Decrease in the Particle Size of Paclitaxel by Increased Surface Area Fractional Precipitation Process Using Hydrophilic Polymer

표면적이 증가된 분별침전에서 친수성 고분자물질을 이용한 paclitaxel의 입자 크기 감소

임용규, 김진현*

공주대학교화학공학부

Received: October 12, 2015 / Revised: October 30, 2015 / Accepted: November 6, 2015

서 론

Paclitaxel

^,

^,

^(Kaposi’s sarcoma)

(non-small cell lung cancer)

⁽

⁾

[16, 19]. Paclitaxel

방법에는주목나무

^{(yew tree)}

^,

나무에서전구체

(baccatin III, 10-deacetylpaclitaxel

⁾

^,

[1, 3, 14].

^,

생산이가능하기때문에일정한품질의

paclitaxel

산할수있다는장점이있다

^.

일반적으로 물에잘녹지않는 원료의약품

(active phar- maceutical ingredient, API)

그활용도를높이고자한다

^.

^,

^,

향상시킬수있다

^{[2, 7].}

분및잔류용매제거에도상당히도움이된다

[12]. Paclitaxel

Biopharmaceutics classification system(BCS) class IV

용도에많은제한이있다

^{[7, 8].}

^,

“top-down(breaking-down)”

“bottom-up(building-up)”

이이용되고있다

^[2].

한방식의

^milling

로분쇄하는

^top-down

요구되고의약품의오염이나분해가발생할수있다

^.

^,

^,

는

bottom-up

수있다

^.

bottom-up

^.

기존문헌에의하면분별침전시다양한종류의이온교환 수지를사용하여반응액부피당표면적을증가시킴으로써

paclitaxel

Decrease in the Particle Size of Paclitaxel by Increased Surface Area Fractional Precipitation Process Using Hydrophilic Polymer

Yong Kyu Lim and Jin-Hyun Kim*

Department of Chemical Engineering, Kongju National University, Cheonan 331-717, Republic of Korea

In this study, we applied an increased surface area fractional precipitation with hydrophilic polymer for decreasing the particle size of the anticancer agent paclitaxel from plant cell cultures. The addition of polymer resulted in a considerable decrease in the size of the paclitaxel precipitate. Among the polymers (HPMC 2910, PVP-K90, PVA) used, PVP-K90 was the most effective for the inhibition of precipitate growth. When PVP-K90 (0.2%, w/v) was used, the paclitaxel particles were four to five times smaller, having less than a 20 µm radius, than those obtained in the absence of the polymer. In addition, the precipitate size was inversely correlated with the absolute value of zeta potential.

Keywords: Paclitaxel, increased surface area fractional precipitation, polymer, particle size, decrease

*Corresponding author

Tel: +82-41-521-9361, Fax: +82-41-554-2640 E-mail: jinhyun@kongju.ac.kr

© 2015, The Korean Society for Microbiology and Biotechnology

(

⁾

^[11].

megestrol acetate

^[2].

적으로작은입자의

paclitaxel

상된다

^.

^,

paclitaxel

^.

(Amberlite IRA 400 OH)

[PVA(polyvinyl alcohol, MW 31,000-50,000), HPMC 2910(hydroxypropyl methylcellulose 2910), PVP-K 90 (polyvinylpyrrolidone-K 90)]

간에따른

paclitaxel

로입자크기를효과적으로감소시킬수있는지를평가하 였다

^.

재료 및 방법

식물재료

본실험에사용된식물세포배양액은

Taxus chinensis

으로부터 얻은 세포주

(cell line)

,

Gamborg’s B5

^[5]

²⁴

^C,

^{(150 rpm)}

^.

^decanter (Westfalia, CA150 Claritying Decanter)

(

-Laval, BTPX205GD-35CDEEP)

(cell debris)

^.

^.

⁽

⁾

^.

Paclitaxel 분석

Paclitaxel

HPLC system(Waters, USA)

Capell Pak C

250

4.6 mm, Shiseido, Japan)

^.

(35/65-65/35, v/v, gradient mode)

1.0 ml/min

^.

^{20 ml}

^{227 nm}

^UV

^{. HPLC}

Sigma-Aldrich

⁽

^{: 95%)}

^[15].

분별침전(fractional precipitation)을 위한 시료준비 식물세포배양액으로부터회수한바이오매스와메탄올의

비율을

^1/1(w/v)

⁴

(

^30%)

^.

^-

에메틸렌클로라이드를첨가

⁽

^25%)

^,

30 min

^.

^-

액추출을수행하여

paclitaxel

로라이드층으로

paclitaxel

^/

^.

(crude extract)

^{20% (v/w)}

sylopute(Fuji Silysia Chemical Ltd.,

Japan)

50% (w/w)

40

C

^{30 min}

^.

³⁰

^C,

^,

⁽

^/

= 1/10, v/v).

paclitaxel

³⁵

^C

²⁴

^,

⁽

paclitaxel

: 0.5%, w/v)

^61.5%

⁴

^C

paclitaxel

^.

35

C

²⁴

^{ODS C}

(50 mm

500 mm, Shiseido, Japan)

^Silica

^{(50 mm}

500 mm, Shiseido, Japan)

^HPLC

^[13].

paclitaxel

^98.0%

^{Fig. 1}

^.

표면적이 증가된 분별침전(increased surface area fractional precipitation)

표면적이증가된분별침전공정의개략도를

^{Fig. 2}

내었다

^.

^{[2, 4]}

효과적인세종류의 고분자물질

(HPMC 2910, PVP-K 90, PVA 31,000

^50,000)

(0.1, 0.2, 0.3%, w/v)

^.

⁽

paclitaxel

^{: 0.3%,}

w/v),

^61.5%

류수를교반

^{(~180 rpm)}

^.

전공정의반응액부피당표면적

^(S/V)

수지

(Amberlite IRA 400 OH)

(S/V = 0.428 mm

)

⁴

^C

(KCL-2000W, EYELA, Japan)

paclitaxel

^[11].

³⁵

^C

^.

간변화에따른

paclitaxel

하여관찰하였다

^.

침전물 형태 및 크기 확인

분별침전동안

paclitaxel

을 위하여 전자현미경

(SV-35 Video Microscope System,

Some Tech., Korea)

^[6].

paclitaxel

⁽

¹⁰⁰⁾

^.

paclitaxel

IT-Plus software(Some Tech., Korea)

paclitaxel

^.

제타전위(zeta potential) 측정

표면적이증가된분별침전공정에서고분자물질을첨가하

지않은경우와동일농도

^{(0.2% w/v)}

질

(PVA, HPMC 2910, PVP-K90)

⁽²⁴

⁾

^{. ELS-Z}

(Photal, Japan)

된제타전위는

ELS-Z software

^.

결과 및 고찰

고분자물질이 입자 형태에 미치는 영향

본연구에서는

paclitaxel

^,

Fig. 1. Preparation of sample for increased surface area fractional precipitation with hydrophilic polymer. The process consists of biomass extraction, liquid-liquid extraction, adsorbent treatment, hexane precipitation, fractional precipitation, ODS-HPLC, and silica- HPLC.

Fig. 2. Schematic diagram of increased surface area frac-

tional precipitation with hydrophilic polymer for decreasing

the particle size of paclitaxel precipitate. The surface area per

working volume (S/V: 0.428 mm

) of the reactor was increased by

addition of ion an exchange resin (Amberlite IRA 400 OH). The

methanol composition in water, pure paclitaxel content, and tem-

perature were 61.5% (v/v), 0.3% (w/v), and 4

C, respectively.

Fig. 3. Electron micrograph of paclitaxel precipitates formed by increased surface area fractional precipitation at 6 −24 h:

(A) Control, (B) 0.1% (w/v) HPMC 2910, (C) 0.2% (w/v) HPMC 2910, (D) 0.3% (w/v) HPMC 2910, (E) 0.1% (w/v) PVP-K90, (F) 0.2%

(w/v) PVP-K90, (G) 0.3% (w/v) PVP-K90, (H) 0.1% (w/v) PVA, (I) 0.2% (w/v) PVA, (J) 0.3% (w/v) PVA. The methanol composition in

water, pure paclitaxel content, and temperature were 61.5% (v/v), 0.3% (w/v), and 4

C, respectively. Scale bar indicates 10 µm.

전공정에고분자물질을첨가하여분별침전을수행하였다

^.

paclitaxel

⁽

⁾

^.

분자물질첨가유무에따른입자형태를

^{Fig. 3}

다

^.

(control, Fig. 3A),

^.

^{HPMC 2910}

^{(0.1, 0.2,} 0.3%, w/v)

(Fig. 3B, C, D),

물질이첨가되지않은경우

^{(Fig. 3A)}

형태

⁽

⁾

^.

paclitaxel

다

^.

^PVP-K90

(0.1, 0.2, 0.3%, w/v)

(Fig. 3E, F, G),

^{HPMC 2910}

^.

^PVA

(0.1, 0.2, 0.3%, w/v)

(Fig. 3H, I, J),

(0.3%, w/v)

^.

고분자물질첨가유무에따른입자크기를

^{Fig. 4}

^.

(control),

6, 12, 18, 24

48.4, 47.2,

45.8, 45.3

^m

미하게감소하는경향을보였다

^.

⁽

⁾

paclitaxel

커지는경향을보였다

^[11].

해표면적이증가된분별침전의경우침전시간이경과함에 따라입자크기가오히려감소하였는데

(Fig. 4, control),

[9]. HPMC 2910

(Fig. 4A),

6, 12, 18, 24

^0.1%

(w/v)

46.3, 37.4, 35.4, 34.8

^m,

^0.2%(w/v)

26.5, 23.0, 21.2, 21.0

^m,

^0.3%(w/v)

41.0, 33.3, 32.8, 32.3

^m

. HPMC 2910

^0.1%(w/v)

0.2%(w/v)

paclitaxel

^0.2%(w/v)

^0.3%(w/v)

가할경우에는

paclitaxel

였다

^.

, HPMC 2910

0.2%(w/v)

paclitaxel

^{. PVP-K90}

(Fig. 4B),

6, 12, 18, 24

각각 농도

^0.1%(w/v)

27.5, 27.8, 27.6, 25.7

^m,

0.2%(w/v)

17.2, 17.1, 17.0, 16.8

^m,

^0.3%(w/v) Fig. 4. Effect of polymer concentration on the particle size of paclitaxel during increased surface area fractional precipita- tion: (A) HPMC 2910, (B) PVP-K90, (C) PVA. The methanol composition in water, pure paclitaxel content, and precipitation temperature were 61.5% (v/v), 0.4% (w/v), and 4

C, respectively.

●: Control; ○: 0.1% (w/v) polymer; ▼: 0.2% (w/v) polymer;△:

0.3% (w/v) polymer.

일때

22.8, 22.4, 22.4, 22.0

^m

. HPMC 2910

PVP-K90 0.2%(w/v)

^{. PVA}

(Fig. 4C),

^6, 12, 18, 24

^0.1%(w/v)

55.2, 44.9, 40.0, 39.6

^m,

^0.2%(w/v)

36.6, 34.4, 30.9, 30.5

^m,

^0.3%(w/v)

48.0, 36.3, 35.1, 31.0

^m

. HPMC 2910

^PVP-K90

PVA 0.2%(w/v)

^.

과적으로표면적이증가된분별침전공정에고분자물질첨 가는

paclitaxel

^,

PVP-K90, HPMC 2910, PVA

. Paclitaxel

^(~98.0%)

^(>85%)

^[11].

paclitaxel

^,

(HPMC 2910, PVP-K90, PVA)

^.

^-

paclitaxel

⁽

⁾

었다

^[4].

^-

생성되는 소수성 표면의 계면장력을 감소시키고 공간적

(steric)

(electrostatic)

성장을저해하게된다

^[4].

^PVP-K90

입자의

paclitaxel

paclitaxel

^.

^PVP-K90

paclitaxel

^(affinity)

^[2].

^,

paclitaxel

paclitaxel

(steric barrier)

^.

paclitaxel

(0.2%, w/v)

^,

antisolvent(nonsolvent)

⁽

탄올

⁾

^[2].

은고분자물질농도에따른입자크기증가현상은다른원 료의약품의경우에도보고되었다

[2, 10, 17, 18].

입자크기와 제타전위의 관계

표면적이증가된분별침전공정에서고분자물질을첨가하

지않은경우와동일농도

^{(0.2% w/v)}

질

(PVA, HPMC 2910, PVP-K90)

^{. Fig. 5}

^(control)

^{-2.35 mV(}

^{: 45.3}

^m)

^,

분자물질

PVA, HPMC 2910, PVP-K90

경우분별침전용액의제타전위값은

-10.90 mV(

름

^{: 30.5}

m), -13.05 mV(

^{: 21.0}

m), -29.54 mV (

^{: 16.8}

^m)

^.

paclitaxel

^,

^.

^,

^.

^,

입자의침전물이얻어지는것으로판단된다

^{[2, 20].}

결과는기존문헌에보고된

paclitaxel

^[15].

요 약

본연구에서는표면적이증가된분별침전공정에친수성 고분자물질을첨가하여식물세포유래항암물질

paclitaxel

^.

(PVA, HPMC 2910, PVP-K90)

^.

^PVP-K90

^{, PVP-}

K90

^0.2%(w/v)

않은 경우보다

⁴

⁵

^(<20

^m

radius)

^.

Fig. 5. Particle size of paclitaxel precipitate and zeta potential

of reacting solution using polymer during increased surface

area fractional precipitation with an ion exchange resin

(Amberlite IRA 400 OH). The methanol composition in water,

pure paclitaxel content, precipitation temperature, and precipita-

tion time were 61.5% (v/v), 0.3% (w/v), and 4

C, 24 h, respec-

tively.

전물의입자크기는분별침전용액의제타전위절대값에반 비례함을알수있었다

^.

Acknowledgments

This research was supported by Basic Science Research Pro- gram through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2015016271).

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