Stability and in vivo evaluation of pullulan acetate as a drug nanocarrier

Stability and in vivo evaluation of pullulan acetate as a drug nanocarrier

Nanoparticles (NPs) have specific physicochemical properties differing from bulk materials of the same composition, and such properties make them very attrac-tive for commercial and medical development (Curtis

et al., 2006; Lanone & Boczkowski, 2006; Medina et al., 2007) However, compared with conventional materials, NPs may have differential toxicity profiles due to their different chemical and structural properties (Vega-Villa

et al., 2008) For example, the interaction between nano-materials and biological components, e.g proteins and cells may lead to their unique bio-distribution, clearance, immune response, and metabolism, and therefore it is

very necessary to study the in vivo toxicity of nanomateri-als (Fischer & Chan, 2007)

Pullulan, a very important neutral and linear natural polysaccharide, has been used as a good biomaterial in drug and gene delivery, tissue engineering, and other fields (Leathers, 2003; Shingel, 2004; Rekha & Sharma, 2007) Many investigations (Akiyoshi et al., 1993; Jeong

et al., 2006) have reported that some hydrophobized pullulan such as cholesterol-modified pullulan can form self-aggregated NPs to be used as the carrier for drug delivery As one of the most conventional hydro-phobized pullulan derivatives (Jung et al., 2003; Na et al., 2003; 2004, Park et al., 2007), pullulan acetate (PA) and its modified materials can form self-aggregated NPs in

Drug Delivery, 2010; 17(7): 552–558

Address for Correspondence: Qi-Qing Zhang, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences, Peking Union Medical College,

The Key Laboratory of Biomedical Material of Tianjin, Tianjin 300192, PR China Tel/Fax: +86 22 87890868 E-mail: zhangqiq@126.com

R E S E A R C H A R T I C L E

Stability and in vivo evaluation of pullulan acetate as a drug nanocarrier

Hong-Bo Tang1, Lei Li1, Han Chen1, Zhi-Min Zhou1, Hong-Li Chen1, Xue-Min Li1, Ling-Rong Liu1, Yin-Song Wang3, and Qi-Qing Zhang1,2

1 Institute of Biomedical Engineering, Chinese Academy of Medical Sciences, Peking Union Medical College, The Key Laboratory of Biomedical Material of Tianjin, Tianjin 300192, PR China, 2 Research Center of Biomedical Engineering, Department of Biomaterials, College of Materials, Xiamen University, Technology Research Center of Biomedical Engineering of Xiamen City, The Key Laboratory of Biomedical Engineering of Fujian Province, Xiamen 361005, PR China, and 3 College of Pharmacy, TianJin Medical University, Tianjin, 300070, PR China

Abstract

To develop pullulan acetate nanoparticles (PANs) as a drug nanocarrier, pullulan acetate (PA) was synthesized and characterized Its acetylation degree determined by the proton nuclear magnetic resonance ( 1 H NMR) was 2.6 PANs were prepared by the solvent diffusion method and characterized by transmission electron

microscope (TEM), size distribution, and ζ potential techniques PANs had nearly spherical shape with a size range of 200–450 nm and low ζ potentials both in distilled water and in 10% FBS The storage stability of PANs was observed in distilled water PANs were stored for at least 2 months with no significant size and ζ potential

changes The safety of PANs was studied through single dose toxicity test in mice, and the result showed that PANs were well tolerated at the dose of 200 mg/kg in mice Epirubicin-loaded PANs (PA/EPI) were also prepared and characterized in this study Moreover, the in vivo pharmacokinetics of PA/EPI was investigated Compared with the free EPI group, the PA/EPI group exhibited higher plasma drug concentration, longer half-life time

(t1/2) and the larger area under the curve (AUC) All results suggested that PANs were stable, safe, and showed

a promising potential on improving the bioavailability of the loaded drug of the encapsulated drug.

Keywords: Pullulan acetate; nanoparticles; pharmacokinetics; toxicity; epirubicin

(Received 26 December 2009; revised 20 April 2010; accepted 28 April 2010)

ISSN 1071-7544 print/ISSN 1521-0464 online © 2010 Informa Healthcare USA, Inc.

2010

17

7

552

558

26 December 2009

20 April 2010

28 April 2010

1071-7544

1521-0464

© 2010 Informa Healthcare USA, Inc.

10.3109/10717544.2010.490250

DRD 490250

aqueous media The hydrophobic core of these

self-assembled NPs formed by the hydrophobic interactions

was considered to act as a reservoir of hydrophobic

substances In our previous report (Zhang et al., 2009),

PANs prepared by the solvent diffusion method had the

potential to be used as a sustained release carrier for

Epirubicin (EPI) in vitro Some investigations (Gu et al.,

1998; Na et al., 2004; Shimizu et al., 2008) showed that

NPs of hydrophobized pullulans had good

morphol-ogy, drug loading, and release properties in vitro, but

the biological effects of hydrophobized pullulan NPs

in vivo have not been investigated deeply up to date

Herein, based on our previous work, we studied the

stability and toxicity of PANs, and further investigated

the sustained release behavior in vivo of drug loading in

PANs PA was firstly synthesized and characterized by

FT-IR and 1H NMR, and then PANs with moderate size

and low potential were prepared by the solvent diffusion

method The storage stability of PANs was studied in the

aqueous medium, and the acute toxicity of PANs was

evaluated in mice Morever, EPI was loaded into PANs

and its pharmacokinetics was also assessed in rats to

compare to the free drug

Methods

Materials

Pullulan (Mw = 200,000) was purchased from Hayashibara

(Tokyo, Japan) Epirubicin·HCl (EPI·HCl) was purchased

from Hisun Pharmaceutical Co (Zhejiang, China) Poly

(vinyl alcohol) (PVA) with an average molecular weight

of 30,000–70,000 was obtained from Sigma-Aldrich (St

Louis, MO) All reagents for high performance liquid

chromatography (HPLC) analysis, including acetonitrile

and methanol, were HPLC grade Other chemical reagents

were of analytical grade and obtained from commercial

sources ICR mice and Wistar rats were purchased from

the Institute of Radiology, Chinese Academy of Medical

Science All animal experiments were performed in

compliance with the Institutional Animal Care and Use

Committee (IACUC) guidelines

Synthesis and characterization of PA

PA was synthesized according to the method described

in previous literature (Jung et al., 2003; Zhang et al.,

2009) Briefly, pullulan (2 g) was suspended in 20 ml of

formamide and dissolved by vigorous stirring at 54°C

Pyridine (6 ml) and acetic anhydride (5.5 ml) were added

to the above solution, and the mixture was subsequently

stirred at 54°C for 48 h The reactant was precipitated with

distilled water, and then washed with distilled water and

methanol The solid material was vacuum-dried at 50°C

for 48 h The final product was identified by Fourier trans-form infrared (FT-IR) (Thermo, Nicolet is10, Los Angeles,

CA ) and 1H NMR (Varian, Varian INOVA 400 M NMR, Palo Alto, CA) spectrometry The degree of substitution (DS) was defined as the number of acetyl groups per glucose unit of pullulan It was determined by 1H NMR (Zhang et al., 2009) The DS values are expressed by the

following equations: DS = 10A/(3B + A), where A is the integration value of acetyl protons at 1.8–2.2 ppm and B

is that of OH protons and H-1 to H-6 protons of pullulan moiety observed at more than 3.5 ppm

Preparation and characterization of PANs and PA/EPI

Nanoparticles were prepared according to a solvent dif-fusion method (Fessi et al., 1989; Govender et al., 1999; Bilati et al., 2005; Zhang et al., 2009) Briefly, PA (100 mg) was dissolved in 10 ml of N, N-Dimethylformamide (DMF) The solution was then added to 0.5% PVA aqueous solution through a syringe under moderate magnetic stirring The produced PANs were collected with centrifugation (Beckman Coulter, Inc Avanti J-25, Fullerton, CA) at 18,000 rpm for 15 min at 4°C Subsequently, PANs were dispersed in the distilled water and 10% fetal bovine serum (FBS), respectively, with PA concentration of 1 mg/ml to carry out the later experiments

EPI-loaded PANs were prepared as follows: EPI·HCl (10 mg) was dissolved in DMF (2 ml) and then triethyl-amine (TEA) was added to this solution to remove hydrochloride The mixed solution was stirred in the dark for 12 h PA (100 mg) dissolved in 8 ml of DMF was added into this mixed solution The PA/EPI were collected with centrifugation (Beckman Coulter, Inc Avanti J-25, Fullerton, CA) at 18,000 rpm for 15 min at 4°C, then the supernatant removed and washed twice with distilled water, at last, dispersed in distilled water

by sonication for several minutes with a probe-type sonifier (Automatic Ultrasonic Processor UH-500A, China) at 100 W

The particle size and ζ potential were determined

by dynamic light scattering (Malvern Instruments Ltd., Zeta sizer 2000, Worcestershire, UK) The morphology

of NPs was observed using TEM (FEI, TECNAI G2F-20, Eindhoven, Holland)

The stability of PANs in water

In order to study the stability of PANs, the above disper-sions were stored for 2 months at 4°C Macroscopic char-acteristics of NPs dispersions such as opalescence and precipitation were observed from time to time Moreover, the size and ζ potential of PANs were also determined by

dynamic light scattering method once a month, and all measurements were performed in triplicate

In vivo toxicity

The toxicity of PANs was evaluated in vivo according to

the previous reported method (Yoksan & Chirachanchai,

2008; Sonaje et al., 2009) Adult male and female ICR mice

(18–22 g) were randomly divided into two groups, each

with 10 mice The experimental group received a single

intravenous (i.v.) dose of blank PANs (200 mg/kg); the

other group was treated with a single i.v dose of normal

saline All animals were fed with normal diet, and water

was provided ad libitum Animals were observed

care-fully for the onset of any signs of toxicity and monitored

for changes in food intake and body weight at 1, 8, and

15 days After being sacrificed at 15 days, internal organs

of each animal were harvested and observed grossly For

histological examinations, specimens of major organs

such as heart, liver, spleen, lung, and kidney were fixed

in 10% phosphate buffered formalin, embedded in

paraf-fin, sectioned, and stained with hematoxylin and eosin

(H&E)

In vivo pharmacokinetics and bioavailability

In vivo pharmacokinetic study was conducted by the

routine method (Mross et al., 1988; Bibby et al., 2005;

Cao & Feng, 2008; Devalapally et al., 2008) The drug

content of the PA/EPI was determined according to

our previous study (Zhang et al., 2009) and PA/EPI with

3.34% was selected to carry out the pharmacokinetics

studies Female Wistar rats of 180~250 g and 4~6 weeks

old were held in an air-conditioned facility, provided

with standard food and filtered water Animals were

ran-domly assigned to two groups, each with six rats, which

received an i.v injection via the tail vein of free EPI and

the PA/EPI solution in saline at 10 mg/kg equivalent

dose, respectively All animals were observed for

mortal-ity, general condition, and potential clinical signs

The blood samples were collected with heparinized

tube at 0 (pre-dose), 0.0167, 0.0833, 0.167, 0.5, 1, 2, 4, 8,

12, 24, and 48 h post-treatment Plasma samples were

harvested by centrifugation at 3000 rpm for 15 min and

stored at −20°C until analysis Liquid–liquid extraction

was performed prior to the HPLC analysis Briefly, the

plasma (100 µl) was mixed with

dichloromethane-meth-anol (4:1, v/v) on a vortex-mixer for 3 min to extract the

drug Upon centrifugation at 10,000 rpm (15,000 g) for

15 min, the upper aqueous layer was removed by

aspi-ration and the organic layer was transferred to a tube

and evaporated under nitrogen at 50°C The residue was

dissolved in 100 µl of anhydrous methanol by vortex For

the HPLC analysis, the C-18 column was used and the

mobile phase (0.02 M KH2PO4/CH3CN/CH3OH = 49:17:34

v/v/v) was delivered at a rate of 1 ml/min Sample (20 μl)

was injected and the column effluent was detected with

a UV detector at 232 nm The main pharmacokinetic

parameters were calculated by DAS 1.0 (Anhui, China) program

Bioavailability (BA) is a measurement of the rate and extent of a therapeutically active drug that reaches the systemic circulation and is available at the site of action When a medication is administered intravenously, its bioavailability is 100% When the standard consists of intravenously administered drug, this is known as relative bioavailability (BAR) The BAR of PA/EPI after administra-tion was calculated using the following formula (Sonaje

et al., 2009):

R

where AUC is the area under the curve

Statistical analysis

All data are presented as a mean value with its stand-ard deviation indicated (mean ± SD) Statistical analysis

was conducted using the Student’s t-test Differences

were considered to be statistically significant when the

p-values were less than 0.05.

Results and discussion

Characterization of PA

Pullulan has three free hydroxyl groups on each glucose unit It is easy to synthesize hydrophobic pullulan deriva-tive, PA, by means of replacing the hydroxyl groups of the glucose unit with acetate groups Figure 1 shows FT-IR spectra of pullulan and PA, which were similar to our previous literature (Zhang et al., 2009) Figure 2 shows

1H NMR spectra of pullulan and PA in DMSO-d6 The acetylation degree of PA calculated by 1H NMR method was 2.6, which is smaller than 2.7 reported by Zhang et al (2009) Based on our previous report, PA with lower DS may form smaller NPs in size Moreover, Li and Huang

a b

Figure 1 FT-IR spectra of PA (a) and pullulan (b).

(2008) also reported that the smaller size of NPs may be

in favor of longer circulation time in vivo Therefore, PA

with acetylation degree of 2.6 was used to prepare PANs

and PA/EPI in our study

Characterization of PANs and PA/EPI

The self-assembled NPs were prepared by solvent

dif-fusion method This method had several advantages

such as a rapid and simple preparation procedure, great

potential for large industrial scale production, and easy

control of the particle size (Zhang et al., 2009) The size

and the size distributions of PANs and PA/EPI in distilled

water were measured by DLS The morphological

char-acteristics were observed by TEM at the same time As

shown in Table 1, the mean diameters of PANs and PA/

EPI were 247.6 ± 34.8 nm and 343.4 ± 87.7 nm, with the

narrow size distributions (the polydispersity indexes

(PDI) < 0.3, Figure 3) Under TEM observations (Figure 4),

PANs and PA/EPI were nearly spherical in shape and

uni-form sized

ζ potential of NPs in different media

The ζ potentials of PANs and PA/EPI in distilled water

were −3.353 ± 1.296 mV and −3.297 ± 1.025 mV To model

the circulation in vivo, we observed the ζ potentials of

NPs in 10% FBS, which had a composition very similar

to the body liquids The ζ potentials of PANs and PA/EPI

in 10% FBS were −1.460 ± 0.297 and −1.902 ± 1.112 mV According to the previous report (Levchenko et al., 2002; Alexis et al., 2008), neutral NPs would exhibit a decreased rate of macrophage phagocytosis system (MPS) uptake However, MPS is the major contributor for the clearance

of NPs, thus the reducing rate of MPS uptake could be considered as the best strategy for prolonging the cir-culation of NPs (Li & Huang, 2008) Therefore, PA/EPI prepared in this study would exhibit the longer blood circulation time than free EPI in vivo

The storage stability of PANs

Stability is an important facet of preparation and a neces-sary step in the development process, it will indicate the potentiality of industry production The possibilities of modulating the loaded drug’s pharmacokinetic param-eters are dependent on physicochemical properties such

as stability, size, and surface characteristics (Lourenco

et al., 1996) PANs dispersions maintained slight opal-escence within 2 months As shown in Table 2, the size, size distribution, and ζ potential of PANs showed no

sig-nificant changes during 2 months Therefore, it could be concluded that PANs in aqueous media were stable for

at least 2 month at 4°C Stabilization of colloidal systems

is traditionally viewed as arising from either electrostatic

or steric effects (Lourenco et al., 1996) A ζ potential of at

least −30 mV for electrostatic stabilized systems is desired

to obtain a physically stable suspension according to the literature (Müller & Jacobs., 2002) The higher zeta poten-tial value indicates the better stability (Dai et al., 2010) The absolute value of ζ potential was lower than 5 mV

in our study The result showed that electrostatic stabi-lization was not provided efficiently in the suspension;

5.0

a

b

Figure 2 1 H NMR spectra of pullulan (a) and PA (b) (DMSO-d6).

1000 100

Size (d.nm)

Size Distribution by intensity

10 1

20 a

15 10 5 0

10000

100 80 60 40 20 0

1000 100

Size (d.nm)

Size Distribution by intensity

10 1

20 b 15 10 5 0

10000

100 80 60 40 20 0

Figure 3 Diameter distributions of PANs (a) and PA/EPI (b).

Table 1 Size and PDI of PANs and PA/EPI ( ± s, n = 6).

therefore, a steric stabilization may occur and provide the

stability of the PANs It may also be important information

for developing a new nanosuspension liquid formulation

about pullulan acetate

In vivo toxicity

To evaluate whether i.v administration (at dose of

200 mg/kg) of PANs was associated with any toxicity in

vivo, animals were treated with a single dose of empty

NPs No significant differences between the PANs group

and the control group in clinical signs, e.g diarrhea, fever,

and other systemic symptoms, and no mortality occurred

throughout the entire study course Additionally, there

were no significant differences in body weight and food

intake for both male and female mice between the two

studied groups (Tables 3 and 4) Figure 5 shows the

microscopic examination of major organs including

heart, liver, spleen, lung, and kidney sections stained with H&E Pathological changes in the major organs including heart, liver, spleen, lung, kidney, stomach, and intestinal segments were scarcely observed at 15 days Moreover,

no evidence of inflammatory reactions was observed in the experimental group All the above results indicated that no apparent toxicity of the PANs was found in the

experimental animals after i.v at a dose of 200 mg/kg.

In vivo pharmacokinetics

The plasma levels of EPI were determined following a

sin-gle i.v injection of EPI or PA/EPI (10 mg/kg EPI equiv.) in

female Wistar rats The plasma levels over 48 h are shown

in Figure 6 and the PK parameters are summarized in Table 5 The peak concentration of total EPI in plasma was 14.65 mg/L at 1 min after injection and then decreased to nearly undetectable levels after 24 h The maximum

con-centration (Cmax) of PA/EPI at 5 min was 9.80 mg/L, which was obviously lower than that of EPI because of sustained release from NPs According to the previous report (Mross

et al., 1988; Jakobsen et al., 1991; 1994), free EPI rapidly

Table 2 Diameter and ζ potential of PANs ( ± s, n = 3).

Table 3 Body weight of PANs i.v injected in mice at 200 mg/kg dose

( ± s, g).

Table 4 Food intake of PANs i.v injected in mice at 200 mg/kg dose

( , g).

Liver

Spleen

Kidney Lung Heart

Figure 5 Representative photomicrographs of the heart, liver, spleen,

lung, and kidney sections (H&E staining) of mice of control group (a) and treated with test NPs (b).

a

b

Figure 4 Transmission electron micrographs (TEM) of (a) PANs and

(b) PA/EPI.

disappeared from the circulation due to its short half-life

In this study, the terminal elimination half-life of free EPI

was 8.129 h (Table 5), which was consistent with that of

7.3 h of doxorubucin (Gustafson et al., 2002; Bibby et al.,

2005) In contrast, PA/EPI showed a much longer

circula-tion time and its eliminacircula-tion half-life time was 17.231 h,

2.12-times that of free EPI The clearance of EPI loaded in

NPs was 0.104 L/h, which is 3.16-times smaller than free

EPI Moreover, the mean residence time (MRT) of PA/

EPI in the plasma was 15.854 h, 1.9-times that of free EPI

Altogether, PA/EPI had the longer t1/2 and MRT, lower Vd

and CL than the free drug in rats At the same time, the

AUC of EPI and PA/EPI from 0–24 h were 48,128.269 and

91,006.508 µg h/L, respectively The bioavailability of free

EPI was 100%, the BAR of PA/EPI calculated through the

equation above by the AUC0–24h was 189%, much higher

than the free drug According to the previous report (Li

& Huang, 2008), we believed that the longer t1/2 of PA/EPI

in plasma may be due to its low ζ potential and

moder-ate size As prolonged plasma circulation is the driving

force for increased tumor targeting (Seymour et al., 1995),

Tsuchihashi et al (1999) prepared long circulating

lipo-somes to improve therapeutic efficacy of doxorubicin

In this study, the slow release of EPI from PANs in the

blood suggested that the bioavailability of EPI improved

when it was loaded into PANs However, whether it could enhance the uptake of passive permeability in targeting tumor tissues or not needs to be further proved by the experiments such as bio-distribution study and pharma-codynamic test

Conclusions

In this study, PA with the DS of 2.6 was synthesized and characterized PANs and PA/EPI with moderate size and low potential were prepared by the solvent diffusion method The nanoparticles were stable in aqueous media for at least 2 months in vitro, furthermore, PANs were safe

in mice at 200 mg/kg and showed the potential to improve the bioavailability of the loaded drug in vivo

Declaration of interest

This work was supported by the Major State Basic Research Program of China (No 2006 CB933300)

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