Poly propyl ether imine (PETIM) dendrimer: A novel non-toxic dendrimer for sustained drug delivery

Article abstract of DOI:10.1016/j.ejmech.2010.08.006

In the present study, an attempt was made to study the acute and sub-acute toxicity profile of G3-COOH Poly (propyl ether imine) [PETIM] dendrimer and its use as a carrier for sustained delivery of model drug ketoprofen. Drug-dendrimer complex was prepared and characterized by FTIR, solubility and in vitro drug release study. PETIM dendrimer was found to have significantly less toxicity in A541 cells compared to Poly amido amine (PAMAM) dendrimer. Further, acute and 28 days sub-acute toxicity measurement in mice showed no mortality, hematological, biochemical or histopathological changes up to 80 mg/kg dose of PETIM dendrimer. The results of study demonstrated that G3-COOH PETIM dendrimer can be used as a safe and efficient vehicle for sustained drug delivery.

Full text of DOI:10.1016/j.ejmech.2010.08.006

European Journal of Medicinal Chemistry 45 (2010) 4997e5005
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Poly propyl ether imine (PETIM) dendrimer: A novel non-toxic dendrimer
for sustained drug delivery
,
a
a
a
b
,
,1
Subheet Jain a , Amanpreet Kaur , Richa Puri , Puneet Utreja , Anubhuti Jain 1, Mahesh Bhide b
,
*
Rakesh Ratnam b 1, Vinay Singh b 1, A.S. Patil b 1, N. Jayaraman c, Gaurav Kaushik d, Subodh Yadav d,
,
,
,
K.L. Khanduja d
a Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala 147002, India
b Research & Development, V.B. Medicare Pvt Ltd, C/o Bioplus Lifesciences Pvt Ltd, SIPCOT Phase II, Krishnagiri Road, Hosur, Tamilnadu 635109, India
c Department of Chemistry, Indian Institute of Science, Malleswaram, Bangalore 560012, Karnataka, India
d Department of Biophysics, PGIMR, Chandigarh, 160012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, an attempt was made to study the acute and sub-acute toxicity profile of G3eCOOH
Poly (propyl ether imine) [PETIM] dendrimer and its use as a carrier for sustained delivery of model drug
ketoprofen. Drug-dendrimer complex was prepared and characterized by FTIR, solubility and in vitro
drug release study. PETIM dendrimer was found to have significantly less toxicity in A541 cells compared
to Poly amido amine (PAMAM) dendrimer. Further, acute and 28 days sub-acute toxicity measurement in
mice showed no mortality, hematological, biochemical or histopathological changes up to 80 mg/kg dose
of PETIM dendrimer. The results of study demonstrated that G3eCOOH PETIM dendrimer can be used as
a safe and efficient vehicle for sustained drug delivery.
Received 6 January 2010
Received in revised form
4 August 2010
Accepted 6 August 2010
Available online 12 August 2010
Keywords:
PETIM dendrimer
Drug delivery
Ó 2010 Elsevier Masson SAS. All rights reserved.
Sustained release
Hemolytic toxicity
Acute and sub-acute toxicity
1. Introduction
accumulation properties [5,6]. Due to this reason, no commercial
dendrimer based formulation for systemic administration is
Dendrimers have generated
a
great deal of interest as
available.
The present investigation was aimed at studying the possibility
controlled and targeted drug delivery system due to their
exceptional structural properties such as low polydispersity (z1),
high density of peripheral functional group, well defined globular
shape (z2.0 nm) and multivalency [1,2]. Dendrimers are
synthesized from branched monomer units in a stepwise manner
so it is possible to conduct precise control on molecular size,
shape, dimension, density, polarity, flexibility and solubility by
choosing different building/branching units and surface func-
tional groups [1,3]. Till now dendrimers have been widely
explored as carrier system in drug delivery [4]. Poly amido amine
(PAMAM), Poly propylene imine (PPI), Polylysine and Triazine are
most widely explored dendrimers for drug delivery and their
major limitation is hematological toxicity and imperfect organ
of using a novel dendrimer carrier system PETIM synthesized by
Pharmaceutical company Bioplus Life Science Pvt. Ltd., India as
a carrier for sustained delivery of drug [7e9]. PETIM was found to
have good water solubility and stability as evaluated in the previous
study [10]. This dendrimer system was also found to have a higher
internal cavity diameter in comparison to PAMAM dendrimer as
measured by Small angle x-ray diffraction techniques [11]. No work
was carried out for evaluation of PETIM dendrimer as a drug carrier
system for sustained delivery of drug. In the present study, an
attempt was made to prepare drug-dendrimer complex using
ketoprofen as a model drug and evaluating its sustained release
potential.
Biocompatibility is a big issue in development of commercial
viable formulation using dendrimer as carrier system [6]. Attempt
was also made for evaluating the acute and short term sub-acute
toxicity profile of PETIM dendrimer by studying the effect of PETIM
administration on hematological, biochemical parameters and vital
organ histopathology of mice. Cytotoxicity assay was also designed
* Corresponding author. Department of Pharmaceutical Sciences and Drug
Research, Punjabi University, Patiala 147002, Punjab, India. Tel.: þ91 175 3046254.
E-mail address: subheetjain@rediffmail.com (S. Jain).
1
Tel.: þ91 4344 261962.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.006

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S. Jain et al. / European Journal of Medicinal Chemistry 45 (2010) 4997e5005
to study cytotoxicity of PETIM in comparison to commercially
available PAMAM dendrimer.
then dried under vaccum to remove methanol (Vaccum oven,
Perfit, India). The complex was dissolved in distilled water and
stirred for another 2 h to extract out the drugedendrimer complex.
The solution was then filtered through the 0.22
mm membrane filter
2. Materials and methods
and lyophilized (Heto, Allerod, Denmark) to remove the water
completely. The drugedendrimer complex was redissolved in PBS
(7.4) and free drug was removed by dialysis method (molecular
weight cut off 1 kD). Drug concentration was determined using
HPLC assay. Characterization of drugedendrimer complex was
carried out by recording the FTIR spectra of drugedendrimer
complex.
2.1. Materials
Polypropyletherimine (PETIM) dendrimer G3eCOOH genera-
tion is synthesized by Bioplus Life Science Pvt Ltd, Hosur, India as
per own patented procedure [7]. Ketoprofen, RPMI 1640 media,
Fetal bovine serum (FBS), G4-OH PAMAM dendrimer and all other
chemicals were procured from Sigma Aldrich Co. Ltd. USA. Dialysis
membrane having molecular weight cut off 1 kD was purchased
from Spectrapor, USA. Glucose, cholesterol, total protein content,
urea, SGPT (Serum glutamic pyruvic transaminase), SGOT (Serum
glutamic-oxaloacetic transaminase), ALP (Alkaline phosphate),
creatinine and bilirubin estimation kit were purchased from Erba
Ltd., India.
2.4. Solubility study
Solubility study was carried out as per method reported by
Higuchi and Connors [13]. Excess of drug (1, 2, 5 and 10 mg/ml) was
added separately into the screw-capped vials containing dendrimer
(20 mg/ml) in PBS (7.4). Vials were shaken for 24 h at 37 ꢀ 1.0 ꢁC on
a horizontal rotary shaker (Remi equipments, Mumbai). The
mixture obtained was filtered on a 0.45 mm membrane filter and
2.2. Synthesis of PETIM dendrimer
drug content in filtrate was determined using HPLC assay.
The PETIM series of dendrimers were synthesized by a divergent
growth methodology as shown in Fig. 1. The 3-Aminopropane-1-ol
was the monomer for the synthesis of PETIM dendrimer that
possess tertiary amine as the branch point and ether as linker
interconnected by propylene spacers. The details of synthetic
procedure for preparation of different generation of PETIM den-
drimers have been described in our previous report [7e10].
2.5. In vitro drug release study
In vitro release behaviour of ketoprofen from the ketopro-
fenePETIM (G3eCOOH) dendrimer complex was investigated by
dialysis method as described by Wang et al. [14]. Pure ketoprofen
was dissolved in methanol at a concentration of 2 mg/ml and used
as control. The complex was dissolved in PBS (pH 7.4) at a concen-
tration of 2 mg/ml. This solution (2 ml in volume) was transferred
to dialysis bag (molecular weight cut off 1 kD) immediately. The
dialysis bag was placed in a 50 ml-beaker containing 40 ml PBS
(pH 7.4). The outer phase was stirred continuously using magnetic
stirrer at 50 rpm. At predetermined time intervals of 0.5, 1, 1.5, 2, 3,
2.3. Formation of drugedendrimer complex
KetoprofenePETIM complex was prepared as per method
reported by Papagiannaros et al. [12]. Ketoprofen was dissolved in
methanol and synthesized G3eCOOH PETIM dendrimer was added.
This solution was stirred overnight by magnetic stirrer (50 rpm),
4, 5, 6, 8, 10, 15, 20 and 24 h, 100
ml sample was withdrawn and
Raney Ni, NH3 gas,
H2, MeOH
NC
CN
2HN
NH2
O
O
400 rpm, 14 bar,RT
G0-NH2
G0-CN
0 tBu
Cat NaOH,
15 hr, RT
CN
RT
MeOH
H2O
O
O
HO
HO
OH
O
O
O
LiAlH4
N
N
O
G1-Ester
O
N
N
O
0oC, THF
G1-OH
O
O
OH
O
O
CH3COCl
DCM
0-5oC
OH
HO
Repeat same path to
G3-COOH PETIM
N
N
O
G1-Acid
OH
HO
O
O
Fig. 1. Scheme for synthesis of PETIM dendrimer.

S. Jain et al. / European Journal of Medicinal Chemistry 45 (2010) 4997e5005
4999
replenished with the same amount of receptor fluid. The concen-
tration of ketoprofen present in the dialysate was monitored using
HPLC and the percent drug release at various time intervals was
calculated.
4000 rpm for 15 min to separate serum. The blood samples con-
taining the blood and anticoagulant were centrifuged at 4000 rpm
for 15 min to separate the plasma. Both plasma and serum were
used for evaluating the different biochemical parameters e glucose,
cholesterol, urea, SGPT, SGOT, total proteins, ALP, Creatinine and
bilirubin content with standard Erba estimation kit using Auto-
anlyzer (Erba, Chem 7, Germany). Standard procedure as specified
in the kit literature was followed. The blood samples collected in
the sodium citrate tubes were also used for analyzing red blood cell
count (RBC), haemoglobin concentration (Hb), Mean Corpuscular
Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean
Corpuscular Hemoglobin concentration (MCHC), Platelets (Plt),
total leukocytes count (TLC), polymorphs, lymphocytes, monocytes
and eosinophils count using auto hematolyzer (PooCH 100, Sys,
Germany).
2.6. Hemolytic toxicity assay
The RBC suspension was obtained as per the well-known and
reported procedure for hemolytic studies [15]. The RBC suspension
was mixed with distilled water, which was considered as producing
100% hemolysis, as normal saline does not produce any hemolysis
thus acting as a blank. The RBC suspension (0.5 ml) was added to
4.5 ml of PBS (pH 7.4) and then 1000,100,10,1, 0.1 and 0.01 mg/ml of
PETIM dendrimer solution in PBS were interacted with RBC
suspension. After 1 h incubation at 37 ꢀ 1.0 ꢁC followed by
centrifugation, the supernatant were taken and diluted with an
equal volume of PBS (pH 7.4) and absorbance was taken at 540 nm
against supernatant of normal saline. The percent hemolysis was
determined for each sample by taking absorbance of water as 100%
hemolytic sample.
2.9. Histopathological study
Animals were sacrificed at the end of 28 days by cervical
dislocation. Heart, kidney and liver were taken out and preserved in
10% formalin for histopathological examination. Sections were
fixed and block was made using the conventional procedure [17].
Sections were cut at 4 microns with the rotary microtome. Paraffin
wax was removed by warming the slide gently, until the wax
melted and then was washed with xylene followed by washings
with absolute alcohol and water respectively to hydrate the
sections and stained with haematoxylin-eosin. Histological
sections were examined using an optical microscope with the
photographic arrangement (DX31, Olympus, Japan).
2.7. Toxicity study
2.7.1. Acute toxicity
The acute toxicity of the PETIM dendrimer was evaluated in
mice using the up and down procedure [16]. Thirty six mice (Swiss
albino mice) of either sex (weight: 28e40 g; age: 12e16 weeks old)
were randomly assigned into six groups (n ¼ 6). Group of six mice
of same sex were housed together. Dendrimer solution in PBS
(pH 7.4) was administered intra-peritoneally. The first group of
animals, serving as control, received PBS (pH 7.4) (5 ml/kg); the
second, third, fourth, fifth and six group received G3eCOOH den-
drimer solution in PBS at dose 10, 20, 40, 80 and 100 mg/kg,
respectively. Animals were observed for toxic symptoms continu-
ously for the first 4 h after dosing. Finally, the number of survivors
was noted after 24 h and these animals were maintained for further
13 days with observations made daily.
2.10. Cytotoxicity assay
Freshly thawed, actively growing small lung cancer cell line
(A-549) was used. Twenty hours old, freshly seeded, actively
growing cultures of the selected cell lines were harvested by
trypsinization/shaking and centrifuged at 3000 rpm for 5 min. The
cells were then washed with PBS and resuspended in RPMI con-
taining 10% FBS at a concentration of 106 cells/mL. Micro titre plates
2.7.2. Sub-acute toxicity
were seeded by transferring 100 mL cells to each well and incubated
Thirty mice (Swiss albino mice) of either sex (weight: 28e40 g;
age: 12e16 weeks old) were randomly assigned into five groups
(n ¼ 6). Group of six mice of same sex was housed together. Den-
drimer solution in PBS (pH 7.4) was administered intra-peritoneally
once a day for 4 weeks. The first group of animals, serving as
control, received PBS (PH 7.4) (5 ml/kg); the second, third, fourth
and fifth group received G3eCOOH dendrimer solution in PBS at
dose 10, 20, 40 and 80 mg/kg, respectively. All animals were
supplied with Fiel Purina ChowÒ and tap water during the testing
periods. Animals were weighed and observed daily for physiolog-
ical and behavioural changes. Animals were examined at the end of
the test period for hematological and biochemical parameters.
Body weight, water and food intake was measured daily. All
investigations were performed after approval of the Institutional
Animal Ethics Committee of the Department of Pharmaceutical
Sciences and Drug Research, Punjabi University, Patiala and in
accordance with the disciplinary principles and guidelines of
committee for the purpose of control and supervision on experi-
ments on animals (CPCSEA).
at 37 ꢀ 0.5 ꢁC for 24 h under 5% CO2 atmosphere (5% CO2 incubator,
New Brunswick Scientific, Ltd. Germany). The final number of cells
seeded in each well was 5000. A stock solution (100 ml) was added
in first well of the 96 well micro titre plate (B.D. Bioscience Ltd.,
USA) followed by serial dilution by 10ꢂ in subsequent well. The
plate was incubated for 48 h and cell viability study was conducted
using MTT assay. MTT solution (0.5% w/v in RPMI medium) was
added to each well, and the plate was incubated at 37 ꢀ 0.5 ꢁC for
4 h. After 4 h the content of each cell was removed and the con-
verted dye was solubilized in 150 mL isopropyl alcohol/dimethyl
sulfoxide mixture (1:1). Absorbance of converted dye was
measured at wavelength of 540 nm using ELISA plate reader
(B.D. Bioscience Ltd., USA).
2.11. HPLC assay
Ketoprofen in the experimental protocol was estimated by the
HPLC method as reported by Satterwhite et al. [18]. Orthophos-
phoric acid (0.1%) & Methanol (40:60) in isocratic mode was used as
mobile phase and was delivered at 1.0 mL/min. The injected fluid
2.8. Blood analysis for hematological and biochemical parameters
(20 mL) was eluted in C18 column (Agilent Technologies, Inc., USA
Blood samples for hematological and biochemical estimations
were collected on the 0 and 28th day from different groups of
animals used for sub-acute toxicity assay. The blood samples were
kept at room temperature for 30 min and then centrifuged at
250 ꢂ 4.6 mm) at room temperature and ketoprofen was monitored
at 258 nm using a PDA detector (Agilent Technologies, Inc., USA).
The calibration curve within a concentration range from 0.05 to
10.0 mg/mL was used to measure the ketoprofen concentration.

5000
S. Jain et al. / European Journal of Medicinal Chemistry 45 (2010) 4997e5005
Table 1
Composition and characterizations of drugedendrimer complex.
Characteristic Parameters
Value
Generation
Terminal group
G3eCOOH
eCOOH
16
2667.3
421
Number of terminal group
Molecular weight (g/mol)
Total no. of atoms (N)
Encapsulation efficacy
aSize (nm)
56.0 ꢀ 2.2
4.14
bSolubilization
Drug release after 24 h
5.0 folds
79.9 ꢀ 3.8
Values Represented as Mean ꢀ SD (n ¼ 3).
a
Calculated by small angle x-ray scattering techniques as reported
in previous publication [11].
b
Ratio of saturation solubility of ketoprofen with PETIM dendrimer
versus intrinsic water solubility.
2.12. Statistical analysis
Data are expressed as the mean ꢀ standard deviation (SD) of
obtained results. The statistical analysis of data was performed
using analysis of variance (ANOVA) (Graphpad, Version 2.01, San
Diego, CA). A value of p < 0.05 was considered as statistically
significant.
3. Results and discussion
3.1. Preparation and in-vitro characterizations
Fig. 2. IR-Spectra of PETIM dendrimer (A), ketoprofen (B) and drugedendrimer
complex (C).
PETIM dendrimer was synthesized using the previously
described method as shown in Fig. 1 [7,9]. The theoretical details of
synthesized dendrimer are summarized in Table 1. Formation of
dendrimer structure was confirmed by FTIR and NMR spectroscopy.
The various peaks and shifts obtained were analyzed, interpreted
and matched with results reported in the previous study [7,9].
Drugedendrimer complex was prepared by using constant amount
of G3eCOOH dendrimer (20 mg/ml) with varying amount of
ketoprofen (1.0e10 mg/ml). Ketoprofen was selected as model drug
due to its low water solubility (0.01% w/w), low bioavailability and
G.I. toxicity [19]. PETIM dendrimer was found to have 5-folds
increase in solubility of ketoprofen. Similarly, Yiyun et al. [20]
studied the solubility enhancement effect of G2-G5 PAMAM den-
drimer and found 2e4 folds increase in ketoprofen solubility.
PETIM dendrimer was found to have a significant increase in
ketoprofen solubility at lower generation probably due to the
towards lower side at 1600 cmꢃ1 and sharpness was retained.
Additionally, characteristic eC¼O peak of dendrimer at 1700 cmꢃ1
is also shifted and merged with the peak of the drug. This is due to
of formation of drugedendrimer complex. The dendrimer may
complex the drug via various mechanisms i.e., hydrophobic
interaction, hydrogen bonding or electrostatic attraction [23]. Low
molecular weight drugs may encapsulate within the void space of
the dendrimer. In the case of ketoprofenePETIM complex, the
encapsulation of ketoprofen may also be due to hydrophobic
interaction. The bond between eCOOH group of ketoprofen and
internal cavities of dendrimer may also contribute for its
encapsulation.
ꢀ
higher internal cavity diameter (14.14 A) [11] in comparison to
3.2. In vitro drug release study
ꢀ
PAMAM dendrimer (8.0 A) [21]. Another reason for solubilization
ability of PETIM dendrimer is the presence of a number of hydro-
philic groups in the periphery, leading to accommodate the larger
number of water molecules as studied previously by water pene-
tration study [11].
The in vitro release behaviour of ketoprofen from the
drugedendrimer complex was examined in PBS 7.4 and results
are summarized in Fig. 3. The release of ketoprofen from the
drugedendrimer complex was appreciably slower as compared
to pure drug. After 2 h, 77.1% of the pure drug was permeated,
whereas, only 19.5% was released from the drugedendrimer
Fig. 2AeC shows the FTIR spectra of drug, dendrimer and
drugedendrimer complex, respectively. Peak around 2500 cmꢃ1
shows the presence of tertiary amine, characteristic feature of
dendrimer. Other peaks around 3100, 1650 and 1200 cmꢃ1 shows
the presence of eOH, eC¼O and CeOeC functional groups,
respectively [7,9]. These peaks are well correlated with G3eCOOH
structure as reported previously. FTIR spectra of pure ketoprofen
showed the 2 characteristic sharp and symmetric carbonyl peaks
at 1700 and 1655 cmꢃ1. These two peaks were ascribed to the
diametric carboxylic and ketonic group stretching vibration,
respectively. Results are well correlated with reported by Blasi
et al. [22] for characterization of ketoprofenePLGA interaction.
The FTIR spectra of drugedendrimer complex showed that char-
acteristic carbonyl group peak of ketoprofen was slightly shifted
complex. The drug release was prolonged to 24
h with
drugedendrimer complex, in comparison to 3 h with free drug,
indicates the sustained release behaviour of PETIM dendrimer.
The release rate was significantly slower than previously repor-
ted with ketoprofenePAMAM dendrimer complex (76% in 10 h)
[24].
3.3. Hemolytic toxicity measurement
Red blood cell (RBC) lysis measurement is a simple and widely
used method to study polymeremembrane interaction. It gives

S. Jain et al. / European Journal of Medicinal Chemistry 45 (2010) 4997e5005
5001
100
90
80
70
60
50
40
30
20
10
0
Ketoprofen alone
Ketoprofen-dendrimer complex
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (h)
Fig. 3. Comparative % drug release of ketoprofen from drugedendrimer complex and plain drug across the cellophane membrane. Mean ꢀ SD (n ¼ 3).
a quantitative measure of haemoglobin (Hb) release. The data
obtained in such assay also give a qualitative indication of potential
damage to RBC’s of dendrimer administered. This is a good indi-
cator of dendrimer toxicity in the in vivo conditions. Fig. 4
summarized the results of the hemolytic toxicity assay of
G3eCOOH PETIM dendrimer after 1 h incubation at concentrations
Electrostatic repulsion among red blood cells prevents their self
aggregation and makes them suspended as colloids in blood.
PAMAM dendrimer have net positive charge on the surface and
interacted with negatively charged surface of red blood cells and
causes hemolysis [26]. In comparison, G3eCOOH PETIM dendrimer
has net negative charge on the surface that minimized the inter-
action with RBCs.
from 0.01 to 1000
dependent hemolysis, 42 ꢀ 1.4 and 47.6 ꢀ 1.6% hemolysis was
observed at 100 and 1000 g/ml. Dendrimers are known for
mg/ml. PETIM dendrimer showed concentration
m
3.4. Cytotoxicity assay
showing the concentration dependent hemolytic toxicity and this is
its major limitation to use as carrier system in drug delivery.
Investigated PETIM dendrimer showed significantly less hemolysis
in comparison with PAMAM dendrimer [6]. Chen et al. [25] found
Cellular toxicity of PETIM dendrimer in comparison to
commercial available PAMAM dendrimer was investigated using
MTT assay, and results are summarized in Fig. 5. PAMAM G4-OH
generation was selected for comparison purpose due to similar
size (13 A) [20] with PETIM G3eCOOH dendrimer (14.14 A). The
MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide assay is simple nonradioactive colorimetric estimation to
measure cell cytotoxicity, proliferation, or viability. MTT is
a yellow, water-soluble, tetrazolium salt. Metabolically active cells
are able to convert this dye into a water-soluble dark blue
60 and 20% hemolysis at 10
mg/ml concentration of amine and
ꢀ
ꢀ
carboxylate terminate Melamine dendrimer, respectively. In
comparison, PETIM dendrimer at 10-folds higher concentration
showed only 42 ꢀ 1.4% hemolysis. This may be due to the chemical
nature of PETIM dendrimer. Hemolytic toxicity of PAMAM den-
drimer is reported due to interaction of positively charge NH2 group
of dendrimer with negatively charged surface of red blood cells.
Fig. 4. Hemolysis induced by PETIM dendrimer at different concentrations incubate at 37 ꢀ 1.0 ꢁC for 1 h. Mean ꢀ SD (n ¼ 3).

5002
S. Jain et al. / European Journal of Medicinal Chemistry 45 (2010) 4997e5005
140
120
100
80
a
a
a
a
a
60
40
20
0
0.1
1
10
100
1000
Concentration (µg/ml)
PETIM PAMAM
Fig. 5. Comparative cytotoxicity of the PETIM (G3eCOOH) Vs commercial available PAMAM (G4-OH) dendrimer in small lung cancer cell line (A-549) after 48 h of incubation.
formazan by reductive cleavage of the tetrazolium ring. Formazan
crystals, then can be dissolved in an organic solvent and quanti-
fied by measuring the absorbance of the solution at 550 nm, and
the resultant value is related to the number of living cells. Results
indicated that PETIM dendrimer are less toxic in comparison to
commercially available PAMAM dendrimer in comparable
The acute study of melamine dendrimer shows that 160 mg/kg
is a lethal dose and 100% mortality was observed 6e12 h after
i.p. injection [28].
3.6. Hematological toxicity evaluation
concentration range (from 1000 to 0.1
of 10ꢂ) (Fig. 5). PETIM dendrimer in concentrations range
1000 g/ml to 0.1 g/ml exhibited % cell viability as 110, 110, 113,
110 and 118%, respectively. PAMAM G4-OH dendrimer showed 95,
91, 82.2, 95 and 102% viability in comparative concentrations
range, respectively. Results of cytotoxicity study revealed the
significantly less toxicity of PETIM dendrimer in comparison to
commercial G4-OH dendrimer. Similarly, Roberts et al. [27]
showed G3 PAMAM dendrimer producing the 90% cell death at
7 ng/ml concentration.
mg/ml with dilution factor
Table 3 shows the results of hematological measurements at
dose 10, 20, 40 and 80 mg/kg of PETIM dendrimer. Dose was
selected based on the results of acute toxicity measurement in
which no mortality was observed to dose 80 mg/kg (Table 2).
PETIM dendrimer treated group didn’t show the significant
m
m
(p
< 0.05) difference in the hematological parameters as
compared to value of control group and value at 0 day for same
group of animals. At higher dose (80 mg/kg) haemoglobin
content was found to have significant difference. Similarly, RBC,
Platelet count and TLC value were also found to have significant
differences. These results are well correlated with % survival/%
changes in body weight results. Changes were observed only at
higher doses of 80 and 100 mg/kg of PETIM dendrimer. Den-
drimer formulation is known for causing the hematological
toxicity. Robert et al. [27] also reported the significant changes in
3.5. Acute and sub-acute toxicity study
Dendrimer as drug carrier should be non-toxic in the in vivo
conditions. However, many reports are available on in vitro
cellular toxicity assay but only few studies reported for evaluation
of chronic, acute and sub-acute toxicity of dendrimer. In the
present study acute and 28 days sub-acute toxicity study of PETIM
dendrimer was performed as per the OECD (The organization of
Economic Co-operation Development) guidelines [15]. Table 2
shows the results of acute and sub-acute toxicity measurement
at different doses i.e. 10, 20, 40, 80 and 100 mg/kg of PETIM
dendrimer. Animals were observed daily for body weight,
behavioral changes, food, water intake and mortality. No
mortality or significant changes in general behaviour or other
physiological activities were observed at any point in case of
animals treated with 10e40 mg/kg PETIM dendrimer. In case of
G3eCOOH 80 mg/kg, no mortality was there up to 21st day but
83.3% survival was observed on 28th day. In the higher dose of
100 mg/kg, no death was observed up to 14th day but 83.3% and
66% survival was observed on 21st and 28th day, respectively.
This mild toxicity is significantly (p < 0.05) less than the other
dendrimers reported in the literature that are currently being
considered as drug delivery vehicles [6]. Preliminary investigations
of PAMAM found that toxicity commenced at 45 mg/kg i.p. [27].
Table 2
Acute and sub-acute toxicity study of PETIM dendrimer.
Group
Treatment
Days after administration
% Survival/% Change in body weight
1 Day
100
0
100
0
100
0
100
0
100
0
100
0
7 Day
100
1.2
100
0
100
0
100
0
100
0
100
0
14 Day
100
1.8
100
1.5
100
1.0
100
1.6
100
1.6
100
3.1
21 Day
100
2.8
100
1.5
100
3.0
100
3.0
28 Day
100
3.1
100
3.0
1
2
3
4
5
6
Control
G3eCOOH (10 mg)
G3eCOOH (20 mg)
G3eCOOH (40 mg)
G3eCOOH (80 mg)
G3eCOOH (100 mg)
100
3.1
100
4.8
100
5.0
83.3a
7.5a
66a
83.3a
5.0
8.0a
The data obtained from various groups were statistically analyzed using one-way
ANOVA followed by Tukey’s Multiple Range Test. P < 0.05, is considered statistically
significant.
a
Data are represent of mean ꢀ SD (n ¼ 6). vs control.

S. Jain et al. / European Journal of Medicinal Chemistry 45 (2010) 4997e5005
5003
Table 3
Effect of PETIM dendrimer on hematological parameters of mice.
Parameters
Control Group
0 Day 28 Day
33 ꢀ 1.3
G3eCOOH 10 mg/kg
0 Day 28 day
33 ꢀ 0.8 32 ꢀ 0.5
G3eCOOH 20 mg/kg
0 Day 28 Day
28 ꢀ 1.2
6.7 ꢀ 3.1
G3eCOOH 40 mg/kg
G3eCOOH 80 mg/kg
0 Day 28 day
0 Day
28 day
Body weight
Hbc (gm/dl)
RBC Million/Cu.m.m
TLCd/Cu.m.m
DLCe
Polymorphs%
Lymphocytes%
Monocytes%
Eosinophils%
32 ꢀ 1.1
29 ꢀ 1.8
30 ꢀ 1.3
29 ꢀ 1.2
29 ꢀ 0.8 30 ꢀ 0.5
9.7 ꢀ 2.7 8.4 ꢀ 0.2
7.1 ꢀ 3.1 5.1 ꢀ 0.3b
10.6 ꢀ 0.4
6.1 ꢀ 1.5
10.0 ꢀ 1.2 10.5 ꢀ 1.1 9.9 ꢀ 0.1 10.8 ꢀ 2.7
10.2 ꢀ 0.7
6.0 ꢀ 2.5
9.5 ꢀ 0.52 9.3 ꢀ 0.8
5.2 ꢀ 0.16 5.2 ꢀ 0.2
5.9 ꢀ 0.9
6.7 ꢀ 2.1
5.1 ꢀ 0.4
7750 ꢀ 494 4800 ꢀ 524 6400 ꢀ 707 8250 ꢀ 494 8233 ꢀ 908 7400 ꢀ 900 6666 ꢀ 2214 7450 ꢀ 1767 6500 ꢀ 173 8400 ꢀ 2121a
32.5 ꢀ 10.6 31.2 ꢀ 4.0 36.5 ꢀ 2.1
39 ꢀ 2.0 34.7 ꢀ 8.4
56 ꢀ 2.7 58.3 ꢀ 10.4
49.3 ꢀ 7.5
46 ꢀ 14.4 46 ꢀ 5.6
45.0 ꢀ 13.2 52.5 ꢀ 28.9a,b
48.3 ꢀ 12.5 44.0 ꢀ 31
3.0 ꢀ 0.6 3.0 ꢀ 0.3
63 ꢀ 9.8
1.5 ꢀ 0.7
3 ꢀ 0.1
44 ꢀ 8.4 58.5 ꢀ 2.2
43 ꢀ 6.5 49.7 ꢀ 15
45.5 ꢀ 2.12
2
6.5 ꢀ 3.5
2 ꢀ 0.6
3 ꢀ 0.6
2.5 ꢀ 0.7
2.5 ꢀ 0.7
3 ꢀ 1.4
2 ꢀ 1.4
5.0 ꢀ 1.4
3.7 ꢀ 2.3
3.0 ꢀ 1.7
4.7 ꢀ 0.4
1.3 ꢀ 0.5
3 ꢀ 1
3.3 ꢀ 2.3 3.0 ꢀ 1.4
Platelet count Lakhs/Cu.m.m 13.6 ꢀ 2.2
12.6 ꢀ 1.3 12.1 ꢀ 0.5 16.6 ꢀ 0.8 15.6 ꢀ 4.1
16.6 ꢀ 5.0 13.6 ꢀ 9.06 15.02 ꢀ 7.2
14.2 ꢀ 6.0 18.2 ꢀ 1.1a
47.6 ꢀ 27.8 53.9 ꢀ 0.1
16.1 ꢀ 12.6 20.7 ꢀ 0.8
26.6 ꢀ 10.2 26.1 ꢀ 1.1
MCVf fl
38.9 ꢀ 12.3 50.9 ꢀ 8.0 46.6 ꢀ 6.6 62.1 ꢀ 7.6 40.6 ꢀ 6.9
61.9 ꢀ 8.2 48.3 ꢀ 1.9
45.6 ꢀ 4.9
11.7 ꢀ 2.0
31.2 ꢀ 1.1
MCHg pg
MCHCh g/dl
13.7 ꢀ 2.0
26.2 ꢀ 4.3
18.0 ꢀ 2.4 13.1 ꢀ 3.5 21.8 ꢀ 2.1 12.0 ꢀ 4.7
28.2 ꢀ 5.1 25.6 ꢀ 7.0 28.5 ꢀ 0.7 25.4 ꢀ 8.8
19.9 ꢀ 1.4
31.6 ꢀ 11
9.3 ꢀ 2.2
18.9 ꢀ 1.4
The data obtained from various groups were statistically analyzed using one-way ANOVA followed by Tukey’s Multiple Range Test. P < 0.05, is considered statistically
significant.
a
Data are represent of mean ꢀ SD (n ¼ 3). vs control group.
b
Data are represent of mean ꢀ SD (n ¼ 3). vs 0 day value.
c
Hemoglobin concentration.
Total leukocyte count.
Differential leukocyte count.
mean corpuscular volume.
mean corpuscular haemoglobin.
mean corpuscular haemoglobin concentration.
d
e
f
g
h
hematological parameters of mice after treatment with G5
PAMAM dendrimer.
were coupled with the histopathological study. Histopathological
study is useful, especially during the anatomical localization of
action of dendrimer and used to identify any alteration in gross
morphology to determine the toxic effect of dendrimer [28].
Fig. 6AeF shows the eosin-haematoxylin stained photomicro-
graphs of liver, kidney and heart of the control group and PETIM
treated group at dose (80 mg/kg). There were no changes in normal
histopathology of organs observed during dosing period with
PETIM dendrimer. Photomicrographs of the liver of control group
and G3eCOOH treated group are comparable. Photomicrograph of
the control group shows normal tabular architecture (Fig. 6A).
Section of PETIM dendrimer treated group also shows normal
tabular architecture of hepatocytes with a slight increase in the
number of congested blood vessels (Fig. 6B). Photomicrograph of
the kidney of the control group shows the presence of glomeruli
and tubular along with congested blood vessels (Fig. 6C). Section of
the treated group also shows the normal presence of glomeruli and
tubules (Fig. 6D). Similarly, no histopathological changes were
observed in the heart of control and treated group. Section of the
control group shows the presence of the normal myocardial bundle
Table 4 shows the effect of PETIM administration on different
biochemical parameters of mice blood. These biochemical param-
eters are the sign of systemic toxicity of carrier i.e. significant
decrease in cholesterol level showed severe hepatotoxicity, alter-
ation in SGPT and SGOT level also indicated the severe hepatotox-
icity, alteration in urea and total proteins level indicated the
nephrotoxicity or alteration in metabolic system and alteration in
triglycerides level indicated atherosclerosis. This test is often
carried out to determine the risk of developing heart diseases.
There was no significant difference (p < 0.05) in biochemical
parameters of treated groups compare to control group. At higher
dose (80 mg/kg) SGOT, cholesterol and urea value of the treated
group were found to have a significant difference with control
group.
Biosafety profile of PETIM dendrimer was also established by
performing the histopathology of vital organs liver, kidney
and heart after 28 days of administration. Functional studies
in toxicology along with biochemical and hematological studies
Table 4
Effect of PETIM dendrimer on biochemical parameters of mice.
Parameters
Control Group
0 Day
G3eCOOH 10 mg/kg
G3eCOOH 20 mg/kg
G3eCOOH 40 mg/kg
G3eCOOH 80 mg/kg
28 Day
0 Day
28 day
0 Day
28 Day
0 Day
28 day
0 Day
28 day
Glucose (mg/dl)
Cholesterol (mg/dl)
Urea (mg/dl)
SGPT (IU/l)
SGOT (IU/l)
Total proteins (g/dl)
ALP (IU/l)
Creatinine (mg/dl)
Bilirubin (mg/dl)
56.6 ꢀ 2.1
80.6 ꢀ 16
62.4 ꢀ 3.7
48.5 ꢀ 2.8
149.3 ꢀ 86
4.9 ꢀ 0.5
57.1 ꢀ 5.6
81.6 ꢀ 9.2
56.2 ꢀ 4.9
36.7 ꢀ 9.5
132.5 ꢀ 12
5.1 ꢀ 0.9
50.8 ꢀ 1.7
92.0 ꢀ 20
52.3 ꢀ 11
32.5 ꢀ 21
52.8 ꢀ 2.4
55.8 ꢀ 1.4
45.7 ꢀ 9.3
27.3 ꢀ 7.2
91.2 ꢀ 8.0
93.6 ꢀ 15
48.2 ꢀ 1.6
48.9 ꢀ 1.7
93.7 ꢀ 2.0
72.2 ꢀ 15
54.7 ꢀ 9.7
32.0 ꢀ 5.2
113.0 ꢀ 3.9
5.7 ꢀ 0.7
82.5 ꢀ 1.9
97.4 ꢀ 8.0
62.2 ꢀ 2.1
45.6 ꢀ 1.6
145.0 ꢀ 8.5
5.2 ꢀ 0.2
75.7 ꢀ 1.2
91.5 ꢀ 2.5
68.9 ꢀ 8.9
26.9 ꢀ 13
88.7 ꢀ 1.7
78.0 ꢀ 2.1
53.9 ꢀ 2.2
38.2 ꢀ 2.3
73.9 ꢀ 1.0a,b
56.1 ꢀ 1.4a,b
41.7 ꢀ 5.6a
48.6 ꢀ 6.4
127.6 ꢀ 23a
4.8 ꢀ 3.3
161.9 ꢀ 14 147.2 ꢀ 4.3 161.4 ꢀ 4.8
134.4 ꢀ 7.4 169.5 ꢀ 12
5.3 ꢀ 1.9
5.6 ꢀ 0.6
5.4 ꢀ 1.1
3.9 ꢀ 0.6
6.1 ꢀ 0.1
148 ꢀ 8.7
200.6 ꢀ 28
0.73 ꢀ 0.05
0.98 ꢀ 0.02
182.4 ꢀ 19
0.95 ꢀ 0.08
0.98 ꢀ 0.02
199.2 ꢀ 11 147.0 ꢀ 33 173.7 ꢀ 18.8 160.4 ꢀ 39
178.0 ꢀ 10.6 187.7 ꢀ 14
148.4 ꢀ 14
0.83 ꢀ 0.1
0.69 ꢀ 0.2
1.0 ꢀ 0.2
0.84 ꢀ 0.1
0.66 ꢀ 0.2
0.55 ꢀ 0.05
0.71 ꢀ 0.1
0.9 ꢀ 0.1
1.0 ꢀ 0.1
0.79 ꢀ 0.02 0.97 ꢀ 0.1
0.82 ꢀ 0.02 0.81 ꢀ 0.02
0.79 ꢀ 0.1
0.79 ꢀ 0.1
0.71 ꢀ 0.1
The data obtained from various groups were statistically analyzed using one-way ANOVA followed by Tukey’s Multiple Range Test. P < 0.05, is considered statistically
significant.
a
Data are represent of mean ꢀ SD (n ¼ 3). vs control group.
b
Data are represent of mean ꢀ SD (n ¼ 3). vs 0 day value.

5004
S. Jain et al. / European Journal of Medicinal Chemistry 45 (2010) 4997e5005
Fig. 6. Eosin-hematoxylin stained photomicrographs of liver, kidney and heart of control (A, C and E) and PETIM treated group (B, D and F) (magnification 450ꢂ) (Scale
bar ¼ 500 m).
m
(Fig. 6E). Section of PETIM treated group also shows the presence of
the normal myocardial bundle with increased congested blood
vessels (Fig. 6F).
dendrimer may be useful as a potentially safe drug delivery vehicle,
having sustained release kinetics.
Acknowledgements
Authors are grateful to Mr. Sundeep Arora, President, BioPlus
Life Science Pvt. Ltd., India for providing PETIM dendrimer as a gift
sample and financial assistance for carrying out this work.
4. Conclusion
Dendrimer as carrier system has many advantages over other
carrier systems like liposomes having the problems of stability and
cost, nanoparticle having the problems of manufacturing process
and batch to batch reproducibility. PETIM is a new development in
the field of dendrimer. Results of present study revealed that this
carrier system has good biocompatibility as determined by acute,
sub-acute and cytotoxicity measurement assay. Cytotoxicity assay
demonstrated significantly less cytotoxicity of PETIM dendrimer in
comparison to commercial PAMAM dendrimer. PETIM dendrimer
was also found to successfully encapsulate the model drug keto-
profen and in vitro drug release study showed its sustained release
potential. PETIM dendrimer seems to be an attractive approach for
sustained delivery of bioactive. Above studies showed that, PETIM
Appendix. Supplementary data
Supplementary data associated with this article can be found in
online version at doi:10.1016/j.ejmech.2010.08.006.
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