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Biomaterials 27 (2006) 5377–5390

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,
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
Institute of Genomics and Integrative Biology, CSIR, Mall Road, New Delhi 110 007, India
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


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

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

0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
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 Over the past several years we have been developing
surface and has been shown that a hydrophilically PEG-based linear cationic polymers which were shown to
stabilized microcontainer can avoid phagocytosis and bind a variety of negatively charged molecules including
achieve a prolonged circulation time [6–8]. Thus the DNA [16–20]. It is very difficult to manipulate the
development of new generation of polymers with various architecture or functionalities of such structures due to
architectures, improved biocompatibility and different the inherent nature of these copolymers. Thus in search of
binding affinities with biomolecules will further enhance new polymers, we have developed a novel class of
our fundamental understanding of such systems and could multivalent hyperbranched cationic polymers based on
potentially lead to new drug delivery systems. hyperbranched polyglycerol (PG) (Scheme 1). In this paper
Dendrimers and hyperbranched polymers hold con- we report the synthesis, characterization and blood
siderable promise as binding agents for drug delivery compatibility studies of such polymers as well as their
including nucleic acid therapeutics due to their three- interactions with nucleic acid. Studying the biological and
dimensional shapes and availability of large number of physiochemical properties of such systems will provide
functional groups for suitable derivatizations [9,10,13,14]. valuable knowledge for future design of more sophisticated
The inherent flexibility and ease of synthesis make the systems.
imperfectly branched hyperbranched polymers of particu-
lar interest. Conformational freedom, allowing a polymer
2. Experimental section
to take up an optimal structure for drug interaction,
appears to be a useful property in this regard. Frechet 2.1. Materials
et al. have detailed in a recent review that for many
biomedical applications perfect dendritic structures may All reagents were purchased from Aldrich and used as received except
not be a prerequisite [14]. Hence, hyperbranched polymers the following. Glycidol (96%) was purified by vacuum distillation and
may prove to be an effective alternative to dendrimers stored in a refrigerator (2–4 1C). 1,1,1-Tris(hydroxymethyl)propane
in areas where a precise structure is not necessary. (Fluka) and potassium methylate solution (25 wt% in methanol, Fluka)
were used as such. Epoxide terminated poly(ethylene glycol) (PEG)
Another advantage they have is their biocompatibility monomethyl ether was synthesized by reaction of MPEG (Mn-350) with
[9,15] as there are only a few synthetic polymers known to epichlorohydrin in presence of sodium hydroxide [21]. Molecular weights
be highly compatible with living cells. and radii of gyration (Rg) were determined by GPC with a DAWN-EOS
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390 5379

multiangle laser light scattering detector in aqueous 0.1 N NaNO3 Table 1

solution; the details have been described earlier [22]. The dn=dc value Reaction conditions for quarternization of PG-PEG-Amine copolymer
for polyglycerol was determined to be 0.12 cm3/g and was used for the
calculation of molecular weight of polymers. z-potential measurements Sample code Amount of PG-PEG Ethyl bromide
were performed in a Coulters Delsa 440SX Zeta Potential Analyzer amine (g) (g)
(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 PG-PEG-Amine-18 1.14 0.021
suspension of polystyrene latex with a nominal mobility of 4 mm cm/V s PG-PEG-Amine-44 1.28 0.075
from Coulter (EMPSC7). Polymer solutions were prepared in 5 mM PG-PEG-Amine-100 1.2 1.5
sodium chloride solution at 1 mg/mL concentration.

2.2. Synthesis of hyperbranched copolymer of glycidol and PEG typical reaction, PG-PEG amine (1.28 g) was dissolved in a mixture of
(PG-PEG) 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
Polymerizations were carried out in a three-necked glass reactor in a rotary evaporator. The final product was dissolved in water, freeze
equipped with a mechanical stirrer and a syringe pump under argon dried and characterized by 1HNMR and conductometric titrations.
atmosphere. Tris(hydroxymethyl)propane (TMP, 120 mg) was stirred with Percentage of quarternization was calculated by 1HNMR and from
0.1 mL potassium methylate solution and the excess methanol was conductometric titrations of the quarternized amine product.
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. 2.5. 1HNMR analysis in D6-DMSO
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 PG-PEG-Amine-18: d 1.24 (methyl protons from –N–CH2–CH3), 2.04
mixture was stirred for an additional 3 h. The viscous polymer was and 2.08 (–N–CH3), 2.16 and 2.22 (–N–CH2–), 3.11 (–N+–CH3), 3.2
dissolved in methanol and passed though cation exchange resin (Amberlite (–OCH3 from PEG), 3.25–3.8 (broad, peaks from glycidol and PEG main
IRC-50) to remove potassium ions. Polymer was precipitated twice from chain protons), 4.2–5.0 (broad, peaks from hydroxyl groups).
diethyl ether to remove unreacted PEG-epoxide and subsequently dried at PG-PEG-Amine-44: d 1.24 (methyl protons from –N–CH2–CH3), 2.04
70 1C in vacuum. Yield was 85%. and 2.08 (–N–CH3), 2.16 and 2.22 (–N–CH2–), 3.07 (–N+–CH3), 3.2
HNMR analysis in d6-DMSO: d 3.2 (OCH3 groups from PEG), (–OCH3 from PEG), 3.25–3.8 (broad, peaks from glycidol and PEG main
3.25–3.8 (broad, peaks from glycidol and PEG main chain protons), chain protons), 4.2–5.0 (broad, peaks from hydroxyl groups).
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
2.3. Amine-terminated PG-PEG (PG-PEG-amine) hydroxyl groups).

PG-PEG (16 g) dissolved in 100 mL THF was reacted with methane

2.6. Conductometric titrations
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 Conductometric titrations were done on a YSI model 35 conductance
100 mL dioxane to which 40 mL Tris(2-aminoethyl)amine was added and meter and 3403 cell with platinum electrode at 25 1C. A syringe pump
refluxed for 24 h. Dioxane was removed by rotary evaporation. After (Harvard Instruments) was used to inject dilute NaOH at constant flow
dissolving in minimum amount of methanol, the polymer was twice rate of 0.102 mL/min. For a typical titration, PG-PEG-Amine (10 mg) was
precipitated in diethyl ether. The obtained polymer (15 g) was dissolved in dissolved in distilled water and titrated first with 0.1 N HCl followed by
100 mL water and added to stirred solution of formic acid (90% w/w) and back titration with 0.1 N NaOH. Conductance of the solution was
formaldehyde (37% w/w) (15 mL each) at 0 1C. The reaction mixture was measured at every 30 s. Potassium hydrogen phthalate solution (0.1 N) was
refluxed overnight. The volatiles were removed in vacuum. Polymer was used for standardizing sodium hydroxide solution.
extracted with dichloromethane after adjusting the pH of the aqueous
solution to 10 with sodium hydroxide. Finally the polymer was purified by 2.7. Blood compatibility analysis
dialysis using regenerated cellulose acetate membrane (MWCO 1000).
Yield 10 g. The product was characterized by 1HNMR, GPC analysis and 2.7.1. Blood and plasma
conductometric titrations. Blood was drawn from healthy unmedicated donors into an evacuated
HNMR analysis: d6-DMSO. siliconized glass tube (Becton Dickinson, Franklin Lakes, NJ) containing
PG-PEG mesylate: d 3.12 (methyl from mesylate), 3.2 (–OCH3 from 3.2% sodium citrate (nine parts blood to one part anticoagulant) or
PEG), 3.25–3.8 (broad-peaks from glycidol and PEG main chain protons), EDTA. Platelet poor plasma (PPP) was isolated by centrifugation at 3000g
4.2–5.0 (broad, peaks from hydroxyl groups): Approximately 20% of the for 15 min at room temperature and used immediately. Platelet Rich
hydroxyl groups have been converted to mesylate groups as calculated Plasma (PRP) was isolated by centrifuging the citrated blood at 800 rpm
from the NMR). for 15 min followed by pipetting out the supernatant plasma containing
PG-PEG-Amine: d 2.08 (–N–CH3), 2.16 and 2.22 (N–CH2–), 3.20 the platelets. The platelet count in the PRP was adjusted to 200  109/L
(–OCH3, from PEG), 3.25–3.8 (broad, peaks from glycidol and PEG main with plasma.
chain protons), 4.2–5.0 (broad, peaks from hydroxyl groups).
GPC analysis: Mn—116 700, Mw/Mn—1.72, Rg—24.5 nm.
2.7.2. Coagulation
Prothrombin time (PT) and activated partial thromboplastin time
2.4. Synthesis of quarternized amines (APTT) were measured with a coagulation analyzer using mechanical end
point determination (ST4, Diagnostica Stago). For the PT determination,
The tertiary amine groups in PG-PEG-Amine were quarternized using the extrinsic and common coagulation activated by incubating plasma
ethyl bromide. Polymers with different degrees of quarternization were with innovins reagent and the clotting time then measured. Innovin is a
synthesized by varying the amount of ethyl bromide used (Table 1). For a lyophilized reagent consisting of recombinant human tissue factor and
5380 R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390

synthetic phospholipids (thromboplastin), calcium ions, a heparin- binding controls. After 30 min incubation, the samples were fixed with
neutralizing compound, buffers and stabilizers (bovine serum albumin) 1 mL of formal saline. Samples were analyzed within 2 h; instrument gates
from Dade Behring. For the APTT determination the intrinsic and were set to count 5000 platelets as defined by their forward scatter profile.
common coagulation pathways were activated by adding a partial Data are reported as the percentage of platelets positive for both of the
thromboplastin reagent (actin from Dade Behring) and calcium chloride bound antibodies and is an average of two separate experiments.
to plasma and the clotting time measured. The effect of each polymer
solution on coagulation was studied after mixing PPP or PRP with the 2.8. Cytotoxicity measurements (MTT assay)
polymer solution (9:1 dilution) in the cuvette-strips at 37 1C for 5 min
before adding the coagulation reagents. Control experiments were done Mouse neuro-2a (N2a) cells were obtained from NCCS, Pune, India
adding identical volumes of 0.15 M saline solution. Each experiment was and grown in MEM media [Gibco-BRL, New York, USA] supplemented
repeated three times. The results were analyzed statistically using a with 10% fetal calf serum [Biological Industries, Israel], 2 mM L-glutamine
Student’s t-test for two group comparisons and using Anova for [Sigma], 1 mM sodium pyruvate [Sigma] and antibiotic-antimycotic
multigroup comparisons at 95% confidence levels. 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-
2.7.3. Plasma recalcification time (PRT)
well plate at a density of approximately 5  103 cells/well. After 24 h of
For PRT measurements, 50 mL of PPP was mixed with polymer
incubation, cationic polymers (95–400 mg/mL) were added to the appro-
solution (5 mL) and recalcified by the addition of 2.5 mL of 0.2 M CaCl2 at
priate wells and were incubated for 48 h. Subsequently, methyl thiazol
37 1C in the cuvette-strips in a coagulation analyzer (ST4, Diagnostica
tetrazolium (MTT) assay was performed. Ten microliters of 5 mg/mL
Stago). A mechanical end point determination was used to measure the
MTT (Sigma) solution [MTT dissolved in Hank’s balanced salt solution
formation of fibrin clot. Each experiment was repeated four times.
(HBSS)] was added to each well and incubated for 4 h. Accumulated
formazan crystals were solubilized in DMSO (Sigma) and placed on a
2.7.4. Red blood cell aggregation in whole blood shaker for 15 min. The absorbance at 560 and 630 nm was recorded in an
EDTA-anticoagulated blood (50 mL) was incubated for 20 min at 37 1C ELISA plate reader (Spectra MAX 190, Molecular Devices).
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 2.9. Gel electrophoresis
saline and polyethyleneimine (PEI, Mn 25 000) solution was used as negative
and positive control, respectively. After incubation, the red blood cells The electrophoretic mobilities of the polymer/DNA complexes at
isolated by centrifugation were resuspended in plasma and examined by different polymer/DNA ratios were determined by gel electrophoresis
transmitted bright field light microscopy (Zeiss Axioskop 2plus) using wet using 1.0% agarose gel in a buffer consisting of 45 mM Tris-borate and
mounted slides. Images were captured with a microscope-mounted black and 1 mM EDTA at pH 8.0. Experiments were run at 80 V for 90 min. DNA
white CCD camera (Qimaging Retiga 1300; exposure times less than 1 ms). was visualized under UV illumination by staining the gels with ethidium
bromide overnight at room temperature.
2.7.5. Complement activation
To assess complement activation, the cleavage of complement 2.10. DNA binding affinity (ethidium bromide exclusion assay)
component C3 was monitored by measuring the formation of its activation
peptides, C3a and C3a des arg, using a commercial C3a enzyme Ethidium bromide (2 mM) and 4 mM DNA solution (one ethidium
immunoassay kit (Quidel, San Diego, CA). Activation studies were bromide per base pair) were mixed in 10 mM phosphate buffer and allowed
performed using pooled citrated plasma isolated by centrifugation from to incubate at 25 1C for 10 min. Various amounts of the hyperbranched
whole blood donations. Equal volumes of plasma and polymer solution in polymers were added to the DNA–ethidium bromide mixture and then
saline were incubated at 37 1C for 1 h. Briefly, the samples were diluted with incubated for 30 min. Fluorescence intensity was measured using a
the dilution buffer provided in the kit and added to a microtiter plate spectrofluorometer (FluoroMax-3, Spex) after diluting to 2 mL with
coated with a monoclonal antibody specific for human C3a and C3a des 10 mM phosphate buffer. The excitation (lex) and emission (lem)
arg. After an hour incubation at room temperature to allow any C3a in the wavelengths were 480 and 600 nm, respectively.
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 2.11. Atomic force microscopy (AFM)
C3a. Absorbance was measured at 450 nm. The sample C3a concentrations
were calculated using a standard curve with net absorbance values plotted A solution of 5 mM DNA in 10 mM phosphate buffer and appropriate
on the y-axis for each C3a concentration indicated on the x-axis. Sample polymer solution in the same buffer were mixed together to obtain a
values were accepted as valid if they fell on the standard curve; sample polyplex solution. The polyplex solution (2–3 mL) was deposited on a
values above the top end of the curve were retested following further freshly split untreated mica strip (Molecular Imaging, Tempe, AZ) and
dilution. The measurements were done in duplicates. 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)
2.7.6. Platelet activation
with a spring constant of 2.8 N/m and a resonance frequency of 65 kHz
To measure platelet activation, 50 mL of PRP was incubated at 37 1C
was used. The cantilever is made to oscillate due to the magnetic force
with an equal volume of polymer dissolved in saline to get a final
resulting from the solenoid placed under the sample plate. The image was
concentration of 10 mg/mL. Ten microliters of the incubation mixture was
generated by the change in amplitude of the free oscillation of the
removed at 30 min to assess the activation state of the platelets using
cantilever as it interacts with the sample.
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 3. Results and discussion
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 3.1. Synthesis and characterization
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 A PG-PEG copolymer was synthesized by a ring
to the same chromophore (FITC or PE) were used as the non-specific opening multibranching anionic sequential polymerization
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390 5381

O p-Tosyl chloride
95 C 1. HO HO
+ OH O O O O
2. H CO ( O) O O O O
MPEG epoxide O O
HO OH HO OTs PG-PEG-Tosylate

O O CH3-CH2-Br

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

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

were compared with the effects of identical volumes of

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
Fig. 1. 1HNMR spectra of PG-PEG-Amine (A) and PG-PEG-Amine-44 of platelets positive for both of the bound antibodies (CD
(B) in d6-DMSO. 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 —
Binding affinity of different polymers in buffered water (pH 7.2) in the presence of 50 mM NaCl at 25 1C.
Zeta potential measurements were done at pH 7.48 in 5 mM NaCl.
Molecular weight properties of parent polymer (PG-PEG-Amine); Mn—116 700, Mw/Mn—1.72 and Rg—24.5 nm.
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390 5383

C3a (ug/ml)




PG PE ine- (1 m L)



(1 mL)

-P min e (1 /mL

(1 mL


-P -Am 8 (1 g/m


lin a c



-A 100 mg

10 mg


EG -A (10

in 1 0





PG -P min

PG EG e-1


Am e-



EG ne-












% platelet activation

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

chloride. All the polymers increased the APTT significantly therefore commonly used as an indicator of blood-
compared to saline control. There was no significant biomaterial interactions. PRT of our polymers are not
difference between amine and the quarternized polymers significantly different from that of control plasma with
(p ¼ 0:28) which again suggest that the coagulation path- buffer (p ¼ 0.18–0.22) with no observation of spontaneous
ways are not affected by the degree of quaternization of the triggering of coagulation. This suggests that these polymers
polymers. These polymers increased the APTT in both PPP do not interfere with the coagulation cascade under these
and PRP and there was no significant difference in presence near physiological conditions. The observed effect of these
and absence of platelets in the plasma (p ¼ 0:64). polymers on APTT might be due to its interference with the
In order to confirm the observed prolongation of APTT ‘activation’ by the thromboplastin reagent. Thus our
by the polymers we measured PRT, which is closer to the in results suggest that APTT alone cannot always be taken
vivo situation than PT and APTT. PRT is a measurement as an indication of biomaterial induced anticoagulant
of intrinsic coagulation cascade activation defined by the activity. It is to be noted that APTT is only a standardized
time required for fibrin clot formation once calcium has modification of PRT performed with the use of a
been introduced into sodium citrate anticoagulated plasma coagulation activator. Further studies are required to
(Fig. 3A). PRT is expressed in minutes required to form better understand these effects. A detailed verification is
fibrin clot. The activation of the intrinsic cascade in plasma however beyond the scope of the present investigation.
can be impaired by the presence of polymers and PRT is 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.
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390 5385

Fig. 4. (Continued)

conditions, we believe our polymers do not change the of macromolecules such as fibrinogen, forming primarily
coagulation pathways significantly. linear aggregates known as rouleaux as seen in control
Erythrocyte interaction with polymers is particularly sample (Fig. 4K) [36]. Our polymers did not cause any
important in the use of polymers for in vivo applications. aggregation and shape of the cells is similar to the control
Any interaction with erythrocytes would negatively influ- sample. However, PEI produced considerable hemolysis,
ence the circulation half-life of the polymer-drug con- crenation and red cell aggregation (not the linear aggre-
jugates. Aggregation, crenation and hemolysis are gates called rouleaux) upon incubation with whole blood
indicators of interaction and incompatibility of cationic under identical conditions (Fig. 4I). At 1.0 and 0.1 mg/mL
polymers with red blood cells [9,12,35]. The newly polymer concentration we did not see much difference
synthesized polymers were incubated with EDTA antic- between PEI and new polymers (Fig. 4B, D, F, H, J). We
oagulated whole blood at different concentrations (0.1, 1, believe that the grafted hydrophilic PEG corona in the
10 mg/mL) and effects on erythrocyte hemolysis, shape and polymers prevents the amine groups from interacting
aggregation were observed microscopically (Fig. 4). The significantly with blood cells.
new polymers did not produce any measurable hemolysis
even at high concentration (10 mg/mL) Moreover the red 3.3. Cytotoxicity studies
cells were morphologically normal (Fig. 4A, C, E, G)
at 10 mg/mL and were similar to that of saline controls Cytotoxicity of the polymers is given in Fig. 5 and is
(Fig. 4K). Erythrocytes aggregate naturally in the presence compared with the values for PEI. The viability of N2a
5386 R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390

Fig. 4. (Continued)

cells decreased abruptly with increase in the concentration 3.4. Interaction with nucleic acid
of PEI. However, the derivatized PG-PEG polymers show
considerably lower toxicity toward the cells even at higher We have studied the interaction of newly developed
concentration ranges (Fig. 5A). This is an important polymers with DNA using ethidium bromide displacement
feature of this polymer, as cytotoxicity is one of the major assay (fluorimetric assay), gel retardation experiments and
barriers in in vitro and/or in vivo applications. At a atomic force microscopy studies.
concentration of 400 mg/mL, only 3% of cells survived in Though several efforts have been made to understand
the presence of PEI whereas cell survival was 81% in the the biophysics of DNA–polycationic interaction of ther-
presence PG-PEG-Amine. However, cytotoxicity increases apeutic interest, much less attention has been paid to study
with the degree of quarternization of the polymers the binding affinity [18,37]. Initial binding studies of new
(Fig. 5B). Although cytotoxicity increases with increase in polymers with DNA were done by an ethidium bromide
the percentage of quaternization, 36% cell viability was displacement assay. This method is commonly used to
still observed in the presence PG-PEG-Amine-100, where study the binding of polycationic compounds to DNA
all the nitrogen atoms are quarternized. This improvement [37,38]. Although it does not provide information about
in the cytotoxicity compared to PEI again is likely due to the absolute binding affinity or stoichiometry of binding, it
the presence of PEG which reduces the interaction of is a very useful comparative method for evaluating the
amine groups with external macromolecules or surfaces. binding efficiency of different polymers. Fig. 6 shows the
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390 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.

Fig. 5. (A) Effect of different hyperbranched polymers and polyethyle-

nimine (Mw—25 000) on the viability of the N2a cells. (B) Effect of unit), whereas all PG-PEG-Amine polymers bind to DNA
quaternization of the polymers on the viability of the N2a cells at the with C50 values are in the nanomolar range (Table 2). We
polymer concentration of 400 mg/mL. Relative viability is expressed could not see any systematic effect of % of quarternization
considering the absorbance at 550 nm of intact cells as 100%. Each data
point is the average (standard deviation of three different experiments).
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
Fig. 6. Fluorescence titration profiles for the addition of spermine and analogues.
hyperbranched polymers to a solution of salmon DNA and ethidium Gel electrophoresis studies allow visualization of the
bromide in buffered water (pH 7.2) in the presence of 50 mM NaCl. 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,
fluorescence titrations of PG-PEG-Amine polymers in and agarose gel electrophoresis was subsequently per-
comparison with spermine (a tetramine) a naturally formed. Representative gel images are presented in Fig. 7.
occurring DNA condensing agent. C50 values report the While free DNA or incompletely neutralized DNA
concentration of polyamine causing a 50% decrease in migrates in the electric field toward the anode,
fluorescence intensity. As seen from Fig. 6, spermine binds full retardation occurred at and above the N/P ratio of 1
to DNA with C50 value of 2.1 mM (in terms of cationic in the case of all the polymers. This study clearly
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

demonstrates that the cationic units of the hyperbranched turbidity of the solution and size of the particles remain
polymer are neutralizing the negative charges of DNA, unchanged over several weeks.
thereby resulting in the formation of stable complexes. We
have noticed that all the polymers form electroneutral 3.5. Conclusions
complexes with DNA irrespective of the inherent amount
of quarternization. This may be due to the fact that all the The synthesis and characterization of novel hyper-
amine groups are charged by abstracting protons from the branched PG-based polymers containing multivalent
buffer. This is quite possible as the normal pKa value of cationic sites is reported. These derivatized polymers are
alkyl amine is around 9.2 which are higher than the buffer shown to be highly blood-compatible as seen by its
pH (8.0) used in this study. Positive zeta potential values of insignificant effects on hemolysis, erythrocyte aggregation,
all the parent polymers at pH 7.48 in 5 mM sodium complement activation, platelet activation and coagula-
chloride (Table 2) also supports this argument. tion. Polymers showed much lower cytotoxicity than
AFM was used to image the nanoparticle complex standard cationic polymers such as PEI and cytotoxicity
formation between DNA (4.6 kb) and PG-PEG-Amine increased with increase in amount of quarternization.
polymers. The PG-PEG-Amine polymers in Tris buffer These new cationic polymers form electroneutral com-
(pH 7.0) was mixed with plasmid DNA (charge ratio 5 (N/P) plexes around charge ratio N/P ¼ 1 as shown by the gel
to obtain aggregates. AFM images were taken after retardation experiments and showed insignificant effect on
depositing DNA–polymer complexes on freshly cleaved the amount of quarternization. High affinity of polymers to
mica and a representative image is given in Fig. 8A. PG- DNA is demonstrated by the ethidium bromide displace-
PEG-Amine polymer condenses DNA to highly stable, ment assay show binding in the nanomolar range. Our
spherical nanoparticles. Average size of the nanoparticles is results show that quaternization of amines is not needed
in the range 60–80 nm (Fig. 8B). These particles have the for DNA binding and should be avoided as this increases
minimum possible size for a DNA/polycation conjugate the cytotoxicity. These new polymers condensed plasmid
based on the partial specific volumes of the constituent DNA to one of the smallest sizes reported and form stable
components and have one of the lowest size reported for a nanoparticles which makes them potential candidates for
high molecular weight branched polymer [39,40]. It has polymer therapeutics. Incorporation of multivalent binding
been shown that dendrimers and linear cationic polymers sites onto the hyperbranched polymers offers a powerful
form large non-uniform particles (50–250 nm) and particle approach for achieving high-affinity DNA binding and
size increases with the generation of dendrimers [20,39–41]. these polymers show similar binding affinities as that of
In the present case, these high molecular weight PG-PEG- cationic multivalent dendrimers which have much lower
Amine condensed DNA to small nanoparticles. At a charge biocompatibility.
ratio of o1.0, the lower charge neutralization of DNA, the
polymer could only partially condense and the extent of Acknowledgments
complexation increased according to the increase of charge
ratio. At and above a charge ratio of 3.0, most DNA was JNK acknowledges the CIHR/CBS for the new investi-
condensed into a particle of spherical shape. Measurements gator award in Transfusion Science and financial support
of turbidity at 550 nm and size measurements showed that from Canadian Institute of Health Research, Canadian
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390 5389

Blood Services and Canada Foundation for Innovation. cationic random copolymer and sodium dodecyl sulfate. Langmuir
SM acknowledges the financial support from the Depart- 2003;19:2947–55.
ment of Science and Technology, Government of India, [17] Nisha CK, Manorama SV, Kizhakkedathu JN, Maiti S. Water-
soluble complexes from random copolymer and oppositely charged
New Delhi (fast track young scientist grant to S.M., project surfactant. 2. Complexes of poly(ethylene glycol)-based cationic
ref. No. SR/FTP/PS-34/2001). The authors acknowledge random copolymer and bile salts. Langmuir 2004;20:8468–75.
the helpful suggestions by the referees. [18] Kizhakkedathu JN, Nisha CK, Manorama SV, Maiti S. Water-
soluble nanoparticles from random copolymer and oppositely
charged surfactant 3. Nanoparticles of poly(ethylene glycol)-based
Supporting Information Available: Conductometric titration cationic random copolymer and fatty acid salts. Macromol Biosci
curves, reaction schemes and 1HNMR spectra of the 2005;5:549–58.
polymers are given. [19] Nisha CK, Manorama SV, Ganguli M, Maiti S, Kizhakkedathu JN.
Complexes of poly(ethylene glycol)-based cationic random copoly-
mer and calf thymus DNA: a complete biophysical characterization.
Appendix A. Supporting Information Langmuir 2004;20:2386–96.
[20] Ganguli M, Jayachandran KN, Maiti S. Nanoparticles from cationic
Supplementary data associated with this article can be copolymer and DNA that are soluble and stable in common organic
solvents. J Am Chem Soc 2004;126:26–7.
found in the online version at doi:10.1016/j.biomaterials. [21] Park J, Cho KY, Kim C. Biodegradable aliphatic polyester grafted
2006.06.021. with polyether and a process for preparing the same. US patent
[22] Kumar KR, Kizhakkedathu JN, Brooks DE. Atom transfer radical
polymerization using multidentate amine ligands supported on
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