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Biomaterials 27 (2006) 5377–5390
Blood compatibility of novel water soluble hyperbranchedpolyglycerol-based multivalent cationic polymers and theirinteraction with DNA
Rajesh Kumar Kainthan
a
, Muthiah Gnanamani
b
, Munia Ganguli
b
, Tanay Ghosh
b
,Donald E. Brooks
a,c
, Souvik Maiti
b,
ÃÃ
, Jayachandran N. Kizhakkedathu
a,
Ã
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 2006Available online 18 July 2006
Abstract
A novel class of hyperbranched polymers based on polyglycerol (PG) and poly(ethylene glycol) (PEG) are synthesized bymultibranching anionic ring opening polymerization. Multivalent cationic sites are added to these polymers by a post-amination andquarternization reactions. Blood compatibility studies using these polymers at different concentrations showed insignificant effects oncomplement activation, platelet activation, coagulation, erythrocyte aggregation and hemolysis compared to branched cationicpolyethyleneimine (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 solublenanoparticles in the range of 60–80nm. Gel electrophoresis studies showed that they form electroneutral complexes with DNA aroundN/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
Polymer-drug conjugates have gained increased atten-tion as polymer therapeutics in recent years due to itsability to improve the blood residence time, bioavailability,reduce toxicities, and enhance the solubilities of parentdrugs[1–12]. However, progress in such therapies involvingcationic polymers depends heavily on better understandingthe electrostatic complexation of polymers with opposi-tively charged drugs and their biological properties[5].Biomaterial development combined with fundamentalstudies of virus function and cellular processes will enablethe molecular level design of modular drug/gene deliverysystems[6]. The term modular systems indicates that drug/gene is complexed with polymers or lipid formulations,which consists of a vector backbone modified withfunctional groups that mediate environmental interactionsto improve the efficiency. The vector backbone shouldenable the formation of stable complexes with drugs/genes,provide low cytotoxicity, blood compatibility and alsoovercome different problems faced by the delivery systems[6,7]. For example, following intravenous administration,potential microcontainer drug delivery systems tend to beremoved from the circulation by phagocytes of thereticuloendothelial system (RES)[8]. Phagocytosis byelements of the RES in the liver is mediated by the
ARTICLE IN PRESS
www.elsevier.com/locate/biomaterials0142-9612/$-see front matter
r
2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.biomaterials.2006.06.021
ÃÃ
Also corresponding author. Tel.: +911127666156;fax: +911127667471.
Ã
Corresponding author. Tel.: +16048227085;fax: +16048227742.
E-mail addresses:
 
adsorption of certain blood components on the particlesurface and has been shown that a hydrophilicallystabilized microcontainer can avoid phagocytosis andachieve a prolonged circulation time[6–8]. Thus thedevelopment of new generation of polymers with variousarchitectures, improved biocompatibility and differentbinding affinities with biomolecules will further enhanceour fundamental understanding of such systems and couldpotentially lead to new drug delivery systems.Dendrimers and hyperbranched polymers hold con-siderable promise as binding agents for drug deliveryincluding 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 theimperfectly branched hyperbranched polymers of particu-lar interest. Conformational freedom, allowing a polymerto take up an optimal structure for drug interaction,appears to be a useful property in this regard. Frechetet al. have detailed in a recent review that for manybiomedical applications perfect dendritic structures maynot be a prerequisite[14]. Hence, hyperbranched polymersmay prove to be an effective alternative to dendrimersin 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 tobe highly compatible with living cells.Over the past several years we have been developingPEG-based linear cationic polymers which were shown tobind a variety of negatively charged molecules includingDNA[16–20]. It is very difficult to manipulate thearchitecture or functionalities of such structures due tothe inherent nature of these copolymers. Thus in search of new polymers, we have developed a novel class of multivalent hyperbranched cationic polymers based onhyperbranched polyglycerol (PG) (Scheme 1). In this paperwe report the synthesis, characterization and bloodcompatibility studies of such polymers as well as theirinteractions with nucleic acid. Studying the biological andphysiochemical properties of such systems will providevaluable knowledge for future design of more sophisticatedsystems.
2. Experimental section
 2.1. Materials
All reagents were purchased from Aldrich and used as received exceptthe following. Glycidol (96%) was purified by vacuum distillation andstored in a refrigerator (2–4
1
C). 1,1,1-Tris(hydroxymethyl)propane(Fluka) and potassium methylate solution (25wt% in methanol, Fluka)were used as such. Epoxide terminated poly(ethylene glycol) (PEG)monomethyl ether was synthesized by reaction of MPEG (Mn-350) withepichlorohydrin in presence of sodium hydroxide[21]. Molecular weightsand radii of gyration (
R
g
) were determined by GPC with a DAWN-EOS
ARTICLE IN PRESS
Scheme 1. Representation of multivalent hyperbranched cationic polymers.
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390
5378
 
multiangle laser light scattering detector in aqueous 0.1N NaNO
3
solution; the details have been described earlier[22]. The d
n
=
d
c
valuefor polyglycerol was determined to be 0.12cm
3
/g and was used for thecalculation of molecular weight of polymers.
z
-potential measurementswere performed in a Coulter
s
Delsa 440SX Zeta Potential Analyzer(Coulter Co. Instrument Co.) with a He–Ne solid-state laser operated at awavelength of 635nm. The instrument was calibrated with an aqueoussuspension of polystyrene latex with a nominal mobility of 4
m
mcm/Vsfrom Coulter (EMPSC7). Polymer solutions were prepared in 5m
M
sodium chloride solution at 1mg/mL concentration.
 2.2. Synthesis of hyperbranched copolymer of glycidol and PEG (PG-PEG)
Polymerizations were carried out in a three-necked glass reactorequipped with a mechanical stirrer and a syringe pump under argonatmosphere. Tris(hydroxymethyl)propane (TMP, 120mg) was stirred with0.1mL potassium methylate solution and the excess methanol wasremoved under vacuum at 50
1
C. Glycidol (6mL) was added drop-wiseusing the syringe pump over 10h to the flask which is kept at 95
1
C. Theratio of glycidol to TMP corresponds to a degree of polymerization of 100.After the addition of glycidol, the mixture was stirred for additional 2h,after which 20mL MPEG-epoxide was added drop-wise over 12h. Themixture was stirred for an additional 3h. The viscous polymer wasdissolved in methanol and passed though cation exchange resin (AmberliteIRC-50) to remove potassium ions. Polymer was precipitated twice fromdiethyl ether to remove unreacted PEG-epoxide and subsequently dried at70
1
C in vacuum. Yield was 85%.
1
HNMR analysis in d6-DMSO
:
d
3.2 (OCH
3
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 (16g) dissolved in 100mL THF was reacted with methanesulfonyl chloride (1.17mL,
$
20% of OH groups) in presence of triethylamine (3mL) for 12h. The salts were filtered off and polymerwas isolated by precipitation in ether. The dried polymer was dissolved in100mL dioxane to which 40mL Tris(2-aminoethyl)amine was added andrefluxed for 24h. Dioxane was removed by rotary evaporation. Afterdissolving in minimum amount of methanol, the polymer was twiceprecipitated in diethyl ether. The obtained polymer (15g) was dissolved in100mL water and added to stirred solution of formic acid (90% w/w) andformaldehyde (37% w/w) (15mL each) at 0
1
C. The reaction mixture wasrefluxed overnight. The volatiles were removed in vacuum. Polymer wasextracted with dichloromethane after adjusting the pH of the aqueoussolution to 10 with sodium hydroxide. Finally the polymer was purified bydialysis using regenerated cellulose acetate membrane (MWCO 1000).Yield 10g. The product was characterized by
1
HNMR, GPC analysis andconductometric titrations.
1
HNMR analysis
: d6-DMSO.
PG-PEG mesylate
:
d
3.12 (methyl from mesylate), 3.2 (–OCH
3
fromPEG), 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 thehydroxyl groups have been converted to mesylate groups as calculatedfrom the NMR).
PG-PEG-Amine
:
d
2.08 (–N–CH
3
), 2.16 and 2.22 (N–CH
2
 –), 3.20(–OCH
3
, from PEG), 3.25–3.8 (broad, peaks from glycidol and PEG mainchain protons), 4.2–5.0 (broad, peaks from hydroxyl groups).
GPC analysis
:
n
 —116700,
w
/
n
 —1.72,
R
g
 —24.5nm.
 2.4. Synthesis of quarternized amines
The tertiary amine groups in PG-PEG-Amine were quarternized usingethyl bromide. Polymers with different degrees of quarternization weresynthesized by varying the amount of ethyl bromide used (Table 1). For atypical reaction, PG-PEG amine (1.28g) was dissolved in a mixture of acetonitrile (16mL) and methanol (8mL) and ethyl bromide (75mg) wasadded. The solution was refluxed over night and the solvent was removedin a rotary evaporator. The final product was dissolved in water, freezedried and characterized by
1
HNMR and conductometric titrations.Percentage of quarternization was calculated by
1
HNMR and fromconductometric titrations of the quarternized amine product.
 2.5.
1
HNMR analysis in D6-DMSO
PG-PEG-Amine-18
:
d
1.24 (methyl protons from –N–CH
2
 –CH
3
), 2.04and 2.08 (–N–CH
3
), 2.16 and 2.22 (–N–CH
2
 –), 3.11 (–N
+
 –CH
3
), 3.2(–OCH
3
from PEG), 3.25–3.8 (broad, peaks from glycidol and PEG mainchain protons), 4.2–5.0 (broad, peaks from hydroxyl groups).
PG-PEG-Amine-44
:
d
1.24 (methyl protons from –N–CH
2
 –CH
3
), 2.04and 2.08 (–N–CH
3
), 2.16 and 2.22 (–N–CH
2
 –), 3.07 (–N
+
 –CH
3
), 3.2(–OCH
3
from PEG), 3.25–3.8 (broad, peaks from glycidol and PEG mainchain protons), 4.2–5.0 (broad, peaks from hydroxyl groups).
PG-PEG-Amine-100
:
d
1.24 (methyl protons from –N–CH
2
 –CH
3
), 3.07(–N
+
 –CH
3
), 3.2 (–OCH
3
from PEG), 3.25–3.8 (broad, peaks fromglycidol and PEG main chain protons), 4.2–5.0 (broad, peaks fromhydroxyl groups).
 2.6. Conductometric titrations
Conductometric titrations were done on a YSI model 35 conductancemeter and 3403 cell with platinum electrode at 25
1
C. A syringe pump(Harvard Instruments) was used to inject dilute NaOH at constant flowrate of 0.102mL/min. For a typical titration, PG-PEG-Amine (10mg) wasdissolved in distilled water and titrated first with 0.1N HCl followed byback titration with 0.1N NaOH. Conductance of the solution wasmeasured at every 30s. Potassium hydrogen phthalate solution (0.1N) wasused 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 evacuatedsiliconized glass tube (Becton Dickinson, Franklin Lakes, NJ) containing3.2% sodium citrate (nine parts blood to one part anticoagulant) orEDTA. Platelet poor plasma (PPP) was isolated by centrifugation at 3000
g
for 15min at room temperature and used immediately. Platelet RichPlasma (PRP) was isolated by centrifuging the citrated blood at 800rpmfor 15min followed by pipetting out the supernatant plasma containingthe platelets. The platelet count in the PRP was adjusted to 200
Â
10
9
/Lwith plasma.
 2.7.2. Coagulation
Prothrombin time (PT) and activated partial thromboplastin time(APTT) were measured with a coagulation analyzer using mechanical endpoint determination (ST4, Diagnostica Stago). For the PT determination,the extrinsic and common coagulation activated by incubating plasmawith innovin
s
reagent and the clotting time then measured. Innovin is alyophilized reagent consisting of recombinant human tissue factor and
ARTICLE IN PRESS
Table 1Reaction conditions for quarternization of PG-PEG-Amine copolymerSample code Amount of PG-PEGamine (g)Ethyl bromide(g)PG-PEG-Amine-18 1.14 0.021PG-PEG-Amine-44 1.28 0.075PG-PEG-Amine-100 1.2 1.5
R.K. Kainthan et al. / Biomaterials 27 (2006) 5377–5390
5379

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