n-acetyl histidine glycol chitosan

8/7/2019 N-acetyl histidine glycol chitosan

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N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles

for intracytoplasmic delivery of drugs: Endocytosis, exocytosis

and drug release

Ji Sun Parka,b, Tae Hee Han a, c, Kuen Yong Lee c, Sung Soo Han d, Jung Jin Hwang a,Dae Hyuk Moon b, Sang Yoon Kim e, Yong Woo Cho a,b,

a Asan Institute for Life Sciences, University of Ulsan College of Medicine, 388-1 Pungnap-2dong, Songpa-gu, Seoul 138-736, Koreab Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, Korea

c Department of Bioengineering, Hanyang University, Seoul 133-791, Koread

School of Textiles, Yeungnam University, Gyeongsan 712-749, Koreae Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, Korea

Received 25 April 2006; accepted 10 July 2006

Available online 20 July 2006

Abstract

Nano-sized vesicular systems (nanoparticles), ranging from 10 nm to 1000 nm in size, have potential applications as drug delivery systems.

Successful clinical applications require the efficient intracellular delivery of drug-loaded nanoparticles. Here we describe N-acetyl histidine-

conjugated glycol chitosan (NAcHis-GC) self-assembled nanoparticles as a promising system for intracytoplasmic delivery of drugs. Because N-

acetyl histidine (NAcHis) is hydrophobic at neutral pH, the conjugates formed self-assembled nanoparticles with mean diameters of 150 250 nm.

In slightly acidic environments, such as those in endosomes, the nanoparticles were disassembled due to breakdown of the hydrophilic/

hydrophobic balance by the protonation of the imidazole group of NAcHis. Cellular internalization and drug release of the pH-sensitive self-assembled nanoparticles were investigated by flow cytometry and confocal microscopy. NAcHis-GC nanoparticles internalized by adsorptive

endocytosis were exocytosed or localized in endosomes. The amount of exocytosed nanoparticles was dependent on the pre-incubation time prior

to removal of free nanoparticles from the culture media. Flow cytometry and confocal microscopy showed that NAcHis-GC nanoparticles released

drugs into the cytosol and cell cycle analysis demonstrated that paclitaxel-incorporated NAcHis-GC nanoparticles were effective in inducing arrest

of cell growth.

2006 Elsevier B.V. All rights reserved.

Keywords: Nanoparticle; Histidine; Chitosan; Endocytosis; Exocytosis; Endosome; Paclitaxel; Cell cycle arrest

1. Introduction

Various polymeric nanoparticles have been studied for the

intracellular delivery of different classes of therapeutic agents,

such as chemicals, oligonucleotides, siRNA, DNA, and proteins

[1]. Nanoparticles are thought to enter cells via an endocytotic

pathway through either specific (e.g., receptor-mediated

endocytosis) or non-specific interactions (e.g., adsorptive endo-

cytosis) with cell membranes. Efficient delivery of therapeutics

requires that delivery systems overcome intracellular barriers,such as endosomes, and release the therapeutics into the cytosol

[2,3]. In the absence of a mechanism for endosomal escape, the

delivery system is localized in endosomes and will ultimately be

trafficked to lysosomes. Supramolecular assemblies, including

polymeric micelles and nanoparticles, have been developed,

which selectively release drugs or genes into the cytosol by

sensing a low pH in endosomes and lysosomes [48].

Histidine has served as a pH-responsive fusogen [911]. The

pKa of the imidazole group of histidine is around 6.5, and

therefore this group is protonated in slightly acidic milieu

(Fig. 1). Since histidine is strongly cationic at acidic pH, it

Journal of Controlled Release 115 (2006) 3745

www.elsevier.com/locate/jconrel

Corresponding author. Asan Institute for Life Sciences, University of Ulsan

College of Medicine, 388-1 Pungnap-2dong, Songpa-gu, Seoul 138-736, Korea.

Tel.: +82 2 3010 4112; fax: +82 2 3010 4182.

E-mail address: [email protected] (Y.W. Cho).

0168-3659/$ - see front matter 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jconrel.2006.07.011
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interacts with negatively-charged endosomal membranes, in-

duces influx of water and ions, and eventually brings about

endosome destabilization and drug release. The gene transfec-

tion efficiency of poly(L-lysine) was significantly enhanced by

the partial substitution of their -amino groups by histidine,

probably due to the destabilization of endosomal membranes

[12

15]. In addition, histidylated oligolysine facilitated thecytosolic delivery of antisense oligonucleotides [11], and poly

(L-histidine)-poly(ethylene glycol) block copolymer micelles

were found effective for the cytosolic delivery of adriamycin by

virtue of their fusogenic activity [8,1618].

Previous reports have described a series of self-assembled

nanoparticles for drug and gene delivery [1926]. Polymeric

amphiphiles with an appropriate hydrophilic/hydrophobic ba-

lance form self-assembled nanoparticles in aqueous milieu.

These self-assembled nanoparticles were prepared by the con-

jugation of hydrophobic groups (deoxycholic acid, lithocholic

acid and adriamycin) to hydrophilic polymers (glycol chitosan

and heparin). In the present study, N-acetyl histidine (NAcHis)was used as the hydrophobic moiety. At neutral pH, NAcHis is

expected to constitute a hydrophobic core of self-assembled

nanoparticles which can serve as a depot for hydrophobic drugs.

At acidic pH, the imidazole group of NAcHis is expected to be

protonated, causing disassembly of the nanoparticles and event-

ual release of drugs into the cytosol. N-acetyl histidine-con-

jugated glycol chitosan (NAcHis-GC) nanoparticles were

labeled with fluorescein isothiocynate (FITC) for intracellular

tracking. The red fluorescent dye DiI was used as a model drug,

and flow cytometric analysis and confocal microscopy were

used to monitor intracellular drug delivery. Paclitaxel, a drug

widely used in the clinical treatment of cancer, was also

incorporated into NAcHis-GC nanoparticles, and the cytotoxicactivity of the latter against cancer cell lines was investigated by

cell cycle analysis.

2. Materials and methods

2.1. Materials

Glycol chitosan (GC, MW 250 kDa, degree of deacetylation

88.7%), purchased from Sigma (St. Louis, MO, USA), was

dissolved in distilled water, filtered to remove insoluble im-purities, and dialyzed against distilled water. N-acetyl histidine

(NAcHis), fluorescein isothiocyanate (FITC), N-(3-dimethyl

aminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-

hydroxysuccimide (NHS), chloroform, dimethyl sulfoxide

(DMSO), paclitaxel (PTX), and bovine serum albumin (BSA,

Fraction V) were purchased from Sigma and used without

further purification. 1,1-Dioctadecyl-3,3,3,3-tetramethindo-

carbocyanine perchloate (DiI, MW = 933.88) was purchased

from Molecular Probes (Carlsbad, CA, USA). Dulbecco's

modified Eagle's medium/F12 (DMEM/F12), fetal bovine

serum (FBS), RPMI-1640 medium (RPMI), trypsin-EDTA

(0.5% trypsin, 5.3 mM EDTA tetra-sodium), and penicillin-streptomycin (100 U/ml) were purchased from Gibco BRL

(Rockville, MD, USA). All other chemicals were of analytical

grade and were used as received.

2.2. Preparation of NAcHis-GC conjugates and FITC-labeling

GC (0.5 g) and different amounts of NAcHis (0.13

0.76 mmol) were dissolved in PBS, EDC (0.392.28 mmol)

and NHS (0.392.28 mmol) were added, and the reaction

mixture was stirred for 24 h at room temperature. The aqueous

suspension of the reaction products (NAcHis-GC conjugates)

was placed in a cellulose membrane (MWCO 12,000, Spectrum

Laboratories, Rancho Dominguez, CA, USA), dialyzed againstdistilled water for 5 days, and freeze-dried. The NAcHis content

in the conjugate was determined by 1H NMR spectroscopy.

Fig. 1. Schematic representation of endosomal escape and drug release of histidylated polymer nanoparticles. The pH-dependent endosomal-membrane destabilization

by histidine correlated with the protonation of its imidazole groups. The pKaof the imidazole group is around 6.5. In a slightly acidic milieu, such as in endosomes, the

imidazole group is protonated, interacts with negatively charged lipid bilayers and induces the influx of water and ions into endosomes, thus causing endosomedestabilization and drug release into the cytosol.

38 J.S. Park et al. / Journal of Controlled Release 115 (2006) 3745


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To label NAcHis-GCconjugates with FITC, NAcHis-GC(0.5 g)

was suspended in 100 ml of bicarbonate buffer (0.1 M Na2CO3,

0.1 M NaHCO3, pH 9.5), FITC was added, and the suspension wasstirred for 24 h at room temperature. The reaction mixture was

dialyzed for 4 days against 50% ethanol and lyophilized. The FITC

content in labeled NAcHis-GC was determined fluorometrically

(ISS K2, Champaign, IL, USA), based on the standard curve

obtained from FITC alone, with excitation and emission wave-

lengths of 495 and 520 nm, respectively.

2.3. Preparation and characterization of NAcHis-GC self-

assembled nanoparticles

NAcHis-GC conjugates were suspended in PBS, and each

suspension was sonicated for 2 min using a probe-type sonicator

(Ultrasonic Processor GEX-600, Sigma) at 90 W, in which thepulse was turned off for 1 s with an interval of 5 s. The nano-

particle suspensions were passed through a cellulose acetate

syringe filter (pore size 0.80 m, Millipore, Billerica, MA,

USA) and freeze-dried. The lyophilized samples were well-

suspended again in aqueous media.

To determine the particle size and size distribution of self-

assembled nanoparticles, dynamic light scattering (DLS) mea-

surements were performed using a helium ion laser system

(Spectra Physics Laser Model 127-35, Mountain View, CA),

which was operated at 633 nm and 251 C. The scattered light

was measured at an angle of 90 and was collected with a

Brookhaven BI-9000AT autocorrelator (Holtsville, NY). Thehydrodynamic diameter of nanoparticles was calculated by the

Stokes-Einstein equation.

2.4. Drug loading into self-assembled nanoparticles

NAcHis-GC nanoparticles (10 mg) were suspended in PBS

(1 ml) with gentle shaking for 3 h, PTX (0.1 mg) in 100 l

chloroform was slowly added, and the suspension was stirred

for 24 h. The chloroform was evaporated, and the suspension

was centrifuged to remove PTX precipitates. The supernatant

was collected, passed through a cellulose acetate syringe filter

(pore size 0.80 m, Millipore) and freeze-dried. DiI-loadednanoparticles were prepared in a similar manner. PTX and DiI

contents in nanoparticles were measured using HPLC and a

fluorometer, respectively [25].

2.5. Cell culture

HeLa (human epithelioid cervical cancer cell line) and A549

(human lung cancer cell line), obtained from the American Type

Culture Collection (ATCC, Manassas, VA, USA), were routine-

ly cultured in RPMI-1640 medium containing 10% heat-inact-

ivated fetal bovine serum (FBS) and 1% penicillin-streptomycin

at 37 C, 5% CO2 and 95% humidity. The MDA-MB231 human

breast cancer cell line (ATCC) was cultured in DMEM/F12supplemented with 10% FBS and 1% penicillin-streptomycin at

37 C, 5% CO2 and 95% humidity.

Table 1

Characteristics of self-assembled nanoparticles

Samplea XHa (wt.%) XF

b (wt.%) Dc (nm) 2/2 d

NAcHis-GC-1 6.8 0.65 250 23 nm 0.19 0.04

NAcHis-GC-2 7.8 0.60 150 18 nm 0.16 0.02

NAcHis-GC-3 8.7 0.55 170 15 nm 0.15 0.03

The sample volume and polymer concentration were 1 ml and 5.0 mg/ml fordynamic light scattering measurement, respectively.a Weight percentage of N-acetyl histidine determined by 1H-NMR spectro-

scopy.b Weight percentage of FITC determined with a UVVis spectrophotometer.c Mean diameter in PBS (pH 7.4), measured by dynamic light scattering.

Each value represents the meanSD (n =5).d Polydispersity factor, calculated from the cumulant method, where 2 is the

second cumulant of the decay function and is the average characteristic line

width. Each value represents the meanSD (n=5).

Fig. 2. Size distribution of NAcHis-GC-2 self-assembled nanoparticles in PBS(pH 7.4), as measured by dynamic light scattering.

Fig. 3. Intracellular uptake of NAcHis-GC-2 nanoparticles. HeLa cells were

incubated in the presence of NAcHis-GC-2 nanoparticles at different

concentrations (0.010.2 mg/ml) for 6 h. After repeated washing to remove

free nanoparticles, cells were analyzed by flow cytometry to quantify cellularuptake.

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2.6. Cellular uptake of FGC nanoparticles

Cells were plated in a 6-well culture dish and cultured at 37 C

in 5% CO2 until 80% confluent. Cells were rinsed twice and pre-

incubated for 1 h with 2 ml of serum-free medium at 37 C. FITC-

labeled NAcHis-GC nanoparticles were added at a particle

concentration of 0.01 mg/ml to 0.2 mg/ml and incubated at 37 C

for 10 min to 6 h. The cells were washed three times with 1 ml of

PBS (pH 7.4) to remove any free FGC nanoparticles, detached

with 0.25% trypsin, and centrifuged at 1200 rpm. Supernatants

were discarded, and the particles were re-suspended with PBS

containing 0.1% bovine serum albumin (BSA). Cells were fixed

in 3.7% paraformaldehyde solution at 4 C, washed three times

with PBS, suspended in PBS, and then introduced into a

FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA,

USA) equippedwith a 488-nm argon ion laser. The data presented

are the mean fluorescent signals for 10,000 cells.

2.7. Confocal microscopy

Cells were seeded into 8-well chambers (Lab-TekR, Nalge

Nunc International, Naperville, IL, USA) (105 cells/cm2, 300 l

media/well) and incubated overnight at 37 C and 5% CO2.

Cells were rinsed twice, pre-incubated at 37 C for 1 h in serum-

free medium, and incubated at 37 C in the presence of FITC-

labeled NAcHis-GC nanoparticles (final particle concentration,

0.2 mg/ml) for 10 min to 6 h. The free nanoparticles were

aspirated, and thecells were washed three times with PBS (pH 7.4)

containing 0.1% BSA and fixed in 3.7% paraformaldehyde

solution at 4 C for 15 min. The samples were mounted in fluor-

escent mounting medium (Dako, Glostrup, Denmark), andfluorescence was monitored in two channels: green (for FITC;

excitation 490 nm, emission 520 nm) and red (for DiI; excitation

549 nm, emission 565 nm). The images were acquired using a

confocal microscope (Nikon Eclipse TE 2000, Tokyo, Japan).

2.8. Cell cycle analysis by flow cytometry [27,28]

Cells were plated in a 6-well culture dish and cultured at 37 C

in 5% CO2 until 80% confluent. Cells were rinsed twice and pre-

incubated for 1 h with 2 ml of serum-free medium at 37 C, and

incubated with PTX-loaded NAcHis-GC nanoparticles at 37 C

for 30min to24 h. The cells werewashed three times with 1 mlof

PBS (pH 7.4), harvested by trypsinization, washed once withPBS, and fixed in ice-cold 70% ethanol. The cells were washed

with PBS to remove residual ethanol, pelleted, and re-suspended

in propidium iodide (PI) stain buffer (50g/mlDNase-freeRNase

A and 50 g/ml PI in PBS) for 30 min, and the samples were

analyzed using a FACSCalibur flow cytometer.

3. Results

3.1. Preparation and characterization of NAcHis-GC self-

assembled nanoparticles

The amphiphilic GC derivative was prepared by conjugationof hydrophobic NAcHis onto hydrophilic GC through the

formation of an amide bond. The NAcHis contents in the con-

jugates were 6.88.7 wt.%, as measured by 1H-NMR spec-

troscopy (Table 1). When the amphiphilic conjugates were

dispersed in phosphate-buffered saline (PBS; pH 7.4) and

sonicated, the conjugates formed self-assembled nanoparticles

with mean diameters of 150250 nm as determined by dynamic

light scattering (Fig. 2 and Table 1). However, when the pH

of the medium was lowered to 6.0, dynamic light scattering did

not give any signal indicating the presence of nanoparticles,

suggesting that these self-assembled nanoparticles dissociate in

acidic pH due to loss of hydrophobicity caused by the pro-

tonation of imidazole groups.

Fig. 4. Endocytosis/exocytosis of NAcHis-GC-2 nanoparticles. HeLa cells were

incubated in the presence of nanoparticles for 10 min to 6 h, washed, and

incubated in fresh medium for an additional 30 min.



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3.2. Time- and concentration-dependent uptake of

nanoparticles

To monitor cellular internalization of NAcHis-GC nanopar-

ticles, they were labeled with FITC. The FITC contents in the

nanoparticles were 0.550.65 wt.% (Table 1). As determined by

fluorescence flow cytometry, cellular internalization of the

FITC-labeled NAcHis-GC nanoparticles was very rapid. Nano-

particle uptake was both time- and concentration-dependent,

and saturation of uptake was achieved in 6 h ( Fig. 3).

3.3. Endocytosis and exocytosis of nanoparticles

To investigate the dynamics of endocytosis and exocytosis,

cells were incubated with FITC-labeled NAcHis-GC nanopar-

ticles for 10 min to 6 h, the free nanoparticles were removed,

and the cells were incubated in fresh medium for an additional

30 min. When the pre-incubation times were relatively short,

10 min and 30 min, a large amount of the nanoparticles wasexocytosed after the removal of nanoparticles from the external

media (Fig. 4). However, the amount of extocytosed nanopar-

ticles decreased with increasing pre-incubation time, up to 6 h.

3.4. Release of model drugs from nanoparticles

The red-fluorescent dye DiI was used as a model drug to

study in vitro drug release from nanoparticles inside cells. DiI

was incorporated into NAcHis-GC nanoparticles by the emul-

sion/solvent evaporation method [26]. When the feed weight

ratio of DiI to NAcHis-GC-2 nanoparticles was 0.01, the load-

ing content of DiI was 0.0094 (0.94 wt.%), representing a 94%

loading efficiency. Flow cytometry analysis (Fig. 5) and

Fig. 5. DiI release from NAcHis-GC nanoparticles. HeLa cells were incubated in the presence of DiI-loaded, FITC-labeled NAcHis-GC-1 (A) and NAcHis-GC-2 (B)

nanoparticles for 10 min to 6 h. After repeated washings to remove free nanoparticles, the cells were analyzed by flow cytometry.



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confocal microscopy (Fig. 6) were used to evaluate the

efficiency of nanoparticles for cellular delivery of DiI.

DiI is a lipophilic tracer. It is weakly fluorescent in aqueous

environment. When DiI is incorporated into nanoparticles in

aqueous environment, it is virtually non-fluorescent. However,

it is highly fluorescent when incorporated into lipid membranes.

That is, DiI undergoes fluorescence enhancement upon in-sertion into lipid membranes such as plasma, endosome, and

lysosome membranes. Ten minutes after the treatment of the

cells with nanoparticles, significant green fluorescence, indic-

ative of the NAcHis-GC nanoparticles, was detected, whereas

red fluorescence, indicative of DiI release from the nanoparti-

cles, was barely detectable (Fig. 5). Red fluorescence intensity

increased with time, indicating that DiI was released from the

nanoparticles and transferred into membranes (Fig. 5). Confocal

imaging (Fig. 6) confirmed the cellular internalization of DiI-

loaded NAcHis-GC nanoparticles and DiI release into the

cytoplasm. When HeLa cells were incubated with DiI-loaded

nanoparticles for 10 min, red fluorescence from DiI was barelydetected in the cells and only weak green fluorescence were

observed in the cell membranes. After 6 h, however, red fluor-

escence was clearly observed in the cytoplasm (Fig. 6C). For

comparison, DiI and FITC-labeled NAcHis-GC nanoparticles

were separately added to the media and incubated with cells for

10 min to 6 h (Fig. 6D-F). At 10 min, selective staining of the

cell membranes by DiI was observed. At 6 h, however, DiI and

FITC-labeled nanoparticles were separately distributed in cells;

most of the DiI was present in the cell membranes, whereas the

nanoparticles were distributed throughout the cytoplasm as well

as in the cell membranes. In contrast, when DiI was delivered

with NAcHis-GC nanoparticles, a large amount of DiI was ob-

served in the cytoplasm.

3.5. Effect of nanoparticles on cell cycle arrest

PTX was incorporated into NAcHis-GC nanoparticles by the

emulsion/solvent evaporation method [26]. At a feed weight ratio

of PTX to NAcHis-GC-1 and NAcHis-GC-2 nanoparticles of

0.01, the loading contents of PTX was 0.64 and 0.61 wt.%,

representing a loading efficiency of 64% and 61%, respectively.

Uptake of PTX-loaded nanoparticles by A549 and MDA-MB231

cells and subsequent PTX release into the cytosol caused cell

cycle arrest in the G2-M phase (Fig. 7, Table 2). In A549 cells, the

G2-M population gradually increased over time, from 10% at zerotime to around 70% at 24 h, indicating that PTX-loaded NAcHis-

GC nanoparticles release PTX, which arrests cell growth. A

similar tendency was observed in MDA-MB231 cells. There was

no significant difference between NAcHis-GC-1 and NAcHis-

GC-2 nanoparticles.

4. Discussion

NAcHis-GC self-assembled nanoparticles were designed to

destabilize endosomal membranes, favoring the delivery of

drugs into the cytosol. A proposed model illustrating the cellular

internalization and drug release of the nanoparticles is shown in

Fig. 8.Cellular internalization is initiated by nonspecific interac-

tions between nanoparticles and cell membranes. A549 cell

uptake of chitosan nanoparticles was reported to occur pre-

dominantly by adsorptive endocytosis, mediated in part by

clathrin, but not by passive diffusion or by fluid-phase endo-

cytosis [29]. Cellular uptake of NAcHis-GC nanoparticles also

seems to occur by adsorptive endocytosis initiated by non-

specific interactions between nanoparticles and cell membranes.

Even in the absence of ligand binding to specific cell membrane

receptors, cellular uptake of NAcHis-GC nanoparticles was

initiated rapidly, within 10 min. Chitosan, which has a positive

zeta potential, can interact with cell membranes expressing ne-gative zeta potential by nonspecific electrostatic interactions

[30], and there have been no reports of chitosan-specific re-

ceptors on cell membranes.

NAcHis-GC nanoparticles internalized by adsorptive endo-

cytosis appear to be exocytosed (Fig. 8A) or localized in

endosomes (Fig. 8B). Unexpectedly, exocytosis of nanoparti-

cles was fairly active following the removal of nanoparticles

from the external media. Even after long pre-incubation time

(6 h), many nanoparticles were still exocytosed. (Fig. 4). Exo-

cytosis of NAcHis-GC nanoparticles was dependent on the pre-

incubation time. The amounts of exocytosed nanoparticles de-

creased with increasing the pre-incubation time, indicating that

exocytosis is dependent on the progress of endocytosis.

Fig. 6. Confocal images of HeLa cells. Cells were incubated in the presence of

DiI-containing, FITC-labeled NAcHis-GC-2 nanoparticles for 10 min (A), 1 h

(B), and 6 h (C). Confocal images were obtained from two channels and

overlaid: green, indicative of FITC-labeled NAcHis-GC-2 nanoparticles

(excitation 490 nm, emission 520 nm) and red, indicative of DiI (excitation

549 nm, emission 565 nm). (DF) DiI and FITC-labeled NAcHis-GC-2

nanoparticles were separately added to the media and cells were incubated for

10 min (D), 1 h (E), and 6 h (F). (For interpretation of the references to colour inthis figure legend, the reader is referred to the web version of this article.)



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Fig. 7. Cell cycle distribution of A549 (A and B) and MDA-MB231 (C and D) cells. Cells were incubated in the presence of PTX-loaded NAcHis-GC-1 nanoparticles

(A, C) or PTX-loaded NAcHis-GC-2 nanoparticles (B, D), and analyzed by flow cytometry. The equivalent PTX concentration was 0.3 g/ml.



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Although about 65% of the internalized fraction of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles were found to un-

dergo exocytosis within 30 min [31], the dependence of exo-

cytosis on the pre-incubation time was contrary to that reported

here. That is, whereas pre-incubation time had little effect on the

exocytosis of PLGA nanoparticles, it has a significant effect on

the exocytosis of NAcHis-GC nanoparticles. This was probably

due to the different natures of the respective nanoparticles.

Once drug-loaded nanoparticles are localized in endosomes,

they should overcome the endosomal membrane and translocate

the drugs to the cytosol. This is especially important if the drugs

are sensitive to lysosomal enzymes. To facilitate escape fromthe endocytotic pathway to lysosomes and transfer more drugs

into the cytosol, we used N-acetyl histidine as the hydrophobic

moiety of self-assembled nanoparticles. N-acetyl histidine is

hydrophobic at neutral pH, and, when conjugated to hydrophilic

glycol chitosan, the amphiphilic conjugate formed nano-sized

self-aggregates, similar to other hydrophobically-modified chi-

tosans such as deoxycholic acid-modified [20,21,32], cholanic

acid-modified [23], and adriamycin-conjugated chitosans [22].

At neutral pH, NAcHis-GC formed self-assembled nanoparti-

cles with mean diameters of 150250 nm, but at acidic pH (4.0

Table 2

Cell cycle distribution (10,000 cells/count)

A549 MDA-MB231

Treatment Cell cycle distribution % Treatment Cell cycle distribution %

G0-G1 S G2-M G0-G1 S G2-M

30 min Control 46.86 43.14 10.01 30 min Control 52.29 38.62 9.09 NAcHis-GC-1 47.77 44.84 7.39 NAcHis-GC-1 56.65 35.70 7.65

NAcHis-GC-2 47.43 44.90 7.67 NAcHis-GC-2 55.77 36.38 7.85

1 h NAcHis-GC-1 50.83 37.46 11.72 1 h NAcHis-GC-1 56.93 35.82 7.26


6 h NAcHis-GC-1 37.78 40.31 21.91 6 h NAcHis-GC-1 39.51 40.30 20.19


12 h Control 66.98 20.9 12.12 12 h Control 58.24 22.21 19.56



24 h Control 74.57 18.64 6.78 24 h Control 74.65 15.64 9.42



Fig. 8. Schematic representation of a proposed model for the cellular internalization and drug release of NAcHis-GC nanoparticles. (A) Internalization of NAcHis-GC

nanoparticles is initiated by nonspecific interactions between nanoparticles and cell membranes, adsorptive endocytosis. (B) A part of the nanoparticles is exocytosed.

(C) Without a specific mechanism for endosomal escape, drug-loaded nanoparticles are trafficked to lysosomes, where a high level of lysosomal enzymes is present.

Drugs sensitive to these enzymes are degraded and lose their activity. (D) Under slightly acidic environments in endosomes, the imidazole group of histidine isprotonated, causing the disruption of endosomal membranes and simultaneous delivery of drugs into the cytosol.



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6.0), light scattering measurements showed no evidence for the

presence of nanoparticles, indicating that the nanoparticles were

disassembled at acid pH due to the protonation of imidazole

groups and subsequent loss of hydrophobicity. The disassembly

of NAcHis-GC nanoparticles at acidic pH may provide a me-

chanism for escape from the acidic endo-lysosome compart-

ments and release of drugs into the cytosol.Flow cytometry analysis and confocal microscopy con-

firmed this chain of cellular events, including the internalization

of NAcHis-GC nanoparticles and drug release into the cytosol.

Moreover, cell cycle analysis demonstrated that PTX was

released into the cytosol from NAcHis-GC nanoparticles and

was effective in reducing cell growth. These findings indicate

that NAcHis-GC nanoparticles may be promising vehicles for

intracytoplasmic delivery of drugs, proteins, and genes.

Acknowledgments

This study was financially supported by the Asan Institutefor Life Sciences (Grant No. 2006-395) and the Ministry of

Science and Technology (Grant No. M10414030002-05N1403-

00240) in Korea.

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n-acetyl histidine glycol chitosan

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