Ligand anchored dendrimers based nanoconstructs for effective targeting to cancer cells

Article abstract of DOI:10.1016/j.ijpharm.2010.04.002

Dendrimers are considered versatile carriers especially for the treatment of diseases like cancer, AIDS, malaria etc. Cancer is a worldwide threat particularly in developing countries. A breakthrough research in this regard is a prime requirement. In the present study, folic acid was conjugated to fifth generation polypropylene imine (PPI) dendrimers and characterized through IR, NMR (¹³C and ¹H), ESI mass spectroscopy as well as electron microscopic studies. Doxorubicin (DOX), an effective anticancer drug, was used in the present study to develop and explore the anticancer potential of the dendrimer based formulations. DOX was loaded (approximately 26 and 65%) to the PPI dendrimers as well as folate conjugated PPI (PPI-FA) dendrimers, respectively. These ligand conjugated dendrimers displayed very less (approximately 3 and 4%, respectively, for PPI-FA and PPI-FA-DOX) hemolysis. The developed formulation PPI-FA-DOX was stable enough. In vitro drug release of the formulation was found to be faster in the acidic media than at the higher pH. The prepared formulation displayed a higher cell uptake in MCF-7 cancer cell lines as evidenced by fluorescence studies. The results suggested that, in future, folic acid conjugated PPI dendrimers may emerge as a better choice for anticancer drug targeting.

Full text of DOI:10.1016/j.ijpharm.2010.04.002

International Journal of Pharmaceutics 393 (2010) 185–196
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical Nanotechnology
Ligand anchored dendrimers based nanoconstructs for effective targeting
to cancer cells
a
b
b
Umesh Gupta , Shailendra Kumar Dhar Dwivedi , Hemant Kumar Bid ,
b
a,∗
Rituraj Konwar , N.K. Jain
Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar 470003 (M.P.), India
Division of Endocrinology, Central Drug Research Institute, Chattar Manzil Palace, Lucknow 226001 (U.P.), India
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Dendrimers are considered versatile carriers especially for the treatment of diseases like cancer, AIDS,
malaria etc. Cancer is a worldwide threat particularly in developing countries. A breakthrough research
Received 13 January 2010
Received in revised form 31 March 2010
Accepted 1 April 2010
in this regard is a prime requirement. In the present study, folic acid was conjugated to fifth genera-
tion polypropylene imine (PPI) dendrimers and characterized through IR, NMR (13C and 1H), ESI mass
Available online 9 April 2010
spectroscopy as well as electron microscopic studies. Doxorubicin (DOX), an effective anticancer drug,
was used in the present study to develop and explore the anticancer potential of the dendrimer based
formulations. DOX was loaded (approximately 26 and 65%) to the PPI dendrimers as well as folate con-
jugated PPI (PPI–FA) dendrimers, respectively. These ligand conjugated dendrimers displayed very less
Keywords:
Dendrimers
Targeting
MCF-7
(approximately 3 and 4%, respectively, for PPI–FA and PPI–FA–DOX) hemolysis. The developed formu-
DOX
Nanocarriers
lation PPI–FA–DOX was stable enough. In vitro drug release of the formulation was found to be faster
in the acidic media than at the higher pH. The prepared formulation displayed a higher cell uptake in
MCF-7 cancer cell lines as evidenced by fluorescence studies. The results suggested that, in future, folic
acid conjugated PPI dendrimers may emerge as a better choice for anticancer drug targeting.
©
2010 Elsevier B.V. All rights reserved.
1
. Introduction
permeability are the key features of tumor (Carmeliet and Jain,
009; Jain, 1987). Complete cure of the cancer is still a chal-
2
Attempts to deliver drug to the desired site in the body are a
lenge to the biomedical research. Available anticancer drug delivery
approaches invariably attack healthy cells along with the can-
cerous cells, which is an important issue. Recent reports of the
last decade showed that nano-particulate carriers displayed higher
accumulation behavior at the tumor site. Higher accumulation of
the anticancer drugs can be achieved by enhanced permeation and
retention effect (EPR) and/or by avoiding reticulo-endothelial sys-
tem (RES) and/or through utilization of ligand mediated targeting.
Polymeric nanocarriers in this context are among one of the best
choices for this purpose. These include nanoparticles, dendrimers,
liposomes etc (Papahadjopoulos and Gabizon, 1990; Kommareddy
et al., 2005).
Dendrimers in the last decade have emerged as a promis-
ing nano-polymeric tool for the drug delivery and targeting.
Dendrimers are the mono-dispersed, three dimensional, highly
branched, macromolecular nanocarriers (1–100 nm). Higher drug
loading capacity, easy synthesis, stability, tailor made function-
ality, transdermal ability are the important advantages of the
dendrimers (Tomalia et al., 1985; Sevenson and Tomalia, 2005).
The carrier has been successfully used for the solubilization of
insoluble drugs, diagnosis, controlled and sustained as well as
transdermal drug delivery approaches and for other nonmedical
thrust area of biomedical research today. Nanotechnology based
drug delivery approaches have proven some exciting results in this
perspective (Brannon-Peppas and Blanchette, 2004; Farokhzad and
Langer, 2009; Gupta and Jain, 2010). Diseases like AIDS, diabetes,
tuberculosis and cancer are among the most disastrous diseases
worldwide today. A recent fact sheet of WHO reports that can-
cer is a leading cause of death worldwide. It accounted for 7.9
million deaths (around 13% of all deaths) in 2007 which is an
alarming figure. Among the different types of cancer lung, stom-
ach, liver, colon and breast cancers cause the most cancer deaths
each year. The projected death figure due to cancer was estimated
to be 12 million in 2030. Deaths due to cancer are more prevalent
in low and middle income countries (WHO). Tumor is a morbid
state of tissue growth, which encompasses its own morphogen-
esis and after proliferation termed as cancer. The unorganized
rapid growth and widened inter-endothelial junctions with higher
∗ Corresponding author. Tel.: +91 7582 264712; fax: +91 7582 264712.
E-mail addresses: umeshgupta175@gmail.com (U. Gupta), jnarendr@yahoo.co.in
N.K. Jain).
(
0
378-5173/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2010.04.002

1
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U. Gupta et al. / International Journal of Pharmaceutics 393 (2010) 185–196
◦
purposes (Chauhan et al., 2003; Satija et al., 2007). Tailor made
surface functionality of the dendrimers has been explored for tar-
geting of anticancer and anti-HIV drugs etc (McCarthy et al., 2005;
Agarwal et al., 2009). Polyamidoamine (PAMAM) and polypropy-
lene imine (PPI) dendrimers are among the most studied classes of
dendrimers especially in the drug delivery. Surface primary amine
group of dendrimers has been successfully conjugated with the
ligands like galactose and mannose for liver targeting and other
potential body areas in the earlier studies (Bhadra et al., 2003,
rise to 38 ± 2 C. After this exothermic effect the reaction mixture
◦
was heated at 80 C for 1 h to complete the addition reaction. The
excess of acrylonitrile was removed as a water azeotrope by vac-
uum distillation (16 mbar, bath temperature 40 C; Superfit, India).
◦
Obtained crystalline solid was 0.5 G PPI dendrimer. Conversion of
half to full generation dendrimers (1.0 G) was carried out in a lab-
oratory scale catalytic hydogenator (Superfit, India). The reaction
was repeated in the same sequence up to fifth generation (5.0 G). PPI
dendrimers were characterized at each step using different spectro-
scopic techniques like IR, 1H NMR as well as transmission electron
microscopy (TEM). IR spectroscopy of dendrimers was carried out
using KBr pellet method after adsorption of smaller amount of den-
drimer on KBr pellets in PerkinElmer IR spectroscope. IR analysis of
5.0 G PPI dendrimers revealed peaks at C–N stretching of CH –NH
(1118.3 cm ), C–H stretching (2957.5 cm , 2824.2 cm ), N–H
bending vibrations of amine (1597 cm ) and, most importantly,
N–H stretching of primary amine (3396.5 cm ). H NMR spectro-
scopic studies of dendrimer samples were carried out at 300 MHz,
after dissolving in D O (Bruker DRX 300 MHz, USA). Important
chemical shifts obtained were between 0.6 and 1.25 ppm (alkane);
1.3 and 1.9 ppm (secondary alkane); 2.3 and 2.9 ppm (alkyl amines);
7.933 ppm (primary amines). TEM was performed after drying den-
drimeric sample on 3 mM forman (0.5% plastic powder in amyl
acetate)-coated copper grid (300 mesh) at 60 kV (Morgani, 268D;
Holland) after staining negatively using uranyl acetate (4%) and
photomicrographs were taken at suitable magnifications (Morgani,
268D; Holland). This analysis was mainly performed to confirm the
size of the synthesized dendrimers.
2
005).
Ligand mediated active targeting of the anticancer drugs
through dendrimers have proven well in the past studies. How-
ever, studies based on PPI dendrimers in this regard as compared
to PAMAM dendrimers are scarce. Earlier reports suggest that the
encapsulation capacity of the PPI dendrimers is comparable to the
PAMAM dendrimers (Richter-Egger et al., 2001). Cationic toxicity is
still an important issue, which creates hurdle in the possible drug
delivery potential of the dendrimers in vivo (Malik et al., 2000).
Surface engineering of the dendrimers can be a solution in this
regard, and if we engineer the surface of dendrimers with some
biocompatible anticancer ligand it would be a beneficial approach,
both in terms of targeting and reduced toxicity. In the present
study, we report design, synthesis and characterization of folic acid
conjugated PPI dendrimers encapsulated with DOX, an effective
anticancer drug, and these conjugated dendrimers were assessed
for their anticancer potential in vitro using human mammary can-
cer MCF-7 cells. It was envisaged that the folic acid conjugation will
shield the cationic charge associated toxicity of the dendrimers in
vivo in general and additionally will target the dendrimeric conju-
gates to the tumor, specifically.
2
2
−
1
−1
−1
−
1
−
1
1
2
2
.3. Folic acid conjugation
2
. Material and methods
Folic acid conjugation was achieved using NHS activated folic
acid and its direct reaction with the PPI dendrimers.
2.1. Materials
2
.3.1. Preparation and characterization of NHS ester of folic acid
Active ester of folic acid was synthesized using slightly modi-
Raney Nickel was purchased from Fluka (USA), while ethy-
lene diamine (EDA) and acrylonitrile were purchased from CDH
India). DOX was obtained as a generous gift from Dabur India Ltd.
Nylon membrane filter (0.45 ␮m) was obtained from Pall Gelman
Sciences, USA. Folic acid, NHS (N-hydroxy succinimide), DCC (N,N-
dicyclohexyl carbodiimide), and DMSO (dimethylsulfoxide) were
purchased from Hi Media (India). MTT [3-(4,5-dimethylthiazol-
fied method reported earlier (Lee and Low, 1994). Briefly, folic acid
1 g) was dissolved in 50 ml of dimethylsulfoxide (DMSO) along
(
(
with 1.1 molar excess of NHS; 1.1 molar excess of DCC was then
added, and the reaction mixture was stirred overnight in dark at
room temperature. The insoluble byproduct, dicyclohexylurea, was
removed by filtration through glass wool (Scheme 1). The filtrate
2
-yl)-2,5-diphenyl tetrazolium bromide] and FITC (fluorocein
containing DMSO solution of the NHS–folate product was stored at
isothiocynate) were purchased from Sigma (Germany). All the other
reagents and chemicals were of analytical grade and were used as
received.
◦
−
20 C until its use in further synthesis. FA–NHS was characterized
through NMR (both C and 1H), IR and ESI mass spectrosocopy.
13
In IR spectroscopic analysis FA–NHS samples were analyzed by
the KBr pellet method in a PerkinElmer 783 IR spectrophotome-
ter. Important peaks obtained in the IR spectra of folic acid were
at 3543.5, 3420.6 cm (N–H stretch of primary amine and amide);
3
2
.2. Synthesis and characterization of EDA core PPI dendrimers
up to 5.0 G
−
1
−
1
−
1
324.9 cm (alkyl C–H and C C stretch); 1696.0 cm (aromatic
−
1
C
C bending and stretching); 1485.3 cm (CH–NH–C O amides
Synthesis of the EDA core PPI dendrimers was according to
−
1
bending); 838.1 cm
disubstitution); and for the NHS conjugated folic acid 3761.0 cm
(aromatic C–H bending and benzene 1,4-
the classical report by Meijer and coworkers in 1993 with some
modifications (Brabander-van den Berg and Meijer, 1993). Mei-
jer and coworkers have used diaminobutane core in dendrimer
but in the present study EDA was selected as dendrimer core
for the synthesis of PPI dendrimers (Brabander-van den Berg and
Meijer, 1993). The detailed synthesis and characterization of PPI
dendrimers has been reported in our previous study (Gupta et al.,
−
1
−
1
(amide N–H and C O stretching); 3446.0 cm (primary aliphatic
−
1
amine N–H stretching); 2927.1 cm
(carboxylic acid C O and
−
1
O–H stretching unconjugated); 1631.8 cm (ketones C O uncon-
jugated stretch); all of which confirmed the synthesis of NHS ester
of folic acid. Further, FA–NHS ester was also characterized with
the H NMR and C NMR. 1H NMR (D O, Bruker DRX300, Ger-
1
13
2
007). Briefly, EDA core PPI dendrimers up to 5.0 G were synthe-
2
many) obtained at 400.1300184 MHz, d 6.99 (d, C C), d 1.9–2.0
sized using repetition of double Michael addition of acrylonitrile
to primary amines, followed by heterogeneously catalyzed hydro-
genation of the nitriles, resulting in double number of primary
amines. Acrylonitrile (8.35 mol, 443 g; 2.5 molar times per NH2)
was added to a solution of ethylenediamine (1.67 mol, 100.34 g) in
(
C
m, CH
C, benzene ring). C NMR revealed important shifts at 166.43
CH COO, conjugated portion of the NHS to folic acid); 169.16
C O ), d 2.4 (t, CH CH , conjugated NHS ring), d 7.6 (d,
13
(
(
2
NC O; NHS ring); 174.16 (HO
C C; folic acid ring); 148 (CH N ;
folic acid ring).
1
.176 kg water. The exothermic reaction caused the temperature to

U. Gupta et al. / International Journal of Pharmaceutics 393 (2010) 185–196
187
Scheme 1. Synthesis of NHS ester of folic acid (intermediate conjugate).
2
.3.2. Conjugation of NHS–folic acid ester to EDA core PPI
dendrimers
The active ester of folic acid in DMSO (25 mg/ml) was mixed with
were recorded on a Micromass Quattro II Triple Quadrupole mass
spectrometer. The samples (dissolved in suitable solvents such
as methanol/acetonitrile/water) were introduced into ESI source
through a syringe pump at 5 ␮l/min. The ESI capillary was set at
3.5 kV and the cone voltage was 40 V. Spectra were collected in 6 s
scans, and the printouts were averaged spectra of 6–8 scans. Spectra
recorded at high mass units were computer deconvoluted. Trans-
mission electron microscopic studies were performed similar to
the procedure explained for the characterization of dendrimers. All
other spectroscopic studies were performed as explained in earlier
section.
EDA core PPI dendrimers in DMSO (10 mg/ml) and stirred for 5 days
at room temperature (20–30 C) in a metabolic shaker (Superfit,
◦
India). Acetone was added to the reaction mixture, and a yellow
precipitate thus formed was collected by filtration and dried under
vacuum. The crude product was dissolved in deionized water, and
the pH was adjusted to 8–10 using 1 N NaOH solution. The solution
was purified using Sephadex G-25 column and deionized water as
effluent. Fractions containing the conjugate were collected, and the
pH of the solution was adjusted to 3 using 1 N HCl (Kono et al., 1999).
The product was precipitated out, collected by filtration, and dried
under vacuum (Scheme 2).
2.5. Drug loading and in vitro release studies
DOX was loaded in the PPI dendrimers through equilibrium dial-
2
.4. Characterization of folic acid conjugates
ysis method as reported earlier with slight modifications (Singhai
et al., 1997; Agarwal et al., 2009). Briefly, DOX was dissolved
in acetone (10 mg/ml) and to this solution approximately 1.4 ml
aqueous triethylamine (TEA) was added in a molar ratio of 2:1
(DOX:TEA). The solution was stirred overnight and was mixed
with the aqueous solution of PPI dendrimers (25 mg/ml). The mix-
ture was magnetically stirred (Remi, India) (50 rpm) for 72 h using
teflon bead and subsequently dialysed through dialysis membrane
(MWCO 3000; Sigma, Germany) for 15 min to remove the unen-
trapped drug under strict sink conditions (sink condition ensures
the complete dissolution of the drug it refers to the excess sol-
ubilizing capacity of the dissolution medium), which was then
Folic acid conjugated PPI dendrimers (PPI–FA) were charac-
terized using different spectroscopic techniques like IR, NMR
1
3
1
(
C and H) and ESI mass spectroscopy. IR spectrum of the
−
1
PPI–FA revealed important peaks at 771.61 cm
bending); 1221.9 cm
1
to attachment of folic acid). H NMR of PPI–FA displayed newer
shift of R–(C O)–NH–CH –CH3 of folic acid linkage appeared
(aromatic C
H
−
1
(ester unconjugated C O stretching) at
−
1
262.5 cm ; 1653.3 (aromatic C C bending and stretching due
1
2
at ı 1.5–2.5 ppm. Synthesis of the folic acid conjugated den-
drimers was further confirmed by 13C NMR. ESI mass spectra

1
88
U. Gupta et al. / International Journal of Pharmaceutics 393 (2010) 185–196
Scheme 2. Schematic synthesis of PPI–FA.

U. Gupta et al. / International Journal of Pharmaceutics 393 (2010) 185–196
189
estimated spectrophotometrically at 490 nm (UV-1601, Shimadzu,
Japan) to determine indirectly the amount of drug loaded within
the system. Similar procedure was followed subsequently for drug
loading to PPI–FA conjugates (aqueous solution of PPI–FA was pre-
pared in a concentration of 25 mg/ml equivalent of PPI dendrimers).
Finally, two different formulations were prepared i.e. PPI–DOX
control. The percent hemolysis was calculated for each sample by
taking the absorbance of water as 100% hemolytic sample, using
the following equation:
AB
S
% Hemolysis = AB
× 100
100
(
DOX encapsulated PPI dendrimers) and PPI–FA–DOX (DOX loaded
where ABS is the absorbance for the sample and AB100 is the
absorbance for control.
folate conjugated PPI dendrimers).
Both PPI–FA as well as PPI–FA–DOX formulations were sub-
jected to in vitro release studies using dialysis tube diffusion
technique in phosphate buffer saline (pH 7.4, 6.4) as well as in
double distilled water (DDW) as recipient medium. Two-milliliter
solution of the above-prepared formulation was placed in the
dialysis sac (MWCO 6000–7000 Da, Sigma), hermetically tied and
immediately suspended in 50 ml of aqueous recipient medium
under perfect sink conditions. The volume of recipient compart-
ment was maintained by replenishing with 1 ml of sink solution,
after each withdrawal of 1 ml aliquot. The same procedure was
followed for both the recipient media.
Similar procedure was followed to determine the hemolytic tox-
icity for the PPI–FA, PPI–DOX and PPI–FA–DOX.
2.7. Hematological studies
Hematological parameters were analyzedin male albinorats(SD
strain) of uniform weight and size. Fifteen animals were selected
and divided into five groups consisting of three rats in each group.
Plain drug (DOX), PPI–FA, PPI–DOX and PPI–FA–DOX formulations
were administered, containing 250 mg/ml equivalent DOX (wher-
ever required), intravenously into first, second, third and fourth
groups of animals, respectively, daily up to 7 days. The fourth group
was kept as control, which was maintained on same regular diet for
2.6. Hemolytic toxicity
7
days. After 15 days blood samples were collected from the ani-
mals and analyzed for RBC count, WBC count and differential count
of monocytes, lymphocytes and neutrophils at a pathology labora-
tory (Agarwal et al., 2009). This study was performed only on the
DOX and both the formulations.
Hemolytic study was performed with a view to monitor reduc-
tion in toxicity of PPI dendrimers due to folic acid conjugation.
The study was performed according to previous reported studies
(
Bhadra et al., 2003; Singhai et al., 1997). Whole human blood was
collected using heparin as anticoagulant in HiAnticlot blood collect-
ing vials (Hi Media, India) and centrifuged at 3000 rpm for 15 min
in an ultracentrifuge (Z36HK, HERMLE LaborTchnik GmbH, Ger-
many). RBCs collected from the bottom were washed with normal
2.8. Stability studies
These studies were performed to assess the stability aspects
of the developed formulations in terms of color change, drug
leakage etc. It was anticipated that the conjugated formulation
(PPI–FA–DOX) would release the drug slowly compared to plain
dendrimer formulation (PPI–DOX) (Agarwal et al., 2009; Singh et
al., 2008). The samples (5 ml) of PPI–FA–DOX formulations were
TM
saline (0.9%, w/v) in reverse osmosis water (Ultra Max 370 Series,
Younglin, Korea) until a clear, colorless supernatant was obtained
above the cell mass. Cells were re-suspended in normal saline.
This RBC suspension was used further for hemolytic studies. To
1
ml of RBC suspension, 5 ml distilled water was added, which was
◦
considered to be 100% hemolytic. Similarly, 5 ml of normal saline
was added to 1 ml of RBC suspension in another tube assumed to
produce no hemolysis, hence acting as control. Half milliliter of for-
mulation was added to the mixture of normal saline (4.5 ml) and
RBC suspension (1 ml). Similarly, 0.5 ml of drug solution and 0.5 ml
of dendrimer solution were taken in separate tubes and mixed with
kept in dark in amber colored vials and in colorless vials at 0 C,
◦
◦
ambient room temperature (RT) (20–30 C) and 60 ± 2 C in con-
trolled ovens for a period of 5 weeks and analyzed every week
for any visual changes like precipitation, turbidity, crystallization,
color, consistency and drug leakage. Drug leakage was determined
by monitoring the release of drug from the formulation after stor-
◦
4
.5 ml of normal saline and 1 ml of RBC. Drug (i.e. DOX) and PPI
age at accelerated condition (60 ± 2 C). Formulation samples (2 ml)
dendrimer in separate tubes were taken in such amount that the
resultant final concentrations of drug and dendrimer were equiva-
lent in all the cases. The tubes were allowed to stand for half an hour
with gentle intermittent shaking and were centrifuged for 15 min
at 3000 rpm in an ultracentrifuge (Z36HK, HERMLE LaborTchnik
GmbH, Germany). The supernatants were taken and diluted with
an equal volume of normal saline, and absorbance was taken at ꢀmax
were dialysed across cellulose tubing. The external medium (50 ml)
was analyzed for the drug content, spectrophotometrically. The
procedure was repeated weekly for up to 5 weeks.
2.9. Cancer cell inhibition assay
MCF-7, an estrogen receptor positive human breast cancer
cell line derived from pleural effusion, the most commonly used
5
40 nm, against supernatant of normal saline diluted similarly, as
Fig. 1. Transmission electron micrograph of (A) PPI dendrimers, where bar shows 200 nm, and (B) folic acid conjugated PPI dendrimers, where bar shows 1 ␮m.

1
90
U. Gupta et al. / International Journal of Pharmaceutics 393 (2010) 185–196
Fig. 2. Infra red spectra of (A) FA, (B) FA–NHS and (C) PPI–FA.
cell line for screening of anticancer breast agents (Soule et al.,
973), was used for evaluation of comparative anticancer activity
200 ␮L DMEM, supplemented with 10% FBS and incubated for 24 h
at 37 C in CO2 incubator. Formulations (PPI, PPI–FA, PPI–FA–DOX,
◦
1
of the prepared formulations. The cytotoxic activity of the com-
PPI–DOX) and DOX alone were diluted to the various concen-
trations in culture medium and added to the wells along with
vehicle treated wells as control group. After 18 h of incubation,
pounds was determined using MTT assay (Sashidharan et al., 2007).
4
1
× 10 cells/well were seeded in 96-well micro culture plates in

U. Gupta et al. / International Journal of Pharmaceutics 393 (2010) 185–196
191
Fig. 3. 1H NMR spectra of (A) FA–NHS and (B) PPI–FA.
1
0 ␮l MTT (5 mg/ml) was added to each well and the plates were
itatively analyzed under inverted fluorescent microscope (Leica,
Germany).
further incubated for 4 h. Then the supernatant from each well
was carefully removed, formazon crystals were dissolved in 100 ␮l
of DMSO and absorbance at 540 nm wavelengths were recorded
2.11. Statistical analysis
(
Molecular Devices, USA). The percentage cell growth inhibitions
for each treatment were calculated by comparing with vehicle
treated group.
All the statistical analyses were performed with Graph Pad Instat
Software (Version 3.0, Graph Pad Software, CA, USA) using either
unpaired t test or one-way ANOVA followed by Tukey–Kramer
multiple comparison test. Difference p < 0.05 was considered as
extremely significant difference.
2.10. Cell uptake assay
The cellular uptake of DOX conjugates was determined with
the following procedure. Briefly, each formulation was labeled
3. Results and discussion
(
(
PPI, PPI–DOX, PPI–FA–DOX, DOX) in 5:1 ratio with FITC solution
10 mg/ml in DMSO). The suspension was incubated at RT for 8 h
3.1. Synthesis and characterization of the EDA core PPI
dendrimers up to 5.0 G
5
with intermittent mixing every 20 min. 2.5 × 10 cells/well were
◦
seeded in six-well plate and incubated for 24 h at 37 C with 5%
CO , and then the medium in each well was replaced with 2 ml
Dendrimer synthesis was performed as reported earlier by De
Brabander and Meijer with some modifications (Brabander-van den
Berg and Meijer, 1993). EDA was used as dendrimer core. Synthesis
was monitored at every step (or generation) up to fifth generation
through the spectroscopic methods. IR and 1H NMR spectroscopy
confirmed the synthesis of PPI dendrimers. The important peaks as
2
of phenol red-free, serum-free, and antibiotic-free medium con-
taining various concentrations of labeled formulations. The cells
were incubated for 4 h and then the medium was removed and cells
were washed three times with PBS. Then the fluorescence due to
uptake of fluorescent labeled formulation of suspension was qual-

1
92
U. Gupta et al. / International Journal of Pharmaceutics 393 (2010) 185–196
Fig. 4. 13C NMR spectra of (A) FA–NHS and (B) PPI–FA.
mentioned in Section 2 of the manuscript indicate that half gener-
of PPI dendrimers reported by Brabander-van den Berg and Meijer
(1993). Electron microscopy was carried out to further authenti-
cate the size of dendrimes (nanometric range or otherwise?). It
was found that the size of the dendrimers was in the nanomet-
ric range and TEM photograph displayed aggregates (Fig. 1A) of the
dendrimers, which was similar to the earlier reports (Gupta et al.,
2007; Bhadra et al., 2005). All these parameters confirmed the syn-
thesis of PPI dendrimers. The dendrimers synthesized were in the
−
1
ation dendrimers displayed strong peak of –CN (1100–1200 cm
)
terminals, while the full generation dendrimers displayed strong
−1
1
peak of –NH2 (3300–3400 cm ). H NMR of the different gener-
ations also supported the synthesis of PPI dendrimers as evident
from the mentioned chemical shift. The detailed synthesis was
reported in our earlier study (Gupta et al., 2007). Results of all the
spectroscopic data were similar and comparable to the synthesis
Fig. 5. ESI mass spectra of PPI–FA.

U. Gupta et al. / International Journal of Pharmaceutics 393 (2010) 185–196
193
3.3. Drug loading, entrapment efficiency and formulation
development
The drug carrying capacity of the formulation was determined
through calculation of the quantity of drug loaded therein and
hence determining the moles of the drug entrapped per mole of the
dendrimer. Drug encapsulation was accomplished through equi-
librium dialysis method. UV–visible spectrophotometry (UV-1601,
Shimadzu, Japan) was used to confirm indirectly the drug load-
ing in PPI dendrimers as well as PPI–FA conjugates, which was
evident from a slight shift in the ꢀmax of DOX (495 nm) (Jansen
et al., 1994). Percent drug encapsulation was measured indirectly
by estimating the un-entrapped drug spectrophotometrically dur-
ing equilibrium dialysis method. Percent DOX encapsulation in
both i.e. PPI–DOX and PPI–FA–DOX was found to be 26 ± 0.7 and
6
4.78 ± 1.3%(datanot reported), respectively. Thehigherdrug load-
ing in case of PPI–FA–DOX conjugate was possibly due to increased
dendritic architecture of folic acid conjugated dendrimers (Agrawal
et al., 2007; Bhadra et al., 2003). Both these formulations i.e.
PPI–DOX and PPI–FA–DOX were selected for the subsequent stud-
ies.
Fig. 6. In vitro drug release from PPI–DOX formulation in double distilled water
(
(
DDW), PBS 6.4 and PBS 7.4 as recipient medium. Values represent mean ± S.D.
n = 6).
3.4. In vitro release kinetics
In vitro drug release studies were carried out in three different
media (i. e. DDW, PBS 7.4 and PBS 6.4) for both the formulations (i.e.
PPI–DOX, PPI–FA–DOX). Drug release from both the formulations
was relatively rapid initially followed by sustained and slow release
pattern in the later half of the experiment. A possible mechanism
of delayed release of the drug molecule may be the encapsulation
of the drug in the hydrophobic cavities of the dendrimer that act as
a sink to retain the drug molecules for extended duration than the
surface of dendrimer molecules. In case of PPI–DOX approximately
5
7.46± 1.4%, 59.46± 0.9% and 74.36 ± 1.04% drug was released,
respectively, in DDW, PBS 7.4 and PBS 6.4 within initial five-hour
5
4
h (Fig. 6). While in the case of PPI–FA–DOX, the drug release was
9.55 ± 0.88, 52.36 ± 1.44 and 68.24 ± 1.21, respectively, in DDW,
PBS 7.4 and PBS 6.4 (Fig. 7). It can be observed that the drug
release was slower and sustained in case of PPI–FA–DOX com-
pared to PPI–DOX. This may be possibly due to the steric hindrance
of the conjugated folic acid on the PPI dendrimer surface limit-
ing the release of drug from dendritic framework. In vitro release
studies at pH 6.4 were carried out in the view that the tumor
vicinity is relatively acidic in nature. The rapid release behavior
of the drug from dendrimeric formulations at pH 6.4 supports the
hypothesis that the dendrimeric formulation will release the drug
in higher concentration, whenever it will reach the target site i.e.
tumor, as the pH in such environment is generally below 7. The
results of the in vitro release as well as drug encapsulation stud-
ies indicate a higher drug loading at the same time delayed release
pattern due to folate conjugation (Singh et al., 2008), our target of
higher drug loading and longer time release pattern is thus accom-
plished.
Fig. 7. In vitro drug release from PPI–FA–DOX formulation in double distilled water
(
(
DDW), PBS 6.4 and PBS 7.4 as recipient medium. Values represent mean ± S.D.
n = 6).
physical state of honey like consistency with dark brownish color
having high viscosity.
3.2. Folic acid conjugation
Similar to the characterization of PPI dendrimers in our earlier
3.5. Hemolytic (ex vivo) and hematological (in vivo) studies
study, folic acid conjugated dendrimer was characterized through
1
various spectroscopic studies (Singh et al., 2008). IR (Fig. 2),
H
Hemolytic toxicity associated with the cationic charge of the
dendrimers poses a hurdle in its usage as drug delivery carrier.
Polycationic charge on the dendrimer surfaces due to primary
amine groups limits their possible applications in vivo as a drug
carrier (Malik et al., 2000). The hemolytic toxicity in the present
study was measured in terms of percent RBC hemolysis. It was
found that free drug DOX exhibited 16.37 ± 0.88%, while PPI and
PPI–DOX displayed 14.38 ± 0.25%, and 18.35 ± 0.63% RBC hemoly-
sis, respectively. Surprisingly the folic acid conjugation on the PPI
dendrimer surfaces drastically reduced the RBC hemolysis. PPI–FA
1
3
NMR (Fig. 3) and C NMR confirmed (Fig. 4) the synthesis of
PPI–folic acid conjugate synthesis. ESI mass spectra of the con-
jugated PPI dendrimers revealed the mass of 11,850 (Fig. 5). The
mass of 5.0 G PPI dendrimers as reported earlier was approxi-
mately 7140. Increase in mass further confirmed the conjugation
of folic acid molecules (about 5–6 folic acid molecules per den-
drimer molecule). Electron microscopy of the PPI–FA conjugate
clearly demonstrated the increase in size which supports (Agarwal
et al., 2009) the conjugation as indirect evidence (Fig. 1B).

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U. Gupta et al. / International Journal of Pharmaceutics 393 (2010) 185–196
Fig. 8. Percent hemolytic toxicity of drug, PPI dendrimers and different formulations
n = 4). Values represent mean ± S.D.
(
and PPI–FA–DOX displayed only 3.19 ± 0.08 and 4.28 ± 0.03% of
RBC hemolysis, respectively, which is about 4 and 3.5 times lesser
than PPI–DOX and PPI dendrimers (Fig. 8). The results clearly
indicate that the folic acid conjugates at the surface of the den-
drimers interferes with the RBC’s biocompatibly, which might be
responsible for the reduced RBC hemolysis. The results are also
similar to the previous reports of surface conjugated dendrimers
Fig. 9. Percent drug leakage from PPI–FA–DOX in conditions of temperature and
light up to 5 weeks.
(
Agarwal et al., 2009; Agrawal et al., 2007). Hemolytic toxicity
of PPI–FA–DOX was approximately 3, 4 and 5 folds less then
PPI, pure DOX and PPI–DOX, respectively. The reduction in the
toxicity was possibly due to shielding/locking of DOX and pri-
mary amine groups in the dendritic architecture due to surface
conjugation with folic acid, making its surface more biocompat-
ible. The dramatic reduction in the hemolytic toxicity gives a
possibility that these may be used in future as a tool for drug deliv-
ery.
Hematological parameters were determined to study the effect
of formulations on the different components of blood. The study
was conducted to analyze the toxic manifestations of the prepared
formulations on the other blood components like WBC, monocytes
etc. Blood samples were analyzed for RBC, WBC and differen-
tial lymphocyte counts at a pathology laboratory (Table 1). It is
clear from the table that RBC count was almost similar for con-
trol group and PPI–FA–DOX receiving animals. However, the RBC
count decreased below normal (i.e. of control group) in case of
DOX and PPI–DOX. Similarly, the WBC count of PPI–DOX increased
significantly (p < 0.05) compared to normal values. In the case of
PPI–FA–DOX, the WBC count increased but to a lesser extent than
for PPI–DOX. Similarly, a relative increase in lymphocyte count
was observed with PPI–DOX than PPI–FA–DOX. The results clearly
Fig. 10. Cell growth inhibition assay in MCF-7 cells treated with various formula-
tions.
indicate the superior biocompatible behavior of the folate conju-
gated than the plain dendrimers. The results were similar and in
accordance with the earlier hematological studies carried out with
nanoparticles and dendrimers (Agrawal et al., 2007). The study was
performed on PPI–DOX and PPI–FA–DOX formulations exclusively
with the objective of exploring their anti-tumor activity in vitro as
well as in vivo.
Table 1
Haematological parameters of dendrimer formulations/drug.
6
3
3
Formulations
RBC count (×10 /␮L)
WBC count (×10 /␮L)
Differential count (×10 ␮L)
Monocytes
Lymphocytes
Neutrophils
Control
DOX
PPI–DOX
PPI–FA–DOX
8.9 ± 0.4
10.2 ± 0.4
1.3 ± 0.6
7.9 ± 0.4
1.6 ± 0.3
a
a1
a2
a3
a4
7.6 ± 0.3
11.6 ± 0.4
1.1 ± 0.3
1.8 ± 0.5
1.2 ± 0.3
9.7 ± 0.4
1.9 ± 0.3
3.1 ± 0.3
3.1 ± 0.5
b
b1
b2
c2
b3
b4
c4
7.1 ± 0.4
17.4 ± 0.3
14.3 ± 0.3
c
c1
c3
8.7 ± 0.3
11.6 ± 0.3
8.6 ± 0.8
n = 6 albino rats per group, tabulated values represent mean ± S.D.
a,a1,a2,a3,a4
P < 0.05 (comparison of DOX with PPI–DOX).
P < 0.05 (comparison of DOX with PPI–FA–DOX).
b,b1,b2,b3,b4
c,c1,c2,c3,c4
P < 0.05 (comparison of PPI–DOX with PPI–FA–DOX).

U. Gupta et al. / International Journal of Pharmaceutics 393 (2010) 185–196
195
Fig. 11. MCF-7 cells (40×) treated with FITC-labeled formulations for 4 h showing representative cellular uptake. (A) PPI–DOX, (B) PPI–FA–DOX, (C) PPI–FA, (D) DOX alone.
3.6. Stability studies
3.8. Cell uptake assay/fluorescence technique
Stability studies are prerequisite in the development and char-
Similar to the results of the percent cell growth inhibition
assay fluorescence study also showed higher uptake of PPI–FA–DOX
(Fig. 11). The higher uptake was possibly due to the folate residues
on the surface of the dendrimer compared to PPI–DOX (p < 0.005).
Among the free DOX and PPI–DOX the higher uptake was observed
with the PPI–DOX, which might be due to higher drug loading
resulting in a higher release. The results again support the strategy
that the ligand mediated anticancer delivery can result in higher
uptake of drug to the MCF-7 cells. The study to develop any possible
in vitro–in vivo correlation is in progress.
acterization of a successful pharmaceutical formulation. In the
present study, stability was assessed in terms of turbidity, color
change, crystallization and other visual changes (Fig. 9). Another
important parameter, which was measured to assess the stability,
was drug leakage. The drug leakage was measured for the formula-
◦
tion PPI–FA–DOX, at various conditions of temperature [0 C, room
◦
◦
temperature (20–30 C) and 60 C] after storage in dark (amber
color glass vials) as well as light (colorless vials) for a period of
5
weeks. PPI–FA–DOX was most stable in dark, at RT (Fig. 9). In
general, drug leakage was higher in light than in dark, which may
be attributed to structure cleavage due to temperature and light.
Percent drug leakage was found to be least at RT compared to at
4
. Conclusion
◦
◦
0
and 60 ± 2 C. At 0 C shrinking of dendrimeric architecture may
The major hurdle with the dendrimer applicability in the drug
be the possible reason leading to decreased cavity enclosing drug
molecules and hence higher leakage. However, the greater leak-
delivery is their inherent toxicity due to cationic surface. Surface
engineering of dendrimers can lead to improved properties, espe-
cially in the context of biomedical applications. The shielding of the
surface charge, however, drastically reduces hemolytic behavior.
The present study was aimed to develop and characterize a ligand
conjugated dendrimer based carrier for the delivery of DOX. The
prepared conjugate was further assessed to target DOX to MCF-7
cancer cell lines. The toxicity profile in terms of hematological as
well as hemolytic was ensured before its in vivo fate. Results clearly
indicate that the folic acid conjugation has drastically reduced the
toxicity. The in vivo result indicates that folic acid conjugation has
provided higher uptake and better targeting.
◦
age at 60 ± 2 C may be due to increased solution kinetics. It can be
inferred from the above results that folate conjugated dendrimer
◦
based formulation is more stable in dark at RT than at 0 and 60 ± 2 C
(
Bhadra et al., 2003, 2005).
3.7. Cancer cell inhibition assay
MCF-7 cells were treated with six equivalent doses ranging from
0.05 to 0.5 ␮g/ml. The result of the cell inhibition assay exhib-
ited significant differences. PPI–FA–DOX exhibited highest percent
cell growth inhibition compared to other formulations as well as
drug itself (Fig. 10). However, all three formulations showed dose-
dependent inhibition of MCF-7 cells. IC50 value of PPI–FA–DOX
was found to be 0.45 ␮g/ml while it was 0.7, 0.75 ␮g/ml, respec-
tively, for DOX and PPI–DOX. The IC50 values of PPI–FA and PPI
alone were undetectable up to the concentration of 0.9 ␮g/ml. The
higher uptake in the case of the PPI–FA–DOX was possibly due to
the ligand specific targeting of dendrimers due to surface conju-
gation of the folic acid. Human mammary carcinoma cells (MCF-7)
were selected for the analysis as it has been investigated several
times in a number of studies. MCF-7 over expresses folate recep-
tors and this was the reason we selected MCF-7 cell line for our
studies (Li et al., 2009).
Acknowledgements
The authors (Umesh Gupta and N.K. Jain) would like to acknowl-
edge Indian Council of Medical Research, New Delhi, India for
providing financial assistance to carry out the present experimental
studies. The authors are grateful to Central Drug Research Institute,
Lucknow, India, for extending facilities for cell line and fluorescence
studies as well as spectroscopic analysis of the synthesized con-
jugates. Additionally, authors would also like to acknowledge M/s
Dabur Research Foundation, New Delhi, India, for providing the gift
sample of DOX.

1
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U. Gupta et al. / International Journal of Pharmaceutics 393 (2010) 185–196
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