Novel Method for Synthesizing Pegylated Chitosan Nanoparticles

7/31/2019 Novel Method for Synthesizing Pegylated Chitosan Nanoparticles

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2011 Malota t al, publi and lin Dov Mdial P Ltd. Ti i an Opn A atilwi pmit untitd nonommial u, povidd t oiinal wok i poply itd.

Intnational Jounal of Nanomdiin 2011:6 485494

International Journal of Nanomedicine Dovepress

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Opn A Full Txt Atil

DOI: 10.2147/IJN.S17190

A novl mtod fo yntizin Pegylatditoan nanopatil: taty, ppaation,and in vito analyi

Mnaki Malota

ciaan Lan

catin

Tomao-Dunau

syamali saa

satya Paka

Biomdial Tnoloy and cllTapy ra Laboatoy,Dpatmnt of Biomdialeninin and Pyioloy, Atifiialcll and Oan ra cnt,Faulty of Mdiin, Mgill Univity,Qub, canada

copondn: satya Paka3775 Univity stt, Montal,Qub, h3A 2B4, canadaTl +1 514 398 2736Fax +1 514 398 7461email [email protected]

Abstract: Preparation o poly (ethylene glycol) (PEG)-grated chitosan is essential or

improving the biocompatibility and water solubility o chitosan. Presently available methods

or this have limitations. This article describes a new method or preparing PEGylated chitosan

nanoparticles. For this chitosan was chemoselectively modied using a novel scheme at the

C6 position o its repeating units by PEG. The amine groups at the C2 position o the chitosanwere protected using phthalic anhydride. Sodium hydride was used to catalyze the etherication

reaction between chlorinated chitosan and methyl-PEG, and PEG-grated chitosan was

successully synthesized. Each step was characterized using 13C nuclear magnetic resonance

and Fourier transorm inrared. Ater PEGylation the phthaloylated chitosan was successully

deprotected using hydrazine monohydrate. The synthetic scheme proposed demonstrates a new

method or grating PEG onto chitosan with a moderate degree o substitution. The potential o

this polymer in nanoparticle preparation using an ionic gelation method and its gene delivery

potentials were investigated by complexing a fuorescently labeled control siRNA. The result

showed that suitable nanoparticles can be synthesized using this polymer and that they have

capacity to carry genes and provide adequate transection ecacy with no toxicity when tested

in neuronal cells.

Keywords: nanoparticles, chitosan, poly (ethylene glycol), polymeric membrane, gene delivery

IntroductionChitosan is a linear polymer composed o-(14)-2-amino-2-deoxy-D-glucopyranose

units. Being a polycationic, nontoxic, biodegradable, and biocompatible polymer,

chitosan has attracted much attention and has wide applications in biotechnology,

pharmaceutical, textile, ood, cosmetics, and agricultural industries.1,2 Current research

with chitosan ocuses on its use as a novel drug, gene, peptide, and vaccine delivery

vehicle, and scaold or targeted delivery and tissue engineering applications.36

Chitosan has achieved much interest compared with chitin and cellulose due to the

presence o primary amine groups present in its repeating units. The presence o primary

amine groups makes chitosan an excellent cell transectant. Like polyethyleneimine,

chitosan exhibits a proton sponge eect, which reers to the swelling behavior

o the polymer on encountering an acidic pH inside the cells endosome, making

it an ecient carrier or therapeutic molecules.79 A number o chitosan derivatives

have been synthesized in the past with modication on the primary amine groups o the

polymer.10,11 Chitosan is soluble only in acidic aqueous solutions due to the presence

o amine groups that become protonated, and thus its poor solubility in other organic

solvents has become a major drawback, limiting its eective utilization.

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This article was published in the following Dove Press journal:

International Journal of Nanomedicine

2 March 2011
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To acilitate the eective use o chitosan in gene delivery

and other biomedical applications, it is necessary to make

chitosan soluble either in water or organic solvents. Such

attempts can be made by grat copolymerization o chitosan

through chemical modications with other polymers, such

as poly (ethylene glycol) (PEG), o dierent molecular

weights.12 This allows chitosan to retain its inherent

characteristics o, or example, molecular structure and

length, and allows or a variety o chemical modications or

reactions on its side chain. However, chemical modication

at amine groups leads to a change in the undamental skele-

ton o chitosan, causing it to lose its original physiochemical

and biochemical activities.12 Recently, modiying hydroxyl

groups o chitosan has gained much importance as it does

not infuence the characteristic structural and unctional

eatures o chitosan.

PEG is widely used as a grat-orming polymer. It is

soluble in both water and organic solvents, has no toxicity,

antigenicity, and immunogenicity, and is biodegradable and

biocompatible.13 PEGylation o chitosan through hydroxyl

groups was rst proposed by Gorochovceva and Makuska.14

PEG has been used mainly as a grat polymer, where it is used

as a crosslinker and orms interconnected channels to enable

drug release.15,16 Various preparation techniques can be used

to prepare chitosan nanocarriers by altering parameters

such as concentration o the polymer or the crosslinker, the

molecular weight o chitosan, the ratio o drug/gene:polymer,

pH, and nally stabilizers or suractants. All these actors

collectively aect the structural and morphological properties

o chitosan nanoparticles and the release rate o the loaded

therapeutic molecule.17

This study aims to achieve acile chemoselective con-

jugation o PEG at the hydroxyl group o chitosan, where

the amine groups o chitosan were rst protected using

phthalic anhydride.18 Nanoparticles were ormed using an

ionic gelation method. The concentration o the polymer

versus crosslinker and the pH o the solution were adjusted

with reerence to our previously optimized study.19 The

current study opens up the uture application o orming

PEG-grated chitosan nanoparticles or ecient gene/drug

delivery.

Materials and methodsMatialLow-molecular-weight chitosan was obtained rom Wako

(Richmond, VA, USA), having a viscosity o 520 cP and

a degree o deacetylation o 80.0%. Poly (ethylene glycol)

monomethyl ether (MW 5000), phthalic anhydride, sodium

hydride (NaH), pyridine, hydrazine monohydrate, sodium

tripolyphosphate (TPP), glacial acetic acid, agarose (low

gelling temperature), and ethidium bromide (10 mg/mL)

o analytical grade were obtained rom Sigma-Aldrich

(Oakville, ON, Canada). Anhydrous tetrahydrouran

(THF), anhydrous N,N-dimethylormamide (DMF), sodium

hydroxide (NaOH) and methanol were obtained rom

Thermo Fisher Scientic (Ottawa, ON, Canada). Thionyl

chloride (SOCl2) was obtained rom VWR (Mississauga,

ON, Canada). For dilution purposes, ultra pure double-

distilled water (ddH2O) was used rom a laboratory-installed

Barnstead Nanopure diamond water supply unit. siGLO

(Green) transection indicator (20 nmole) was obtained

rom Dharmacon (Laayette, IN, USA). CellTiter 96

AQueous One Solution Reagent was purchased rom Pro-

mega (Madison, WI, USA) to perorm cell viability assay.

TrackIt 10 bp DNA ladder (0.5 g/L) was obtained rom

Invitrogen (Burlington, ON, Canada). Neuro2a cell line

and Eagles minimum essential medium (EMEM) were

obtained rom Cedarlane (Burlington, ON, Canada) and

supplemented with 10% etal bovine serum (FBS) rom

Invitrogen (Burlington, ON, Canada).

Datylation of itoanCommercially available low-molecular-weight chitosan (5 g)

was added to a 40% (w/v) aqueous NaOH solution. The

mixture was stirred or 4 hours at 110C under a nitrogen

atmosphere. Ater 4 hours the mixture was vacuum ltered,

A B

C

DE

F

Figure 1 smati of O-Pegylatd itoan polym ppaation uin Nah and

ThF: A) itoan; B) datylatd itoan; C) ptaloyl itoan; D) loinatd

ptaloyl itoan intmdiat; E) Pegylatd ptaloyl itoan; and F) Pegylatd

itoan.

Abbreviations: Nah,odium ydid; ThF, ttaydofuan.
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syntizin Pegylatd itoan nanopatil

pulverized, and was again treated with 40% (w/v) NaOH

under similar conditions. The resultant product (b) in Figure 1

was reeze-dried.20

Ptaloylation of itoanDeacetylated chitosan (1.00 g) was added to a solution o

phthalic anhydride (2.76 g) in 20 mL o DMF. The mixture

was stirred or 8 hours at 120C under a nitrogen atmosphere.

The resultant product (c) in Figure 1 was cooled to room

temperature and precipitated in ice cold water. The precipitate

was ltered, washed with methanol overnight, and vacuum

dried.18,21

synti of Pegylatd itoan(itoan-O-Peg)We employed a novel scheme to PEGylate chitosan chemose-

lectively. The scheme depicted in Figure 1 utilizes the

activation o PEG by NaH, used as a catalyst to conjugate

PEG and chitosan. The synthesis was accomplished in 3 steps:

1) chlorination o chitosan, 2) activation o OH-PEG-OCH3

(mPEG) with NaH, and 3) grating activated mPEG onto

chlorinated chitosan. To prepare chlorinated chitosan, SOCl2

was added in 10-old excess compared to phthaloylated

chitosan (0.1 g) in 20 mL o pyridine. The reaction was stirred

at 80C or 30 minutes under a nitrogen atmosphere. Ater

30 minutes, the reaction was cooled to room temperature,

precipitated in ice cold water, ltered, and vacuum dried to

yield chlorinated-phthaloyl chitosan, product (d) in Figure 1.

PEG was activated by adding 4 g o OH-PEG-OCH3(mPEG)

to a suspension o NaH (10 mg) in 50 mL o anhydrous THF.

The reaction was stirred at 60C or 2 hours under nitrogen

atmosphere. Ater 2 hours, chlorinated-phthaloyl chitosan

(60 mg) was added to the reaction mix and stirred or another

16 hours under similar conditions. Ater 16 hours the reaction

was allowed to cool at room temperature and was precipitated

in methanol, ltered, and vacuum dried to yield product (e)

in Figure 1.

Dpottion of Pegylatd-ptaloylitoanPEGylated-phthaloyl chitosan (100 mg), hydrazine mono-

hydrate (15 mL), and distilled water (30 mL) were mixed

and heated at 100C or 16 hours under constant magnetic

stirring. Excess hydrazine monohydrate was removed by

evaporating the mixture using a rotary evaporator until a

viscous solution was let. The process was repeated 3 times

by reconstituting the mix with distilled water each time and

then rotary evaporating it until a solid residue was let. The

nal product was dried under vacuum to obtain the desired

PEGylated chitosan.

Ppaation of Pegylatd itoannanopatilPEGylated chitosan nanoparticles were prepared by an

ionic gelation procedure, as explained in previous studies.22

Ater the deprotection o PEGylated-phthaloyl chitosan, the

polymer was dissolved in 1% (v/v) acetic acid solution to yield

a concentration o 0.5 mg/mL and the pH was adjusted to 5.

TPP was dissolved in ddH2O to obtain a concentration o

0.7 mg/mL and the pH was adjusted to 3.19 Nanoparticles were

ormed ater the addition o TPP (drop-wise) to the chitosan

solution under constant magnetic stirring or 1 hour at room

temperature. The nanoparticles obtained were characterized

by TEM or their morphology and size.

Gene loading efciency by the gel

tadation aayThe encapsulation/gene loading eciency o PEGylated

chitosan polymer was determined by complexing it with

siGLO, a transection indicator, to orm nanoparticles

ollowing similar conditions as described in section 2.6.

The ree siGLO in PBS and siGLO complexed with

nanoparticles were run in duplicates on a 4% w/v agarose

gel electrophoresis or 4 hours at 55 V in Tris/Borate/EDTA

(TBE) buer (pH 8.3). The siGLO complexation with nano-

particles was perormed at a ratio o 200:1 (polymer:gene)

(w/w), as determined in our previous study.19 The TBE

buer used contained ethidium bromide at a concentration

o 0.5 g/mL, which is required or the visualization o

the RNA bands under UV transilluminator at a wavelength

o 365 nm using a ChemiDoc XRS System (Bio-Rad,

Hercules, CA).

Transfection efciency and cell viabilityaayMouse neuroblastoma cells (Neuro2a) were seeded in

a 96-well plate at a density o 20,000 cells per well in

complete growth media (antibiotics-ree EMEM with

10% serum). The transection study was perormed ater

24 hours o initial seeding. The dierent tested samples

were: chitosanTPP nanoparticles and ree siGLO as

controls, chitosanTPP:siGLO nanoparticles, and PEGy-

lated chitosanTPP:siGLO nanoparticles (200:1 w/w) as

treatment samples. The cells were then incubated at 37C,

5% CO2

or 4 hours, ater which the cells were observed

under fuorescence microscope (Nikon Eclipse TE2000-U)


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at a wavelength o 490 nm. The same treatment and control

samples were also used or a cytotoxicity assay. The

cytotoxicity assay was perormed ater 4 hours using Cell-

Titer 96 AQueous One Solution Reagent (MTS assay), as

per manuacturers protocol. Ater an additional incubation

o 4 hours with the MTS assay the absorbance in each well

was recorded at 490 nm using a microtiter plate reader (Perkin

Elmer 1420 Multilabel Counter Victor 3TM V). The cell

study was perormed in triplicate.

statitial analyiValues are expressed as means standard deviations (SD).

The statistical signiicance was determined by using an

independent t-test to compare the values o chitosan-TPP

nanoparticles with PEGylated chitosanTPP nanopar-

ticles (n = 3 or each group). P values o,0.05 were

considered signiicant. Statistical analysis was carried

out using Minitab (Minitab, Version 14; Minitab Inc,

State College, PA).

ResultsDatylation of itoanThe 13C nuclear magnetic resonance (NMR) o deacetylated

chitosan conrmed the absence o an acetyl peak (-CH3)

at 23.88 ppm and a carbonyl peak (-C=O) at 175.04 ppm

present in the commercially available chitosan. NMR:

(topological substructural molecular design (TOSS mode)

o commercially available chitosan: C

23.88 (CH3), 59.01

(C-2), 61.76 (C-6), 75.64 (C-5, 3), 82.51 (C-4), 105.68

(C-1), 175.04 (C=O). 13C CP/MAS NMR: (TOSS mode) o

deacetylated chitosan: C

60.76 (C-2, 6), 75.84 (C-4, 5, 3),

102.80 (C-1).

Ptaloylation of itoanThe 13C NMR in TOSS mode o phthaloylated chitosan

conrmed the appearance o peaks Phth phenylene and Phth

C=O at 134.42 ppm and 169.66 ppm, respectively, and the

appearance o Phth C-1,2 and Phth C=O peaks at 131.63

ppm and 169.89 ppm, respectively, in TOSDL mode.13C CP/MAS NMR: (TOSS mode):

C58.19 (C-2), 61.76

(C-6), 72.44 (C-3), 75.42 (C-5), 83.91 (C-4), 101.24 (C-1),

124.62, 131.70, 134.42 (Phth phenylene), and 169.66

(Phth C=O); (TOSDL mode): C

131.63 (Phth C-1,2) and

169.89 (Phth C=O). Fourier transorm inrared (FTIR) o

phthaloylated chitosan as represented in Figure 2b:max

/cm-1

32003400 (OH), 1774 (imide C=O), 1710 (imide C=O),

11501000 (pyranose), and 720 (arom). FTIR o commercially

available chitosan as shown in Figure 2a: max

/cm-1 1630

(amide I), 1542 (amide II), and 1024 (pyranose).

synti of Pegylatd itoanThe FTIR spectra presented in Figure 2d represent PEGylated

chitosan with the characteristic peaks atmax

/cm-1: 2871 (C-H

stretching), 1066 (C-O stretching), 1290, 1251, 950 and

837, conrming the PEG5000

substitution when compared to

Figure 2c that represents the FTIR spectra o PEG only with

the characteristic peaks atmax

/cm-1: 2878 (C-H stretching),

1100 (C-O stretching), 1466, and 1278.

Dpottion of Pegylatd-ptaloylitoanThe FTIR spectra presented in Figure 2e represent depro-

tected PEGylated chitosan with the characteristic peaks at

max

/cm-1: 2878 (C-H stretching), 1066 (C-O stretching)

belonging to PEG, the appearance o 1633 (amide I), 1582(amide II), and 1024 (pyranose) belonging to chitosan, and

the disappearance o 1774 (imide C=O), 1710 (imide C=O)

belonging to the phthaloyl group, as shown in Figure 2b.

Ppaation of Pegylatd itoannanopatilDeprotected PEGylated chitosan polymer was cross-

linked with TPP by the electrostatic interaction between

the cationic charges o the primary amine groups on

chitosan with the anionic charges o TPP. The crosslink-

ing results in the ormation o nanoparticles ranging rom

100 to 150 nm in size as determined by transmission

electron microscopy (TEM). Figure 3a represents the

TEM image o the PEGylated chitosan polymer (magni-

cation: 57000X). Figure 3b represents the chitosanTPP

nanoparticles (magnication: 22000X) and Figure 3c and

3d represents the PEGylated chitosan-TPP nanoparticles

at magnication 57000X and 135000X respectively. It was

observed that PEGylation yielded a spherical shape to the

nanoparticles.

Gene loading efciency of PEGylateditoan nanopatilIn order to evaluate the complexation o siGLO with

PEGylated chitosan-TPP nanoparticles, a gel retardation

assay was perormed, wherein a 10 bp DNA ladder was used

as a reerence, ree siGLO at a concentration o 6 g/mL

o PBS solution was used as the control and PEGylated

chitosanTPP nanoparticles complexing siGLO at a ratio

o 200:1 (w/w), as determined in our previous study,


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was used as the treatment sample.19 The gel, under a UV

transilluminator, shows bands indicating the presence o

unbound siGLO in the suspension, as shown in Figure 4,

or the control sample (ie, ree siGLO in PBS), whereas

there is no band in a treatment sample, which conrms

the complete complexation o the siGLO with PEGylated

chitosan nanoparticles. The strong interaction pertains to

the positive charges available on the chitosan chain ater

the deprotection procedure on the polymer allowing the

interaction with the negatively charged siGLO and TPP.

A

B

C

D

E

2871

2871

1633

1582

1066

1024

1024

950

837

1066

1251

1290

1774

1710

1466

1278

1100

7201630

1542

1024

35003200

35003200

2878

3600 3100 2600 2100 1600 1100 600

Figure 2 Foui tanfom infad pta of t itoan intmdiat and O-Pegylatd itoan: A) datylatd itoan; B) ptaloylatd itoan: pak at1774 m-1and 1702 m-1. T Oh oup of ptaloylatd itoan wa loinatd uin tionyl loid, pntd by t dution in pak: fom 3500 to 3200 m -1;

C) Oh-Peg-Och3, M.W. 5,000; D) 6-O-Peg--2-N-ptaloyl itoan; E) Peg-aftd itoan: diappaan of pak 1774 m-1 and 1702 m-1.


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Figure 3 TeM ima ofa) Pegylatd itoan polym (ma. 57,000); b) itoan

TPP nanopatil (ma. 22,000); c) Pegylatd itoanTPP nanopatil

(ma. 57,000) and d), Pegy latd itoan-TPP nanopatil (ma. 135000x).

Abbreviation: TPP,odium tipolypopat.

330

100

40

20

10

10 bp

DNA

ladder

Free siGLOPegylatedchitosan

with siGLO

Figure 4 gl tadation aay: lan 1) ontol 10 bp DNA ladd; lan 2 and 3

ontol igLO, and lan 4 and 5 a Pegylatd itoan omplxd igLO at a atio

of 200:1 (w/w). In all oup t igLO onntation wa kpt ontant at 6 /mL.

T diappaan of t band iz at 40 bp in Pegylatd itoan omplxd igLO

(lan 4 and 5), ompad to iglO ontol (lan 2 and 3), indiat t omplt

omplxation of t igLO.Transfection efciency and cell viabilityaayThe transection eciency was analyzed by measuring the

fuorescence intensity o the siGLO-loaded nanoparticles.

siGLO is a transection indicator, which is a fuorescent

oligonucleotide that is localized in the nucleus o the

mammalian cell (Figure 5). Free siGLO, chitosanTPP

nanoparticles, and cells without any treatment sample were

used as negative controls. An ecient transection e-

ciency was observed with both chitosanTPP:siGLO and

PEGylated chitosanTPP:siGLO nanoparticles prepared

at a weight ratio o 200:1 (Figure 5). The cell viability

assay was perormed on these cells ater 4 hours, wherein

it was observed that cells treated with PEGylated chitosan

TPP:siGLO nanoparticles showed signicant increase in cell

viability (70%) compared with the chitosanTPP:siGLO

nanoparticles (20%) (independent t-test, P, 0.05), as

observed in Figure 6.

DiscussionThe degree o N-acetylation o chitin dierentiates it rom

chitosan. The lower degree o N-acetylation o chitosan

widens its application or chemical modications with the

amine groups, imparts greater solubility to the polymer, and

enhances its biological properties. Unlike chitin, chitosan is

soluble only in acidic aqueous solutions. The solubility o

the polymer is an important parameter or having a homoge-

neous reaction mixture in order to obtain successul chemical

modications. The solubility o chitosan also depends on

its molecular weight, its degree o neutralization o amine

groups, the ionic strength o the solvent, and the distribution

o N-acetyl glucosamine residues along the backbone o

the chain.23 In these experiments complete deacetylation o

chitosan was achieved ater treating it twice with a 40% (w/v)

NaOH solution. Products (a) and (b) in Figure 1 represent

the schematic o acetylated chitosan (commercially available)

and deacetylated chitosan, respectively.

The complete deacetylation is required to achieve a chi-

tosan with ully activated amine groups allowing or ur-

ther chemical reactions but renders crystalline character to

the polymer, making it insoluble in most organic solvents.21

In order to solubilize chitosan in organic solvents and to

avoid the intererence o amine groups while perorming

chemical modications on the backbone, the amine groups

o chitosan were protected using phthalic anhydride.21

Since it has been observed that phthaloylation o chito-

san in 100% DMF yields partial O-phthaloylation along

with N-phthaloylation, we attempted to use DMF/water


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and 169.89 ppm assigned to C 1, 2 and carbonyl o phthaloyl

group, respectively. The IR data show distinct sharp peaks at

1774 cm-1 and 1702 cm-1 corresponding to imide o phthaloyl

group (Figure 2b). The phthaloyl chitosan prepared by this

method becomes gel-like when precipitated in water, which

supports the data shown by Kurita et al o ormation o a

uniorm structure o phthaloylated chitosan.21

The 2-N-phthaloylated chitosan ormed was urther

reacted with thionyl chloride to obtain chlorinated chitosan

as represented by product (d) in Figure 1. The IR data in

Figure 2b show the disappearance o the OH peak in the range

o 3200 cm-1 to 3400 cm-1 in chlorinated phthaloyl chitosan,

conrming the replacement o hydroxyl groups with chlorine

and the peaks pertaining to imide bond o phthaloyl group

(1774 cm-1 and 1710 cm-1) remain intact.

Simultaneously, mPEG (OH-PEG-OCH3) was activated by

NaH in THF to orm PEG alkoxide (-O-PEG-OCH3). To this

intermediate, chlorinated phthaloyl chitosan was added to

orm PEG-grated phthaloyl chitosan. The desired product was

precipitated in methanol, with unreacted PEG being soluble

in this solvent. This method is the most direct and the easi-

est way to obtain PEG-grated chitosan and does not require

any intensive purication procedures. Product (e) in Figure 1

represents the schematic o PEGylated phthaloyl chitosan.

The deprotection o the chitosan was successully

achieved by treating the PEGylated phthaloyl chitosan,

product (e) in Figure 1, with hydrazine monohydrate, which

c)b)

)e)d

a)

Figure 5 Transfection efciency of nanoparticles on Neuro2a cells after 4 hours of incubation at 37c and 5% cO2: a) Pegylatd-itoan-TPP:igLO nanopatil; b)itoan-TPP:igLO nanopatil; c) igLO only (nativ ontol); d) itoan-TPP nanopatil (nativ ontol); e) ll only (nativ ontol)

Abbreviation: TPP,odium tipolypopat.

(95:5 v/v) as a solvent.21 However, it was observed that the

product obtained did not solubilize homogeneously in most

o the organic solvents, which in turn was a hindrance to

perorm urther chemical reactions. Thus, in this chemical

scheme we did not incorporate the use o DMF/water (95:5

v/v) as a solvent to synthesize phthaloylated chitosan.13C NMR data conrm the successul N-phthaloylation

o the product. Product (c) in Figure 1 shows the schematic

representation o the N-phthaloyl chitosan. The total sup-

pression o side bands (TOSS mode) exhibits peaks at

124.62 ppm, 131.70 ppm, 134.42 ppm corresponding to

phenylene, and 169.66 ppm and carbonyl group o phthaloyl

group and the TOSS dipolar dephasing (TOSDL) mode

conrms the absence o CH and CH2peaks due to their short

relaxation time and presence o only 2 peaks 131.63 ppm

%c

ellviability

120

100

80

60

40

20

0C on tr ol C S- TP -P EG

-siGLO

CS-TPP

-siGLO

siGLOCS-TPP

Treatment samples

Figure 6 Dind cs-TPP-Peg-igLO nanopatil ytotoxiity wa invtiatd

uin Nuo2a ll. Fo t xpimnt, vaiou ontol nanopatil fomulation

w ud, t Nuo2a ll w xpod fo 4 ou and ll viability wa

valuatd uin ptopotomt at 490 nm uin tandad MTs aay (n = 3).


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causes destabilization o the phthaloyl moiety by creating an

excess alkaline condition o pH. 12. Product () in Figure 1

represents the nal product achieved: PEG-grated chitosan.

The absence o peaks at 1774 cm-1 and 1710 cm-1 in product

(e) conrms the complete dissociation o phthalimido group

rom chitosan and the appearance o peaks 1633 cm-1

(amide I) and 1582 cm-1 (amide II) corresponds to the

presence o primary amine groups in chitosan. The peak

at 2871 cm-1 reers to the presence o PEG in product (e),

Figure 1. The data on 1H NMR are not shown since it was

observed that the multiple peaks o oxymethyl groups in

PEG obtained at 3.3 to 3.7 cover over the signals o the

pyranose ring o chitosan.

The reaction conditions used in the scheme or O-PE-

Gylating chitosan are less intensive in terms o operation

and purication. However, NaH, used as a catalyst, makes

the reaction conditions alkaline which can potentially have a

deleterious eect on the phthaloyl group o chitosan. But, a

correct proportion o NaH, PEG, and chitosan can optimize

the reaction conditions. In our study we used 2:4:1 molar

ratio o NaH:PEG:chitosan. The studies conducted by

Makuskas group used Ag2O as a catalyst, producing less

alkalinity in the solution, suitable or the etherication

reaction between chitosan and PEG. The reaction yielded a

high degree o substitution o PEG on chitosan, but as stated

by the researchers, the residual silver salts that remain in

the reaction lead to degradation o chitosan polymer while

undergoing deprotection with hydrazine.24 Also, the overall

yield o the PEGylated chitosan was less and involved 28 to

30 hours o reaction time. Even ater implementing several

cycles o purication on PEGylated chitosan intermediate,

it could not completely remove the silver particles. Thus,

the procedure involves intensive and multiple steps to obtain

the desired product. The use o NaH as a catalyst is more

promising than using Ag2O as a catalyst. In our case, the

degree o substitution o PEG on chitosan can be increased

by using low molecular weight PEG, but not ,1000 due

to the high solubility o PEG in aqueous as well as organic

solvents. We also tried PEGylating chitosan through a scheme

ollowed by Jian and You-Lo, in which phthaloylated chitosan

was rst swelled in pyridine overnight.25 Other researchers

have used NaOH to swell chitosan prior to the reaction

in order to achieve homogeneous conditions. NaOH can

deprotect the phthaloyl group on chitosan, and thereore its

use was avoided and using pyridine as a solvent was avored

due to the solubility o phthaloylated chitosan in pyridine.

We obtained similar results in terms o yield o PEGylated

chitosan by ollowing the protocol o Jian and You-Lo with

the exception o the use o PEG acyl chloride ollowed by

multiple steps that take more time (3640 hours) in terms

o reaction and purication at each step to obtain the nal

product. The total reaction time employed in this scheme,

using NaH, was 18 to 20 hours.

The proposed scheme yielded a moderate (15%50%)

degree o substitution o PEG on chitosan, which was

soluble in acetone, toluene, chloroorm, DMF, and ethyl

acetate at 50C. Also, as reported earlier, O-PEGylated

chitosans with ree amino groups have a wider application

in biotechnology and biomedical systems because o the

unchanged poly (glucosamine) skeleton.14 As PEG separates

the chitosan backbone, thereby decreasing intermolecular

hydrogen bonding and making it water soluble, it acts as

an important intermediate to carry out urther chemical

modications.

Chitosan nanoparticles were prepared by ionic gelation

that involves a mixture o 2 aqueous phases, o which one

is the polymer chitosan and the other is a polyanion TPP.

The positively charged amino group o chitosan interacts

with negatively charged TPP to orm complex coacervates

with a size o about 100 nm.2627 The nanoparticles ormed

can have a signicant application as a drug/gene carrier

system or biomedical applications both in vitro and in

vivo. In this study, we have complexed siGLO, a scrambled

siRNA with a fuorescent tag with the nanoparticles prepared

rom the proposed ormulation o PEGylated chitosan poly-

mer. Complete complexation o the siGLO was achieved

as conrmed by the gel retardation study. The optimal

concentration o siGLO to be used or complexation was

initially determined by our group and has been published

previously as a characterization study.19 The PEGylated

chitosan polymer complexing siGLO was used as a nanocar-

rier to transect a neuronal cell line in order to determine

the transection eciency o the nanoparticle/carrier. It has

been reported by many researchers that PEGylation reduces

the transection eciency o the nanoparticles since PEG,

being neutral in charge, does not eciently interact with

the negatively charged cell membrane. However, in this

study, the acile chemoselective substitution o PEG on the

C6 position was avored, especially so as not to aect the

primary amine groups on chitosan that play a major role is

gene complexation and cell transection.

The transection study, as shown in Figure 5, conrmed

the ecient uptake o PEGylated chitosan nanoparticles (a)

compared with chitosan nanoparticles alone (b). This proves

that conjugating PEG on the C6 position o the chitosan

polymer did not hinder the inherent character o the chitosan


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polymer to complex and to deliver the siGLO to neuronal

cells. To urther investigate the eect o nanoparticles on the

cells, a cell viability assay was perormed. This conrmed the

stressed conditions o cells transected with chitosan nano-

particles alone. The results indicated that although primary

amine groups contribute signicantly to transection they

may also disrupt the cellular integrity o the cell membrane

due to an excessive positive charge on the nanoparticles.

The presence o PEG on the nanoparticles reduces steric

hindrance and makes nanoparticles more hydrophilic. Also,

as observed in Figure 6, the cytotoxicity was greater or the

chitosan nanoparticles complexing siGLO than the chitosan

nanparticles alone. This could be inerred rom a reduction

in size o the nanoparticles ater complexing siGLO, which

in turn allows more particle interaction with the cell mem-

brane, thereby increasing the surace area. The optimization

study based on the particle size and zeta potential o chitosan

nanoparticles with and without siGLO has previously been

published by our group.19

ConclusionWe achieved successul grating o PEG on chitosan poly-

mer through a novel scheme o orming PEG alkoxide using

NaH as a catalyst. The PEGylated chitosan polymer obtained

was easily soluble in water and other organic solvents. The

overall procedure is easier and requires less intensive reac-

tion conditions and purication steps to obtain the desired

product in a short period o time than the other methods

used by other protocols reported in the literature. Also, the

procedure does not impart any deleterious eect on the

chitosan chain. The PEGylated chitosan nanoparticles suc-

cessully complexed siRNA and transected into the neuronal

cell line with minimal cytotoxicity eects. The proposed

ormulation could have a major implication or biomedical

drug targeting strategies involving nanoparticle-mediated

targeted delivery.

AcknowledgmentsThe authors would like to acknowledge the Canadian

Institutes o Health Research (CIHR) grant (MOP-93641)

to Dr S Prakash, the support o McGill Major Scholarship to

M Malhotra, the support o Industrial Innovation Scholarship

(IIS) BMP Innovation NSERC, FQRNT scholarship and

Micropharma Limited Scholarship to Catherine Tomaro-

Duchesneau. We would like to acknowledge the support

o the Centre o Biorecognition and Biosensors, the NMR

Facility at Quebec/Eastern Canada High Field NMR Center,

the Centre or sel-assembled Chemical Structures (CSACS)

and the acility o Electron Microscopy Research at McGill

University. We would like to acknowledge the guidance o

Dr Hani Al-Salami or statistical analysis.

DisclosureThe authors declare no conficts o interest.

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Novel Method for Synthesizing Pegylated Chitosan Nanoparticles