Cl
Cl
N
N
O
Cl
N
N
N
N
N
Cl
Cl
Cl
N
O
O
O
O
O
N
N
4
000
3000
2000
Wavenumber [cm–1
1000
500
N
ClN
Cl
N
N
N
N
N
N
N
Cl
N
N
]
N
Cl
N Cl Cl
Cl Cl
Cl Cl
N
1
00
9
8
7
6
5
4
3
2
0
0
0
0
0
0
0
0
4
000
3000
2000
1000
500
Wavenumber [cm–1
]
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
4000
3000
2000
1000
500
Wavenumber [cm–1
]
Fig. 1. FTIR spectra of triazine dendrimers.
Fig. 2. 1H NMR Investigation of G2.5 Dendrimers.
J. Nanosci. Nanotechnol. 10, 8395–8404, 2010
8
400

Bansal et al.
Development and Characterization of Triazine Based Dendrimers for Delivery of Antitumor Agent
G3 dendrimers showed O–H streching at 3364, 3405, and
Additionally we characterized G3 triazine dendrimers by
C NMR and by ESI mass spectroscopy for confirming
−
1
13
3
413 cm and C–Cl stretching was absent whereas in
G1.5 and G2.5 O–H stretching was not detected and C–Cl
carbon atoms present in the structure of dendrimer and for
determining its exact mass respectively. Carbon atoms in
triazine ring are all in same environment and therefore sin-
gle peak for triazine ring was detected at 158 ppm. Carbon
−
1
stretching was detected at 767 and 766 cm (Fig. 1).
1
In H NMR spectroscopy two triplets of methylene
groups were observed in G1, G1.5 and G2.5 dendrimers
due to the presence of CH groups in two different envi-
ronments (Fig. 2). In G2 and G3 dendrimers four triplets
of CH
2
groups are present in three different environment
2
i.e., in first and second ring N–CH , CH -Triazine whereas
2
2
in third ring first methylene group was present in same
environment i.e., N–CH but another carbon was in differ-
were detected because CH groups were present in four
2
different environments (Fig. 3). In the structure of den-
drimer it was clearly seen that protons were present in the
form of methylene group hence triplets were detected by
NMR spectroscopy. The instrument detected single triplet
for the numbers of methylene groups present in the same
environment whereas chemical shifts of the peaks were
changed. The number of protons detected in NMR was
matching with the theoretical numbers. Absence of side
2
ent environment i.e., CH –OH. Therefore three peaks were
2
detected by NMR spectroscopy for the carbon of methy-
lene groups present in the G3 triazine dendrimers. By ESI
mass spectra exact mass was found to be 8950 Dalton
(Fig. 4).
The results of elemental (CHN) analysis showed that the
experimental percent of elements were matching with the
theoretical percentage (Table II). By this result we con-
cluded that the generation of dendrimers increases because
in even generation the percent hydrogen is more (because
of addition of diethanolamine) compared with the odd
generation (because of addition of cyanuric chloride). All
the data suggests that the synthesis of dendrimers up to
third generation is feasible in step-by-step manner.
1
products in H NMR during these experiments should be
conspicuously noted.
HOHO HOHO HO OH OHOH
OHOH
HOHO HO
HO
HO
HO
HO
HO
N
OH
OH
N
N
N
N
N
OH
OH
OH
OH
N
N
HO
HO
HO
HO
HO
HO
HO
N
O
N
N
N
N
N N
O
N
N
O N
N
N
N
N N
N
N
O N
N
N
O
OH
OH
OH
OH
OH
N
O
O
N
N
d
N
N
N
N
N
N
N
N N
N
c
N
N N
N N
O
N
N
O
O
N
N
(A)
HO
Deliver
ed by
N
Publishing Technology to: Deakin University Library
N N
N
OH
HO
N
N
a
N
O
IP: 72.9.2.
O
16
N
4
N
On:OHMon, 04 Jan 2016 19:19:30
HO
N
OH
N
N
b
HO
HO
N
N
Cop
N
yright: Amer
O
ic
H
OH
OH
an Scientific Publishers
O
N N
N
N N
N
O N
HO
HO
HO
N
N
N
N O
N N
N
N
N
N
N
OH
OH
OH
N
N
O
N
N
O
N
N
N
O N
N N
HO
HO
HO
N
O
N
N
N
O
N
N
N
N
N
O
N
N
OH
OH
OH
O
N
HO
N
N
N
N
N
N
HO
HO
HO
N
N O
N
N
N
N
OH
OH
OH
OH
OH
OH
N
N
O N
N
N
HO
HO
HO
HO
HO
HO
HO
HO
N
N ON
N
N
O
N
N
N
N
N
O
N
O
O
N
N
N
O
N N
N
N
N
N
N
N N
N
N
OH
N
N
OH
OH
N
N
N
N
N
HO
N
OH
OH
HO
HO
OH
OH
HO OH
HOHO HO
OH HO OH OH
(
B)
Fig. 3. 1H NMR investigation of G3 dendrimers.
J. Nanosci. Nanotechnol. 10, 8395–8404, 2010
Fig. 4. 13C NMR (A) and ESI mass spectra (B) of G3 Dendrimers.
8401

Development and Characterization of Triazine Based Dendrimers for Delivery of Antitumor Agent
Bansal et al.
Table II. Elemental analysis data of triazine dendrimers.
Percentage of element present
Dendrimers
Generation
Theoretical
H
Experimental
H
C
N
C
N
G1.0
G1.5
G2.0
G2.5
G3.0
46.14
31.01
46.28
32.51
46.30
7.74
1.89
6.90
2.14
6.76
21.52
26.30
23.99
26.76
24.41
46.12
31.03
46.28
32.50
46.30
7.72
1.86
6.89
2.12
6.75
21.50
26.30
24.00
26.76
24.40
3
.3. Solubilization Efficiency of Dendrimers
Dendrimers as unimolecular micelles are extensively used
as a solubilizing tool to improve the aqueous solubility of
poorly soluble agents thus increase their bioavailability as
well as reduced toxicity. Novel triazine dendrimers have
internal hydrophobic region (triazine rings) and external
hydrophilic region (hydroxyl groups) thus act as micelles
and use to enhance the solubility. Paclitaxel aqueous sol-
ubility was found to be enhanced with the use of differ-
ent generations and concentrations of triazine dendrimers.
Aqueous solubility of PTX increases with increase in
dendrimers concentration. Solubility of PTX in water
Fig. 6. Effect of pH of solvent on solubilizing ability of triazine den-
drimers (n = 3).
The order of the ability of triazine dendrimers to solubi-
lize PTX at different pH was pH 4ꢃ0 > pH 7ꢃ4 > pH 10.0.
The reason behind this order might be due to degradation
36
of drug at higher pH.
(
0.0003 mg/ml) was enhanced up to 0.562 mg/ml with
3.4. Encapsulation Efficiency and In Vitro
−2
the use of triazine G3 (2ꢃ23 × 10 Mm concentration)
Release Profile
dendrimers. It was show nD teh l ai vt ei nr ec dr e ab sy in Pg u db el ins dh r ii nm ge rTs e gc ehn n- ology to: Deakin University Library
IP: 72.9.2.164 On: Mon, 0 N4 o Jv ae nl 2t r 0i a1z 6i n 1e 9 d: 1e 9n :d3r 0i mers showed 24.34 wt% of drug
eration increased the aqueous solubility of PTX (Fig. 5).
Copyright: American S cl oi ea nd it ni f gi c( PP uT bX l i) s ihn es ri ds e its hydrophobic region. PTX loaded
Thus, for further solubilization studies only G3 triazine
dendrimers was used. Triazine ring imparts hydrophobic
interaction whereas hydroxyl groups present on the periph-
ery take part in hydrogen bonding thus, the mechanism
involved in the solubility enhancement could be either
hydrophobic interaction or hydrogen bonding or both.
While performing the solubility study of PTX at differ-
ent pH, it was concluded that at pH 4 triazine dendrimers
displayed maximum enhancement in aqueous solubility
compared to pH 7.4 and 10 (Fig. 6).
in dendrimers showed 81% of drug release after 24 hr,
whereas nearly similar quantity of free PTX (85%) was
released only after 4 hr, suggesting the controlled release
property of dendrimers (Fig. 7). The release behavior
of PTX from triazine dendrimers exhibited a non-linear
release profile by showing initial faster release followed by
slower release. The reason behind this phenomenon was
possibly the ꢆ−ꢆ interaction between aromatic triazine
ring and PTX.
1
20
0
0
0
0
.8
.6
.4
.2
0
.55×10–2 mM
.11×10–2 mM
(Free drug)
(Drug loaded in dendrimer)
0
100
80
1
1.67×10–2 mM
.23×10–2 mM
2
60
4
2
0
0
0
1
2
3
0.25 0.5
1
2
4
8
12 16 20 24 28 30
Dendrimer generation
Time (hr)
Fig. 5. Effect of the generations of triazine dendrimers on aqueous sol-
ubilization of PTX (n = 3).
Fig. 7. Cumulative release patterns of free PTX and PTX loaded in
dendrimers (n = 3).
8
402
J. Nanosci. Nanotechnol. 10, 8395–8404, 2010

Bansal et al.
.5. Toxicity Studies
Development and Characterization of Triazine Based Dendrimers for Delivery of Antitumor Agent
5
4
4
3
3
2
2
0
5
0
5
0
5
0
3
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Cationic macromolecules in general cause destabiliza-
tion of the cell membrane resulting in cell lysis. Thus
amino-terminated dendrimers were shown to be generally
cytotoxic comparatively anionic and neutral dendrimers.
Previously hydroxyl- or methoxy-terminated dendrimers
based on a polyester scaffold were shown to be non toxic
3
7
15
both in vitro and in vivo. Hemolytic study gives quan-
titative measure of haemoglobin (Hb) release from RBCs
and the data obtained in such assays gives a qualitative
indication of potential damage to RBCs on dendrimers
administration. Many researchers’s used this study as a
preliminary tool to determine the membrane polymer inter-
action. The novel triazine dendrimer was found to be non-
hemolytic up to concentration of 5 mg/mL after 1 hr
incubation but hemolytic after 24 hr showed concentration-
dependent and time dependent hemolysis (Fig. 8). This
may be due to the aromatic interior of dendrimers which
causes haemolysis through hydrophobic membrane con-
tact. In contrast, toxicity study on melamine dendrimers-
derivatives showed that the cationic dendrimers were more
haemolytic (at both 1 and 24 h) than anionic or PEGylated
melamine dendrimers.38 In acute toxicity study effects of
triazine dendrimers on renal function were evaluated by
change in BUN level, all the groups of mice (except group
1
0
5
0
BUN level in serum
SGPT activity in serum
Fig. 9. Change in serum enzyme level post dosing in mice (n = 3).
that synthesized triazine dendrimers may also be a possi-
ble candidate for biomedical and clinical applications. We
concluded that the in vivo toxicity of this molecule to liver
and kidneys is low enough to justify that this dendrimer is
suitable for tumor inhibition studies.
4
. CONCLUSION
Our laboratory is constantly engaged in exploring den-
drimers potentiality as drug delivery vehicle. By taking
5
) did not show any significant change in BUN level when
Delivered by Publishing Technolog tyh it so :b Da ce kag kr ion u nU dn i ivn e ros ui tr y mL ii bn dr a rwy e synthesized cost effec-
compared to the control group af It Pe r: 74 28 . 9h . r2 . o1 f6 4i n Oj e cn t :i oMn o. n, 04 Jan 2016 19:19:30
tive triazine based dendrimer up to third generation with
3% yield using divergent method because the convergent
Copyright: American Scientific Publishers
The effects of triazine dendrimers on hepatic function were
6
evaluated by changes in SGPT activity in serum. None of
the groups of mice (except group 5) showed any signifi-
cant change in SGPT activity when compared to the control
group after 48 hr of injection (Fig. 9). However, a statis-
tically significant increase in BUN and SGPT level was
observed in those mice which are in group 5th after 48 hr of
injection. The results showed by novel triazine dendrimers
in toxicity study suggested that this dendrimer was safe in
BALB/c mice up to dose of 200 mg/kg via IP route. The
inherent toxicity of this dendrimers was less compared to
method of dendrimer synthesis is not feasible for large
scale synthesis. This triazine dendrimer can be synthesized
without using protecting groups thus it must be somewhat
economical and easy to synthesize compared to other exist-
ing triazine dendrimers. The synthesis was accomplished
in five steps from commercially available materials, requir-
ing extraction, evaporation or recrystallization to purify
the intermediate dendrimer generations. Thus this synthetic
strategy will overcome the problems associated with the
synthesis of other classes of dendrimers like tedious and
lengthy method of preparation, low yield, high synthesis
cost and cytotoxicity associated with cationic dendrimers
because the proposed triazine dendrimers have hydroxyl
group on the surface so it may be biocompatible and thus
this carrier system may be useful in various clinical appli-
cations. Further exploration of this novel class of dendrimer
is underway and its further possible utility in drug delivery
applications is being examined in our laboratory.
1
3
Simanek’s cationic triazine dendrimers which concluded
2
2
1
1
5
0
5
0
5
0
0
1
.5 mg/mL
.5 mg/mL
1.0 mg/mL
2
5
.0 mg/mL
.0 mg/mL
Acknowledgments: This research was financially sup-
ported by All India Council for Technical Education, New
Delhi, INDIA. The authors acknowledge sophisticated
analytical instrumentation facility (SAIF), Punjab Univer-
sity, Chandigarh, and SAIF, Central Drug Research Insti-
tute, Lucknow, (India) for providing Spectroscopic and
elemental analysis.
1
24
Time (hr)
Fig. 8. Percent hemolysis by 3.0G triazine dendrimers of different con-
centration on human RBCs after 1 and 24 hr exposure (n = 3).
J. Nanosci. Nanotechnol. 10, 8395–8404, 2010
8403

Development and Characterization of Triazine Based Dendrimers for Delivery of Antitumor Agent
Bansal et al.
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2
0. K. Huthmacher and D. Most, Ullmann’s Encyclopedia of Industrial
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. M. Enomoto and T. Aida, J. Am. Chem. Soc. 124, 6099 (2002).
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(
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2007/0098676 A1 (2007).
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IP: 72.9.2.164 On: Mon, 04 Jan 2016 19:19:30
Copyright: American Scientific Publishers
Received: 10 November 2009. Accepted: 15 January 2010.
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