ARTICLE IN PRESS

Biomaterials 27 (2006) 5377–5390

www.elsevier.com/locate/biomaterials

Blood compatibility of novel water soluble hyperbranched

polyglycerol-based multivalent cationic polymers and their
interaction with DNA
Rajesh Kumar Kainthana, Muthiah Gnanamanib, Munia Gangulib, Tanay Ghoshb,
Donald E. Brooksa,c, Souvik Maitib,, Jayachandran N. Kizhakkedathua,
a
Centre for Blood Research and Department of Pathology and Lab Medicine, 2350 Health Sciences Mall, Life Science Centre,
University of British Columbia, Vancouver, BC, Canada V6T 1Z3
b
Institute of Genomics and Integrative Biology, CSIR, Mall Road, New Delhi 110 007, India
c
Department of Chemistry, 2350 Health Sciences Mall, Life Science Centre, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

Received 9 March 2006; accepted 29 June 2006

Available online 18 July 2006

Abstract

A novel class of hyperbranched polymers based on polyglycerol (PG) and poly(ethylene glycol) (PEG) are synthesized by
multibranching anionic ring opening polymerization. Multivalent cationic sites are added to these polymers by a post-amination and
quarternization reactions. Blood compatibility studies using these polymers at different concentrations showed insignificant effects on
complement activation, platelet activation, coagulation, erythrocyte aggregation and hemolysis compared to branched cationic
polyethyleneimine (PEI). The degree of quarternization does not have large influence on the blood compatibility of the new polymers.
Cytotoxicity of these polymers is significantly lower than that of PEI and is a function of quarternized nitrogen present in the polymer.
Also, these polymers bind DNA in the nanomolar range and are able to condense DNA to highly compact, stable, water soluble
nanoparticles in the range of 60–80 nm. Gel electrophoresis studies showed that they form electroneutral complexes with DNA around
N/P ratio 1 irrespective of the percentage of quarternization under the conditions studied.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Hyperbranched polymers; Cationic polymers; Blood-compatibility; Drug delivery; DNA; AFM

1. Introduction Biomaterial development combined with fundamental

Polymer-drug conjugates have gained increased atten-
tion as polymer therapeutics in recent years due to its
ability to improve the blood residence time, bioavailability,
reduce toxicities, and enhance the solubilities of parent
drugs [1–12]. However, progress in such therapies involving
cationic polymers depends heavily on better understanding
the electrostatic complexation of polymers with opposi-
tively charged drugs and their biological properties [5].
Also corresponding author. Tel.: +91 11 2766 6156;
fax: +91 11 2766 7471.
Corresponding author. Tel.: +1 604 822 7085;
fax: +1 604 822 7742.
E-mail addresses: souvik@igib.res.in (S. Maiti), jay@pathology.ubc.ca
(J.N. Kizhakkedathu).
studies of virus function and cellular processes will enable
the molecular level design of modular drug/gene delivery
systems [6]. The term modular systems indicates that drug/
gene is complexed with polymers or lipid formulations,
which consists of a vector backbone modified with
functional groups that mediate environmental interactions
to improve the efficiency. The vector backbone should
enable the formation of stable complexes with drugs/genes,
provide low cytotoxicity, blood compatibility and also
overcome different problems faced by the delivery systems
[6,7]. For example, following intravenous administration,
potential microcontainer drug delivery systems tend to be
removed from the circulation by phagocytes of the
reticuloendothelial system (RES) [8]. Phagocytosis by
elements of the RES in the liver is mediated by the

0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2006.06.021
 ARTICLE IN PRESS
5378 R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390
Scheme 1. Representation of multivalent hyperbranched cationic polymers.
adsorption of certain blood components on the particle
surface and has been shown that a hydrophilically
stabilized microcontainer can avoid phagocytosis and
achieve a prolonged circulation time [6–8]. Thus the
development of new generation of polymers with various
architectures, improved biocompatibility and different
binding affinities with biomolecules will further enhance
our fundamental understanding of such systems and could
potentially lead to new drug delivery systems.
Dendrimers and hyperbranched polymers hold con-
siderable promise as binding agents for drug delivery
including nucleic acid therapeutics due to their three-
dimensional shapes and availability of large number of
functional groups for suitable derivatizations [9,10,13,14].
The inherent flexibility and ease of synthesis make the
imperfectly branched hyperbranched polymers of particu-
lar interest. Conformational freedom, allowing a polymer
to take up an optimal structure for drug interaction,
appears to be a useful property in this regard. Frechet
et al. have detailed in a recent review that for many
biomedical applications perfect dendritic structures may
not be a prerequisite [14]. Hence, hyperbranched polymers
may prove to be an effective alternative to dendrimers
in areas where a precise structure is not necessary.
Another advantage they have is their biocompatibility
[9,15] as there are only a few synthetic polymers known to
be highly compatible with living cells.
Over the past several years we have been developing
PEG-based linear cationic polymers which were shown to
bind a variety of negatively charged molecules including
DNA [16–20]. It is very difficult to manipulate the
architecture or functionalities of such structures due to
the inherent nature of these copolymers. Thus in search of
new polymers, we have developed a novel class of
multivalent hyperbranched cationic polymers based on
hyperbranched polyglycerol (PG) (Scheme 1). In this paper
we report the synthesis, characterization and blood
compatibility studies of such polymers as well as their
interactions with nucleic acid. Studying the biological and
physiochemical properties of such systems will provide
valuable knowledge for future design of more sophisticated
systems.
2. Experimental section
2.1. Materials
All reagents were purchased from Aldrich and used as received except
the following. Glycidol (96%) was purified by vacuum distillation and
stored in a refrigerator (2–4 1C). 1,1,1-Tris(hydroxymethyl)propane
(Fluka) and potassium methylate solution (25 wt% in methanol, Fluka)
were used as such. Epoxide terminated poly(ethylene glycol) (PEG)
monomethyl ether was synthesized by reaction of MPEG (Mn-350) with
epichlorohydrin in presence of sodium hydroxide [21]. Molecular weights
and radii of gyration (Rg) were determined by GPC with a DAWN-EOS
 ARTICLE IN PRESS
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390
multiangle laser light scattering detector in aqueous 0.1 N NaNO3
solution; the details have been described earlier [22]. The dn=dc value
for polyglycerol was determined to be 0.12 cm3/g and was used for the
calculation of molecular weight of polymers. z-potential measurements
were performed in a Coulters Delsa 440SX Zeta Potential Analyzer
(Coulter Co. Instrument Co.) with a He–Ne solid-state laser operated at a
wavelength of 635 nm. The instrument was calibrated with an aqueous
suspension of polystyrene latex with a nominal mobility of 4 mm cm/V s
from Coulter (EMPSC7). Polymer solutions were prepared in 5 mM
sodium chloride solution at 1 mg/mL concentration.
2.2. Synthesis of hyperbranched copolymer of glycidol and PEG
(PG-PEG)
Polymerizations were carried out in a three-necked glass reactor
equipped with a mechanical stirrer and a syringe pump under argon
atmosphere. Tris(hydroxymethyl)propane (TMP, 120 mg) was stirred with
0.1 mL potassium methylate solution and the excess methanol was
removed under vacuum at 50 1C. Glycidol (6 mL) was added drop-wise
using the syringe pump over 10 h to the flask which is kept at 95 1C. The
ratio of glycidol to TMP corresponds to a degree of polymerization of 100.
After the addition of glycidol, the mixture was stirred for additional 2 h,
after which 20 mL MPEG-epoxide was added drop-wise over 12 h. The
mixture was stirred for an additional 3 h. The viscous polymer was
dissolved in methanol and passed though cation exchange resin (Amberlite
IRC-50) to remove potassium ions. Polymer was precipitated twice from
diethyl ether to remove unreacted PEG-epoxide and subsequently dried at
70 1C in vacuum. Yield was 85%.
1
HNMR analysis in d6-DMSO: d 3.2 (OCH3 groups from PEG),
3.25–3.8 (broad, peaks from glycidol and PEG main chain protons),
4.2–5.0 (broad, peaks from hydroxyl groups).
2.3. Amine-terminated PG-PEG (PG-PEG-amine)
PG-PEG (16 g) dissolved in 100 mL THF was reacted with methane
sulfonyl chloride (1.17 mL,
20% of OH groups) in presence of
triethylamine (3 mL) for 12 h. The salts were filtered off and polymer
was isolated by precipitation in ether. The dried polymer was dissolved in
100 mL dioxane to which 40 mL Tris(2-aminoethyl)amine was added and
refluxed for 24 h. Dioxane was removed by rotary evaporation. After
dissolving in minimum amount of methanol, the polymer was twice
precipitated in diethyl ether. The obtained polymer (15 g) was dissolved in
100 mL water and added to stirred solution of formic acid (90% w/w) and
formaldehyde (37% w/w) (15 mL each) at 0 1C. The reaction mixture was
refluxed overnight. The volatiles were removed in vacuum. Polymer was
extracted with dichloromethane after adjusting the pH of the aqueous
solution to 10 with sodium hydroxide. Finally the polymer was purified by
dialysis using regenerated cellulose acetate membrane (MWCO 1000).
Yield 10 g. The product was characterized by 1HNMR, GPC analysis and
conductometric titrations.
1
HNMR analysis: d6-DMSO.
PG-PEG mesylate: d 3.12 (methyl from mesylate), 3.2 (–OCH3 from
PEG), 3.25–3.8 (broad-peaks from glycidol and PEG main chain protons),
4.2–5.0 (broad, peaks from hydroxyl groups): Approximately 20% of the
hydroxyl groups have been converted to mesylate groups as calculated
from the NMR).
PG-PEG-Amine: d 2.08 (–N–CH3), 2.16 and 2.22 (N–CH2–), 3.20
(–OCH3, from PEG), 3.25–3.8 (broad, peaks from glycidol and PEG main
chain protons), 4.2–5.0 (broad, peaks from hydroxyl groups).
GPC analysis: Mn—116 700, Mw/Mn—1.72, Rg—24.5 nm.
2.4. Synthesis of quarternized amines
The tertiary amine groups in PG-PEG-Amine were quarternized using
ethyl bromide. Polymers with different degrees of quarternization were
synthesized by varying the amount of ethyl bromide used (Table 1). For a
5379
Table 1
Reaction conditions for quarternization of PG-PEG-Amine copolymer
Sample code Amount of PG-PEG Ethyl bromide
amine (g) (g)
PG-PEG-Amine-18 1.14 0.021
PG-PEG-Amine-44 1.28 0.075
PG-PEG-Amine-100 1.2 1.5
typical reaction, PG-PEG amine (1.28 g) was dissolved in a mixture of
acetonitrile (16 mL) and methanol (8 mL) and ethyl bromide (75 mg) was
added. The solution was refluxed over night and the solvent was removed
in a rotary evaporator. The final product was dissolved in water, freeze
dried and characterized by 1HNMR and conductometric titrations.
Percentage of quarternization was calculated by 1HNMR and from
conductometric titrations of the quarternized amine product.
2.5. 1HNMR analysis in D6-DMSO
PG-PEG-Amine-18: d 1.24 (methyl protons from –N–CH2–CH3), 2.04
and 2.08 (–N–CH3), 2.16 and 2.22 (–N–CH2–), 3.11 (–N+–CH3), 3.2
(–OCH3 from PEG), 3.25–3.8 (broad, peaks from glycidol and PEG main
chain protons), 4.2–5.0 (broad, peaks from hydroxyl groups).
PG-PEG-Amine-44: d 1.24 (methyl protons from –N–CH2–CH3), 2.04
and 2.08 (–N–CH3), 2.16 and 2.22 (–N–CH2–), 3.07 (–N+–CH3), 3.2
(–OCH3 from PEG), 3.25–3.8 (broad, peaks from glycidol and PEG main
chain protons), 4.2–5.0 (broad, peaks from hydroxyl groups).
PG-PEG-Amine-100: d 1.24 (methyl protons from –N–CH2–CH3), 3.07
(–N+–CH3), 3.2 (–OCH3 from PEG), 3.25–3.8 (broad, peaks from
glycidol and PEG main chain protons), 4.2–5.0 (broad, peaks from
hydroxyl groups).
2.6. Conductometric titrations
Conductometric titrations were done on a YSI model 35 conductance
meter and 3403 cell with platinum electrode at 25 1C. A syringe pump
(Harvard Instruments) was used to inject dilute NaOH at constant flow
rate of 0.102 mL/min. For a typical titration, PG-PEG-Amine (10 mg) was
dissolved in distilled water and titrated first with 0.1 N HCl followed by
back titration with 0.1 N NaOH. Conductance of the solution was
measured at every 30 s. Potassium hydrogen phthalate solution (0.1 N) was
used for standardizing sodium hydroxide solution.
2.7. Blood compatibility analysis
2.7.1. Blood and plasma
Blood was drawn from healthy unmedicated donors into an evacuated
siliconized glass tube (Becton Dickinson, Franklin Lakes, NJ) containing
3.2% sodium citrate (nine parts blood to one part anticoagulant) or
EDTA. Platelet poor plasma (PPP) was isolated by centrifugation at 3000g
for 15 min at room temperature and used immediately. Platelet Rich
Plasma (PRP) was isolated by centrifuging the citrated blood at 800 rpm
for 15 min followed by pipetting out the supernatant plasma containing
the platelets. The platelet count in the PRP was adjusted to 200  109/L
with plasma.
2.7.2. Coagulation
Prothrombin time (PT) and activated partial thromboplastin time
(APTT) were measured with a coagulation analyzer using mechanical end
point determination (ST4, Diagnostica Stago). For the PT determination,
the extrinsic and common coagulation activated by incubating plasma
with innovins reagent and the clotting time then measured. Innovin is a
lyophilized reagent consisting of recombinant human tissue factor and
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5380 R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390
synthetic phospholipids (thromboplastin), calcium ions, a heparin-
neutralizing compound, buffers and stabilizers (bovine serum albumin)
from Dade Behring. For the APTT determination the intrinsic and
common coagulation pathways were activated by adding a partial
thromboplastin reagent (actin from Dade Behring) and calcium chloride
to plasma and the clotting time measured. The effect of each polymer
solution on coagulation was studied after mixing PPP or PRP with the
polymer solution (9:1 dilution) in the cuvette-strips at 37 1C for 5 min
before adding the coagulation reagents. Control experiments were done
adding identical volumes of 0.15 M saline solution. Each experiment was
repeated three times. The results were analyzed statistically using a
Student’s t-test for two group comparisons and using Anova for
multigroup comparisons at 95% confidence levels.
2.7.3. Plasma recalcification time (PRT)
For PRT measurements, 50 mL of PPP was mixed with polymer
solution (5 mL) and recalcified by the addition of 2.5 mL of 0.2 M CaCl2 at
37 1C in the cuvette-strips in a coagulation analyzer (ST4, Diagnostica
Stago). A mechanical end point determination was used to measure the
formation of fibrin clot. Each experiment was repeated four times.
2.7.4. Red blood cell aggregation in whole blood
EDTA-anticoagulated blood (50 mL) was incubated for 20 min at 37 1C
with polymer solutions (50 mL) in 0.15 N NaCl to get a final concentration of
10, 1 and 0.1 mg/mL of polymer in the blood. Whole blood incubated with
saline and polyethyleneimine (PEI, Mn 25 000) solution was used as negative
and positive control, respectively. After incubation, the red blood cells
isolated by centrifugation were resuspended in plasma and examined by
transmitted bright field light microscopy (Zeiss Axioskop 2plus) using wet
mounted slides. Images were captured with a microscope-mounted black and
white CCD camera (Qimaging Retiga 1300; exposure times less than 1 ms).
2.7.5. Complement activation
To assess complement activation, the cleavage of complement
component C3 was monitored by measuring the formation of its activation
peptides, C3a and C3a des arg, using a commercial C3a enzyme
immunoassay kit (Quidel, San Diego, CA). Activation studies were
performed using pooled citrated plasma isolated by centrifugation from
whole blood donations. Equal volumes of plasma and polymer solution in
saline were incubated at 37 1C for 1 h. Briefly, the samples were diluted with
the dilution buffer provided in the kit and added to a microtiter plate
coated with a monoclonal antibody specific for human C3a and C3a des
arg. After an hour incubation at room temperature to allow any C3a in the
sample to bind to the monoclonal antibody, the plates were washed and
incubated with peroxidase-conjugated rabbit anti-C3a for 15 min. Follow-
ing a final wash step, the chromogenic substrate was added to detect bound
C3a. Absorbance was measured at 450 nm. The sample C3a concentrations
were calculated using a standard curve with net absorbance values plotted
on the y-axis for each C3a concentration indicated on the x-axis. Sample
values were accepted as valid if they fell on the standard curve; sample
values above the top end of the curve were retested following further
dilution. The measurements were done in duplicates.
2.7.6. Platelet activation
To measure platelet activation, 50 mL of PRP was incubated at 37 1C
with an equal volume of polymer dissolved in saline to get a final
concentration of 10 mg/mL. Ten microliters of the incubation mixture was
removed at 30 min to assess the activation state of the platelets using
fluorescence flow cytometry. Expression of the fluorescently labeled platelet
activation marker anti-CD62P and the platelet pan-marker anti-CD42a
were detected using a Coulter Epics-XL (Miami, FL) and a double staining
method. Briefly, 10 mL of post-incubation polymer/platelet mix was diluted
in HEPES buffer and incubated with 5 mL of monoclonal anti-CD42-FITC
and 5 mL of monoclonal anti-CD62P-PE (both from Immunotech,
Marseilles, France). The addition of 1 U/mL of bovine thrombin (Sigma)
was used to activate platelets as a positive control. Mouse IgGs conjugated
to the same chromophore (FITC or PE) were used as the non-specific
binding controls. After 30 min incubation, the samples were fixed with
1 mL of formal saline. Samples were analyzed within 2 h; instrument gates
were set to count 5000 platelets as defined by their forward scatter profile.
Data are reported as the percentage of platelets positive for both of the
bound antibodies and is an average of two separate experiments.
2.8. Cytotoxicity measurements (MTT assay)
Mouse neuro-2a (N2a) cells were obtained from NCCS, Pune, India
and grown in MEM media [Gibco-BRL, New York, USA] supplemented
with 10% fetal calf serum [Biological Industries, Israel], 2 mM L-glutamine
[Sigma], 1 mM sodium pyruvate [Sigma] and antibiotic-antimycotic
solution (100X)[Sigma] at 37 1C in a humidified incubator with 5% CO2.
Cells were detached from the culture flask using trypsin-EDTA (Sigma)
when they became 70–80% confluent. The N2a cells were seeded on 96-
well plate at a density of approximately 5  103 cells/well. After 24 h of
incubation, cationic polymers (95–400 mg/mL) were added to the appro-
priate wells and were incubated for 48 h. Subsequently, methyl thiazol
tetrazolium (MTT) assay was performed. Ten microliters of 5 mg/mL
MTT (Sigma) solution [MTT dissolved in Hank’s balanced salt solution
(HBSS)] was added to each well and incubated for 4 h. Accumulated
formazan crystals were solubilized in DMSO (Sigma) and placed on a
shaker for 15 min. The absorbance at 560 and 630 nm was recorded in an
ELISA plate reader (Spectra MAX 190, Molecular Devices).
2.9. Gel electrophoresis
The electrophoretic mobilities of the polymer/DNA complexes at
different polymer/DNA ratios were determined by gel electrophoresis
using 1.0% agarose gel in a buffer consisting of 45 mM Tris-borate and
1 mM EDTA at pH 8.0. Experiments were run at 80 V for 90 min. DNA
was visualized under UV illumination by staining the gels with ethidium
bromide overnight at room temperature.
2.10. DNA binding affinity (ethidium bromide exclusion assay)
Ethidium bromide (2 mM) and 4 mM DNA solution (one ethidium
bromide per base pair) were mixed in 10 mM phosphate buffer and allowed
to incubate at 25 1C for 10 min. Various amounts of the hyperbranched
polymers were added to the DNA–ethidium bromide mixture and then
incubated for 30 min. Fluorescence intensity was measured using a
spectrofluorometer (FluoroMax-3, Spex) after diluting to 2 mL with
10 mM phosphate buffer. The excitation (lex) and emission (lem)
wavelengths were 480 and 600 nm, respectively.
2.11. Atomic force microscopy (AFM)
A solution of 5 mM DNA in 10 mM phosphate buffer and appropriate
polymer solution in the same buffer were mixed together to obtain a
polyplex solution. The polyplex solution (2–3 mL) was deposited on a
freshly split untreated mica strip (Molecular Imaging, Tempe, AZ) and
allowed to adsorb for 5 min at room temperature. The mica surface was
then imaged using a PicoSPM system (Molecular Imaging) operating in
MAC mode. A 225 mm long magnetically coated cantilever (MAC lever)
with a spring constant of 2.8 N/m and a resonance frequency of 65 kHz
was used. The cantilever is made to oscillate due to the magnetic force
resulting from the solenoid placed under the sample plate. The image was
generated by the change in amplitude of the free oscillation of the
cantilever as it interacts with the sample.
3. Results and discussion
3.1. Synthesis and characterization
A PG-PEG copolymer was synthesized by a ring
opening multibranching anionic sequential polymerization
 ARTICLE IN PRESS
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390 5381

OPEG OPEG
OPEG OPEG
HO O HO O
O O
O HO OH OHO OTs
O p-Tosyl chloride
95 C 1. HO HO
CH3O-K
+ OH O O O O
pyridine
O O O OH O O O OTs
O
2. H CO ( O) O O O O
O O O O O
3 7 OPEG OPEG
MPEG epoxide O O
OPEG OH OPEG OTs
HO OH HO OTs PG-PEG-Tosylate
PG-PEG N
1.

H2N 2. HCHO/HCOOH
H2N NH2
OPEG
OPEG OPEG
HO O
O OPEG
OHO AMINE HO O
O
HO OHO AMINE
O O CH3-CH2-Br
HO
O O O AMINE 50 C O O
O O O O O AMINE
O O N
OPEG O O O
O N
O N OPEG
OPEG N O N
HO AMINE N+ - OPEG N
Br AMINE N
HO
Quarternized PG-PEG-AMINE PG-PEG-AMINE

Scheme 2. Synthesis of polyglycerol-based multivalent cationic polymers.

of glycidol and glycidyl methoxypoly(ethylene glycol) ether
(MPEG) initiated from partially deprotonated tris(hydrox-
ymethyl)propane using potassium methylate (Scheme 2).
Control over branching and polydispersity was achieved by
slow monomer addition [23]. In a second step multivalent
cationic sites were added to the hyperbrached polymers by
an amination reaction with tris(2-aminoethyl)amine and
subsequent methylation and quarternization (Scheme 2).
All the synthetic steps were high yielding and all the
polymers were characterized by 1HNMR (Fig. 1 and
Fig. S2 (supporting info)), conductometric titration and by
GPC analysis. Characteristics of these polymers are given
in Table 2.
We were not able to calculate the absolute amine content
of the polymer from 1HNMR analysis as intensity of peaks
varied largely upon changing the solvent as shown in
Fig. S1 (supporting info). So we adopted a conductometric
titration method using HCl and NaOH for this purpose.
Representation of reactions occurring during conducto-
metric titrations are given in Scheme S1 (supporting info)
and conductometric titration curves are given in Fig. S3
(supporting info). Values obtained from conductometric
titrations agree very well with the charge neutralization
point as measured from DNA binding. In the case of
quarternized polymers we took the advantage of the
difference in protonation behavior of parent amine and
quarternized nitrogen for the determination of quarter-
nized nitrogen in the polymer.
An advantage of these polymers is that one can fine-tune
the composition to achieve optimal binding efficiency and
blood compatibility. This design will also allow residual
primary hydroxyl groups (see Scheme 1), distinct from
cationic sites, to be used to attach cell binding receptors or
labels (fluorescent or radiolabel) to polymers without
affecting its binding properties. With this methodology
one could possibly create a library of compounds by
changing the nature of the amine groups (mono, di or
multivalent) and the concentration of cationic groups
within the polymer. Thus several modifications may be
added to these systems to address additional challenges
without any substantial alteration of existing properties.
3.2. Blood compatibility of polymers
Hemocompatibility of the newly developed polymers
was evaluated by measuring complement activation,
platelet activation, coagulation (PT, APTT and PRT)
and erythrocyte aggregation under in vitro conditions. One
of the limitations to the use of cationic polymers in
therapeutics is the limited stability of delivery vehicles in
blood due to their interactions with blood components
[9,24–27]. Such interactions could minimize the half-life,
targetability of complexes and reproducibility of such
therapies. All blood compatibility experiments reported in
this manuscript were done by the addition of polymer
solutions in buffer to whole blood or plasma and the results
 ARTICLE IN PRESS
5382 R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390

were compared with the effects of identical volumes of

Fig. 1. 1HNMR spectra of PG-PEG-Amine (A) and PG-PEG-Amine-44
(B) in d6-DMSO.
buffer (control) in order to offset any dilution effects.
Complement activation is one of the major barriers
preventing the use of synthetic cationic polymers as drug
delivery vehicles [27]. Activation of components of the
complement system could produce anaphylatoxins which
will activate the immune system. Oposonization of poly-
mer-drug conjugates with complement components could
eventually lead to the clearance of such particles by the
RES. We have used an enzyme immunoassay kit (C3a kit)
for measuring the complement activation. C3a is an
anaphylatoxin produced during the complement cascade
and its concentration in the plasma is a measure of the
extent of complement activation [27]. Polymer samples
were incubated with citrate anticoagulated plasma at
different concentrations at 37 1C for 1 h and the amount
of C3a produced was measured (Fig. 2A). Our new cationic
polymers were found to be neutral to the complement
system in that the amount of C3a produced by the
polymers even at high concentration (10 mg/mL) was not
significantly different from that in the control plasma
sample. With increase in the amount of quarternization,
polymers (PG-PEG-Amine-44 and PG-PEG-Amine-100)
show a slight decrease in the total C3a measured at high
polymer concentration. PG-PEG-Amine-18 behaved simi-
larly to the parent PG-PEG-Amine polymer at the different
concentrations studied. Platelet activation upon interaction
with polymers is another indication of blood incompat-
ibility and could lead to thrombotic complications under in
vivo conditions [27]. It is known that polycations induce
aggregation and activation of platelets which could impair
the platelet function [28,29]. We have measured the platelet
activation after incubating polymers in platelet rich plasma
at 10 mg/mL concentration for 30 min at 37 1C using flow
cytometry. Platelet activation is expressed as the percentage
of platelets positive for both of the bound antibodies (CD
62P and CD 42). PG-PEG-Amine polymers did not

Table 2
Characteristics and DNA binding affinities of multivalent hyperbranched polymers

Sample code Nitrogen (amine) content by % of quarternization Kb (M1) Zeta potential

condutometric titration (mole/g of (ethidium bromide (mV)b
polymer) assay)a

Direct titration Back titration Direct titration Back titration

(with HCl) (with NaOH) (with HCl) (with NaOH)

PG-PEG-Aminec 0.019670.0014 0.021570.001 0 0 9.00  107 11.47 2.9

PG-PEG-Amine-18 0.015970.0005 0.017770.0005 18.9 17.7 8.00  107 13.173.9
PG-PEG-Amine-44 0.01170.0009 0.01270.0011 43.8 44.2 9.60  107 18.374
PG-PEG-Amine-100 0 0 100 100 6.00  107 25.172.2
Spermine — — 0 0 3.60  105 —
a
Binding affinity of different polymers in buffered water (pH 7.2) in the presence of 50 mM NaCl at 25 1C.
b
Zeta potential measurements were done at pH 7.48 in 5 mM NaCl.
c
Molecular weight properties of parent polymer (PG-PEG-Amine); Mn—116 700, Mw/Mn—1.72 and Rg—24.5 nm.
 ARTICLE IN PRESS
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390 5383

20
18
16
C3a (ug/ml)

14
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(1 mL)

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(1 mL
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-P -Am 8 (1 g/m

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10 mg
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m
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EG -A (10

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in
PG -A

EG Am
G

m
i
EG

PG -A

PG -A

-
G
-P

EG

-
PG

-P

-P
-
-P
PG

(A)

100
87.7
90
80
% platelet activation

70
60
50
29.55
40
30
20
10
0.1 0.63 0.14 0.25
0
PRP positive saline PEI PG- PG-
contrl contrl PEG- PEG- PEG- PEG-
Amine Amine- Amine- Amine-
(B) 18
Fig. 2. (A) Complement activation upon interaction of polymers with
platelet poor plasma (10 mg/mL) at 37 1C for 1 h at 1:1 dilution of PPP.
Plasma incubated with saline as a control and inulin (a potent complement
activator) as positive control are also given. (B) Platelet activation upon
interaction of polymers (10 mg/mL) with platelet rich plasma for 30 min at
37 1C. Expression of the platelet activation marker anit-CD62P-FITC on
platelets is given as % of platelet activation.
activate platelets and the values were similar to that of
saline control (Fig. 2B). We did not observe any significant
difference among the different polymers but PEI, a
branched cationic polymer which is extensively used in
drug/gene therapies, caused significant platelet activation
under identical polymer concentration (Fig. 2B). Differ-
ence between our polymers and PEI might be due to the
presence of PEG which protects the cationic sites from
interacting with the platelets and the relatively lower
amount of amine present.
A change in plasma coagulation properties upon
incubation with the polymers is an indication of its
interaction with blood components which might lead to
thrombosis [27,30–32]. Polymer samples were tested for
this using conventional clinical coagulation assays and
measuring PRT at 37 1C. The blood coagulation cascade
includes an intrinsic pathway, an extrinsic pathway, and a
common pathway which is usually examined by measuring
1.93
0.23
PG- PG-
Fig. 3. Effect of polymers on coagulation times (A) prothrombin time
44 100 (PT) and plasma recalcification time (PRT). (B) Activated partial
thromboplastin time (APTT) in platelet-rich plasma (PRP) and platelet
poor plasma (PPP). A final polymer concentration of 10 mg/mL is used at
9:1 dilution of plasma at 37 1C. Control experiments were done adding
identical volumes of saline solution.
the PT and APTT. The PT is used to evaluate the extrinsic
and common coagulation pathway whereas APTT is used
to evaluate the intrinsic pathway.
The results are summarized in Fig. 3A. The PT results
are expressed in seconds required for a fibrin clot to form
after tissue thromboplastin (innovin) was added to a
citrated PPP and PRP. The presence of any of the four
polymers in plasma did not produce any statistically
significant difference in PT compared to control plasma
(saline control) in both PPP (p ¼ 0:15) and PRP (p ¼ 0:82)
suggesting that the extrinsic pathway of coagulation is not
affected by these polymers. No statistical difference in PT
was observed in both PPP and PRP (p40:5).
Biomaterial-induced coagulation is usually initiated by
the activation of intrinsic coagulation factor XII which
results in kallikrein and factor XI activation [33,34].
This pathway usually is measured by APTT. We studied
the effects of our polymers on APTT using PPP and PRP.
The results (Fig. 3B) are expressed in seconds required
for a fibrin clot to form in the plasma after the addition
of partial thromboplastin reagent (actin) and calcium
 ARTICLE IN PRESS
5384 R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390

chloride. All the polymers increased the APTT significantly
compared to saline control. There was no significant
difference between amine and the quarternized polymers
(p ¼ 0:28) which again suggest that the coagulation path-
ways are not affected by the degree of quaternization of the
polymers. These polymers increased the APTT in both PPP
and PRP and there was no significant difference in presence
and absence of platelets in the plasma (p ¼ 0:64).
In order to confirm the observed prolongation of APTT
by the polymers we measured PRT, which is closer to the in
vivo situation than PT and APTT. PRT is a measurement
of intrinsic coagulation cascade activation defined by the
time required for fibrin clot formation once calcium has
been introduced into sodium citrate anticoagulated plasma
(Fig. 3A). PRT is expressed in minutes required to form
fibrin clot. The activation of the intrinsic cascade in plasma
can be impaired by the presence of polymers and PRT is
therefore commonly used as an indicator of blood-
biomaterial interactions. PRT of our polymers are not
significantly different from that of control plasma with
buffer (p ¼ 0.18–0.22) with no observation of spontaneous
triggering of coagulation. This suggests that these polymers
do not interfere with the coagulation cascade under these
near physiological conditions. The observed effect of these
polymers on APTT might be due to its interference with the
‘activation’ by the thromboplastin reagent. Thus our
results suggest that APTT alone cannot always be taken
as an indication of biomaterial induced anticoagulant
activity. It is to be noted that APTT is only a standardized
modification of PRT performed with the use of a
coagulation activator. Further studies are required to
better understand these effects. A detailed verification is
however beyond the scope of the present investigation.
Since PRT experiments are more close to in vivo

Fig. 4. Optical micrographs of human blood red cells after 10 min incubation with polymers at different concentrations in whole blood. All images are at
400  magnification. (A) PG-PEG-Amine (10 mg/mL); (B) PG-PEG-Amine (1 mg/mL); (C) PG-PEG-Amine-18 (10 mg/mL); (D) PG-PEG-Amine-18
(1 mg/mL); (E) PG-PEG-Amine-44 (10 mg/mL); (F) PG-PEG-Amine-44 (1 mg/mL); (G) PG-PEG-Amine-100 (10 mg/mL); (H) PG-PEG-Amine-100
(1 mg/mL); (I) PEI (10 mg/mL); (J) PEI (1 mg/mL) and (K) saline control.
 ARTICLE IN PRESS
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390
Fig. 4. (Continued)
conditions, we believe our polymers do not change the
coagulation pathways significantly.
Erythrocyte interaction with polymers is particularly
important in the use of polymers for in vivo applications.
Any interaction with erythrocytes would negatively influ-
ence the circulation half-life of the polymer-drug con-
jugates. Aggregation, crenation and hemolysis are
indicators of interaction and incompatibility of cationic
polymers with red blood cells [9,12,35]. The newly
synthesized polymers were incubated with EDTA antic-
oagulated whole blood at different concentrations (0.1, 1,
10 mg/mL) and effects on erythrocyte hemolysis, shape and
aggregation were observed microscopically (Fig. 4). The
new polymers did not produce any measurable hemolysis
even at high concentration (10 mg/mL) Moreover the red
cells were morphologically normal (Fig. 4A, C, E, G)
at 10 mg/mL and were similar to that of saline controls
(Fig. 4K). Erythrocytes aggregate naturally in the presence
5385
of macromolecules such as fibrinogen, forming primarily
linear aggregates known as rouleaux as seen in control
sample (Fig. 4K) [36]. Our polymers did not cause any
aggregation and shape of the cells is similar to the control
sample. However, PEI produced considerable hemolysis,
crenation and red cell aggregation (not the linear aggre-
gates called rouleaux) upon incubation with whole blood
under identical conditions (Fig. 4I). At 1.0 and 0.1 mg/mL
polymer concentration we did not see much difference
between PEI and new polymers (Fig. 4B, D, F, H, J). We
believe that the grafted hydrophilic PEG corona in the
polymers prevents the amine groups from interacting
significantly with blood cells.
3.3. Cytotoxicity studies
Cytotoxicity of the polymers is given in Fig. 5 and is
compared with the values for PEI. The viability of N2a
 ARTICLE IN PRESS
5386 R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390
Fig. 4. (Continued)
cells decreased abruptly with increase in the concentration
of PEI. However, the derivatized PG-PEG polymers show
considerably lower toxicity toward the cells even at higher
concentration ranges (Fig. 5A). This is an important
feature of this polymer, as cytotoxicity is one of the major
barriers in in vitro and/or in vivo applications. At a
concentration of 400 mg/mL, only 3% of cells survived in
the presence of PEI whereas cell survival was 81% in the
presence PG-PEG-Amine. However, cytotoxicity increases
with the degree of quarternization of the polymers
(Fig. 5B). Although cytotoxicity increases with increase in
the percentage of quaternization, 36% cell viability was
still observed in the presence PG-PEG-Amine-100, where
all the nitrogen atoms are quarternized. This improvement
in the cytotoxicity compared to PEI again is likely due to
the presence of PEG which reduces the interaction of
amine groups with external macromolecules or surfaces.
3.4. Interaction with nucleic acid
We have studied the interaction of newly developed
polymers with DNA using ethidium bromide displacement
assay (fluorimetric assay), gel retardation experiments and
atomic force microscopy studies.
Though several efforts have been made to understand
the biophysics of DNA–polycationic interaction of ther-
apeutic interest, much less attention has been paid to study
the binding affinity [18,37]. Initial binding studies of new
polymers with DNA were done by an ethidium bromide
displacement assay. This method is commonly used to
study the binding of polycationic compounds to DNA
[37,38]. Although it does not provide information about
the absolute binding affinity or stoichiometry of binding, it
is a very useful comparative method for evaluating the
binding efficiency of different polymers. Fig. 6 shows the
 ARTICLE IN PRESS
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390
Fig. 5. (A) Effect of different hyperbranched polymers and polyethyle-
nimine (Mw—25 000) on the viability of the N2a cells. (B) Effect of
quaternization of the polymers on the viability of the N2a cells at the
polymer concentration of 400 mg/mL. Relative viability is expressed
considering the absorbance at 550 nm of intact cells as 100%. Each data
point is the average (standard deviation of three different experiments).
Fig. 6. Fluorescence titration profiles for the addition of spermine and
hyperbranched polymers to a solution of salmon DNA and ethidium
bromide in buffered water (pH 7.2) in the presence of 50 mM NaCl.
fluorescence titrations of PG-PEG-Amine polymers in
comparison with spermine (a tetramine) a naturally
occurring DNA condensing agent. C50 values report the
concentration of polyamine causing a 50% decrease in
fluorescence intensity. As seen from Fig. 6, spermine binds
to DNA with C50 value of 2.1 mM (in terms of cationic
5387
Fig. 7. Analysis of complex formation between linearized DNA and
hyperbranched polymers by agarose gel electrophoresis. An increasing
amount of copolymer was added to 5 ng of pSV linearized DNA (4.7 kbp)
in 45 mM Tris-borate and 1 mM EDTA at pH 8.0. The mixture was then
analyzed on a 1% agarose gel in Tris-borate buffer (45 mM, pH 8.0)
containing 1 mM EDTA. The plasmid was finally stained with ethidium
bromide to visualize it. (a) PG-PEG-Amine, (b) PG-PEG-Amine-18,
(c) PG-PEG-Amine-44, (d) PG-PEG-Amine-100.
unit), whereas all PG-PEG-Amine polymers bind to DNA
with C50 values are in the nanomolar range (Table 2). We
could not see any systematic effect of % of quarternization
of the polymers on the C50 values. However, much lower
C50 value for all the polymers in comparison to the C50
value of spermine under the same condition accounts for
the higher binding affinity of the polymer towards the
DNA chain. The higher DNA affinity of these polymers is
presumably due to the multivalent nature of the binding
sites. Recently, we reported that the apparent binding
affinity for linear PEG-based cationic random copolymers
and ctDNA was in the order of 104 M1 in terms of
cationic units [19]. Higher DNA binding affinities were
recently reported by Kostiainen et al. for spermine-based
dendrimers [37]. PEG conjugated hyperbranched polymers,
which are more biocompatible, can nonetheless interact as
strongly with DNA as do naked dendrimers. Our results
are therefore consistent with the fact that higher generation
dendrimers bind DNA more tightly than lower generation
analogues.
Gel electrophoresis studies allow visualization of the
interaction of DNA and hyperbranched polymers. Com-
plexes of a linearized plasmid DNA (4.7 kbp) with PG-
PEG-Amine polymers were formed at different N/P ratios,
and agarose gel electrophoresis was subsequently per-
formed. Representative gel images are presented in Fig. 7.
While free DNA or incompletely neutralized DNA
migrates in the electric field toward the anode,
full retardation occurred at and above the N/P ratio of 1
in the case of all the polymers. This study clearly
 ARTICLE IN PRESS
5388 R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390
Fig. 8. (A) Atomic force microscopy image of nanoparticles obtained from hyperbranched PG-PEG-Amine-100 and DNA. Polymer solutions in 10 mM
Tris buffer (pH 7.0) were mixed together to obtain polymer/DNA nanoparticles of charge ratio ¼ 5 (N/P). Five nanograms DNA in 25 mL was used. (B)
Histogram showing the nanoparticles of PG-PEG-Amine-100 and DNA from AFM image. Histogram was generated by measuring sizes of at least 100
particles.
demonstrates that the cationic units of the hyperbranched
polymer are neutralizing the negative charges of DNA,
thereby resulting in the formation of stable complexes. We
have noticed that all the polymers form electroneutral
complexes with DNA irrespective of the inherent amount
of quarternization. This may be due to the fact that all the
amine groups are charged by abstracting protons from the
buffer. This is quite possible as the normal pKa value of
alkyl amine is around 9.2 which are higher than the buffer
pH (8.0) used in this study. Positive zeta potential values of
all the parent polymers at pH 7.48 in 5 mM sodium
chloride (Table 2) also supports this argument.
AFM was used to image the nanoparticle complex
formation between DNA (4.6 kb) and PG-PEG-Amine
polymers. The PG-PEG-Amine polymers in Tris buffer
(pH 7.0) was mixed with plasmid DNA (charge ratio 5 (N/P)
to obtain aggregates. AFM images were taken after
depositing DNA–polymer complexes on freshly cleaved
mica and a representative image is given in Fig. 8A. PG-
PEG-Amine polymer condenses DNA to highly stable,
spherical nanoparticles. Average size of the nanoparticles is
in the range 60–80 nm (Fig. 8B). These particles have the
minimum possible size for a DNA/polycation conjugate
based on the partial specific volumes of the constituent
components and have one of the lowest size reported for a
high molecular weight branched polymer [39,40]. It has
been shown that dendrimers and linear cationic polymers
form large non-uniform particles (50–250 nm) and particle
size increases with the generation of dendrimers [20,39–41].
In the present case, these high molecular weight PG-PEG-
Amine condensed DNA to small nanoparticles. At a charge
ratio of o1.0, the lower charge neutralization of DNA, the
polymer could only partially condense and the extent of
complexation increased according to the increase of charge
ratio. At and above a charge ratio of 3.0, most DNA was
condensed into a particle of spherical shape. Measurements
of turbidity at 550 nm and size measurements showed that
turbidity of the solution and size of the particles remain
unchanged over several weeks.
3.5. Conclusions
The synthesis and characterization of novel hyper-
branched PG-based polymers containing multivalent
cationic sites is reported. These derivatized polymers are
shown to be highly blood-compatible as seen by its
insignificant effects on hemolysis, erythrocyte aggregation,
complement activation, platelet activation and coagula-
tion. Polymers showed much lower cytotoxicity than
standard cationic polymers such as PEI and cytotoxicity
increased with increase in amount of quarternization.
These new cationic polymers form electroneutral com-
plexes around charge ratio N/P ¼ 1 as shown by the gel
retardation experiments and showed insignificant effect on
the amount of quarternization. High affinity of polymers to
DNA is demonstrated by the ethidium bromide displace-
ment assay show binding in the nanomolar range. Our
results show that quaternization of amines is not needed
for DNA binding and should be avoided as this increases
the cytotoxicity. These new polymers condensed plasmid
DNA to one of the smallest sizes reported and form stable
nanoparticles which makes them potential candidates for
polymer therapeutics. Incorporation of multivalent binding
sites onto the hyperbranched polymers offers a powerful
approach for achieving high-affinity DNA binding and
these polymers show similar binding affinities as that of
cationic multivalent dendrimers which have much lower
biocompatibility.
Acknowledgments
JNK acknowledges the CIHR/CBS for the new investi-
gator award in Transfusion Science and financial support
from Canadian Institute of Health Research, Canadian
 ARTICLE IN PRESS
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390
Blood Services and Canada Foundation for Innovation.
SM acknowledges the financial support from the Depart-
ment of Science and Technology, Government of India,
New Delhi (fast track young scientist grant to S.M., project
ref. No. SR/FTP/PS-34/2001). The authors acknowledge
the helpful suggestions by the referees.
Supporting Information Available: Conductometric titration
curves, reaction schemes and 1HNMR spectra of the
polymers are given.
Appendix A. Supporting Information
Supplementary data associated with this article can be
found in the online version at doi:10.1016/j.biomaterials.
2006.06.021.
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