Novel polymeric micelles with cinnamic acid as lipophilic moiety for 9-Nitro-20(S)-camptothecin delivery

Materials Letters 97 (2013) 4–7

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Novel polymeric micelles with cinnamic acid as lipophilic moiety for 9-Nitro-20(S)-camptothecin delivery Yan Liang, Yusi Lai, Dong Li, Bin He n, Zhongwei Gu n National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 October 2012 Accepted 20 January 2013 Available online 25 January 2013

9-Nitro-20(S)-camptothecin (9-NC) is a broad-spectrum anticancer drug but its application was limited by its poor solubility in aqueous medium. Novel polymeric micelles were developed for 9-NC delivery. The micelles were fabricated from the self-assembly of amphiphiles with cinnamic acid (CIN) as lipohilic moiety and methoxy poly(ethylene glycol) (mPEG) as hydrophilic segment. The micelles were nontoxic to NIH 3T3 fibroblasts. 9-NC was trapped efficiently in mPEG–DCIN micelles. The release of 9-NC from the micelles with one cinnamic acid molecule as lipophilic moiety (mPEG– CIN) was much faster than that from the micelles with two cinnamic acid molecules as lipophilic moiety (mPEG–DCIN). The in vitro anticancer inhibition study demonstrated that the anticancer activity of 9-NC loaded mPEG–DCIN micelles was much better than that of 9-NC loaded mPEG–CIN micelles. Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.

Keywords: 9-Nitro-20(S)-camptothecin Polymeric micelle Drug delivery Cinnamic acid

1. Introduction 9-Nitro-20(S)-camptothecin (9-NC), an analog of camptothecin, is a promising broad-spectrum anticancer drug with high potency against human cancers, it has achieved remarkable success in early clinical trials [1]. However, the clinical application of 9-NC is largely hampered because of its poor solubility and stability [2]. The lactone form of 9-NC is easily changed to carboxylate form, which leads to the loss of bioactivity. In order to avoid these drawbacks, drug delivery systems are developed. Polymeric micelles are potential carriers for 9-NC delivery, they have exhibited significances in enhancing solubility and maintaining bioactivity [3]. The hydrophobic cores of polymeric micelles are considered as drug repositories, and the hydrophilic shells effectively prolong the circulation time in blood. In polymeric micelles, the lipophilic moieties are generally hydrophobic macromolecules. Biodegradable hydrophobic polymers such as poly(lactic acid), poly(e-caprolactone) and their copolymers are extensively studied as lipophilic moieties in polymeric micelles for anticancer drug delivery [4–7]. Recently, some new strategies have been used in fabricating polymeric micelles. Stereocomplexation [8], host–guest interaction [9], hydrogen-bonding [10], electrostatic interaction [11] and p–p stacking interaction [12] are utilized to generate polymeric micelles.

n

Corresponding authors. Tel.: þ 86 28 85412923; fax: þ 86 28 85410653. E-mail addresses: [email protected] (B. He), [email protected] (Z. Gu).

In this paper, new polymeric micelles with cinnamic acid as lipophilic moiety were synthesized. The micelles with one and two cinnamic acid molecules as lipophilic moieties were used for 9-NC delivery. The size distribution, morphology, release profiles and in vitro anticancer activity of the 9-NC loaded micelles were studied.

2. Experimental Materials. Cinnamic acid, a-methoxyo-amino-poly (ethylene glycol) (mPEG–NH2, Mw¼2000 g/mol), N, N-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP) were purchased from Sigma-Aldrich Co. and used as received. L-lysine methyl ester dihydrochloride (H-Lys-OMe  2HCl) was purchased from GL Biochem. Ltd. (Shanghai, China). 9-NC was purchased from Shanghai future reagent Co., Ltd. (China). Dulbecco’s modified Eagle’s medium (DMEM), 100x mycillin, and fetal bovine serum (FBS) were used for cytotoxicity test. All the solvents were purchased from Kelong Chemical Co. (Chengdu, China) and used as received. Synthesis of the amphiphiles. The synthesis of the two cinnamic acid molecules substituted L-lysine (DCIN–Lys) was according to the procedure previously reported [13]. The synthesis of mPEG– DCIN was as follow: mPEG–NH2 (0.34 g, 0.17 mmol), DCIN–Lys (0.3 g, 0.68 mmol) and DMAP (0.02 g, 0.18 mmol) were dissolved in 10 mL CH2Cl2 in an ice bath. DCC (0.70 g, 3.39 mmol) was dissolved in CH2Cl2 (10 mL) and added dropwise. The solution was stirred at room temperature for 48 h. The white precipitate was filtrated. The filtrate was concentrated, precipitated in cold

0167-577X/$ - see front matter Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matlet.2013.01.093

Y. Liang et al. / Materials Letters 97 (2013) 4–7

anhydrous diethyl ether and recrystallized in ethanol. The crude product was dialyzed in dialysis membrane tube (MWCO 2000). After three days’ dialysis, the product (mPEG–DCIN) was freezedried. The mPEG–CIN was synthesized with the same procedure. Preparation of drug-loaded micelles. The amphiphiles (10 mg) and 9-NC (2 mg) were dissolved in 20 mL CH2Cl2. The solution was concentrated to spread film and vacuum-dried overnight. Five milliliters of distilled water was dropped for rehydration. The solution was stirred violently at room temperature for 4 h. The solution was subsequently lyophilized after centrifugation and filtration. The content of 9-NC was determined by UV (Lambda35, Perkin–Elmer, USA) excitation at 370 nm in a mixture of methanol-acetate buffer solution (70:30 v/v, pH¼4.5) using calibration curve. The drug loading content (DLC) and encapsulation efficiency (EE) were calculated from the following formulae: DLCð%Þ ¼ ðweight of loaded drug=weight of drug loaded micelleÞ 100% EEð%Þ ¼ ðweight of loaded drug=weight of drug in feedingÞ  100% In vitro release study. Ten milligram of 9-NC loaded micelles were dispersed in 3 mL PBS (pH¼7.4). The solution was transferred in dialysis membrane tubes (MWCO¼1000). The tubes were immersed in vials containing 25 mL PBS solution and put in a shaking bed at 37 1C. 1 mL PBS solution was taken out and the same volume PBS was added to the vials at prescribed time intervals. The released 9-NC was detected by UV (Lambda35, PerkinElmer, USA) with excitation wavelength at 370 nm. Three replicated samples at each time point were recorded and the mean value was presented. Cytotoxicity test. The cytotoxicity of blank micelles was tested by CCK8 assay against NIH 3T3 fibroblasts. The NIH 3T3 fibroblasts were separately inoculated into 96-well plates with 5  103 cells per well in 100 mL of medium. After 24 h incubation, the medium was removed and replaced with 100 mL of medium containing blank micelles. The cells were incubated for 48 h. The medium was removed and the wells were washed with PBS (pH¼7.4). Ten microliters of 5 mg/mL CCK8 solution in PBS (pH¼7.4) was added to each well. After incubated for 4 h, the

H2N

NH2 SOCl2 H2N O

OH CH3OH

O

medium containing unreacted CCK8 was removed carefully and the absorbance was measured. In vitro anticancer activity study. HepG2 liver cancer cells were separately inoculated into 96-well plates with 5  103 cells per well in 100 mL of medium for 24 h. 9-NC and 9-NC loaded micelles solutions in DMEM were added to the plates and incubated for 48 h. The cell viability was measured by CCK8 assay.

3. Results and discussion The balance between hydrophobicity and hydrophilicity in the amphiphiles was important for the formation of polymeric micelles [14,15]. The amphiphiles with one and two cinnamic acid molecules as lipophilic moieties were synthesized and the encapsulating of 9-NC was shown in Scheme 1. The critical micelle concentration (CMC) is as important parameter for the micellization. The CMCs of the mPEG–CIN and mPEG–DCIN micelles were 780 and 40 mg/mL, respectively. The 1H NMR spectra of the two amphiphiles were presented in Fig. S1 in supporting information. The DLC and EE of the two 9-NC loaded micelles were shown in Table S1. Both the DLC and EE of mPEG– DCIN micelles were higher than those of mPEG–CIN micelles. Fig. 1 showed the size and morphology of the drug loaded mPEG–DCIN micelles. The drug loaded micelles were monodisperse and the calculated mean diameter was around 95 nm. In the TEM photograph (Fig. 1B), the dark nanoparticles were the drug loaded micelles. The size of the micelles was around 80 nm, which was smaller than that in DLS result. As the DLS size was hydrodynamic diameter, the micelles suspended in aqueous solution tested by DLS were bigger than those in dry state in TEM photograph. Fig. 2 showed the release profiles of 9-NC loaded micelles. The release rate of 9-NC loaded mPEG–DCIN micelles was much slower than that of mPEG–CIN micelles. In the first 5 h, the release rates of mPEG–DCIN and mPEG–CIN micelles were 25% and 60%, respectively. The fast burst release was attributed to the drug deposited at the region near or within the mPEG shells. The difference release rate of the two micelles was attributed to the interaction between 9-NC and the hydrophobic moieties of the micelles. The drug-carrier interaction in mPEG–DCIN micelles

O OH

NH2

5

O

O

O

N H

N H

OCH3 HOBT HBTU DIEA

O

O O

NaOH/H2O

CH3OH

O N H

OH O N H

O

n

NH2

DCC DMP

NH2

+

O N H O

O O

O

OH

DMAP DCC

N H

n

N HO N H

O

Scheme 1. Synthetic route of amphiphiles and the self-assembly of 9-NC loaded micelles.

CH3

6

Y. Liang et al. / Materials Letters 97 (2013) 4–7

25

95 ± 32

Number (%)

20 15 10 5 0 10

100

1000

Size (nm) Fig. 1. DLS (A) result and TEM photograph (B) of drug loaded mPEG–DCIN micelles.

70

100 50

Cell Viability (%)

Cumulated 9-NC release (%)

120 60

40 30 20 mPE EG-C CIN Mic celle es mPEG-DCIN Micelles

10

15

45

30

60 40 20

0 0

80

60

75

0 1E-4

9-NC 9-NC/mPEG-DCIN 9-NC/mPEG-CIN 1E-3

0.01

0.1

1

10

Concentration(µg/mL)

Time (h) Fig. 2. Release profiles of 9-NC loaded mPEG–CIN and mPEG–DCIN micelles in physiological condition (pH¼ 7.4), means7 SD (n¼ 3).

Fig. 3. In vitro anticancer activities of 9-NC loaded micelles against HepG2 liver cancer cells, the incubation time was 48 h.

was stronger than that in mPEG–CIN micelles, which retarded the release of 9-NC, thus led to the slower release rate. The cytotoxicity of the blank mPEG–CIN and mPEG–DCIN micelles was evaluated with NIH/3T3 fibroblasts and HepG2 cells, the results were presented in Fig. S2 in supporting information. The cell viabilities of the micelles incubated with the two cell lines were around 100%, which indicated that the two micelles were non-toxic to NIH/3T3 fibroblasts and HepG2 cells. The 9-NC loaded micelles were incubated with HepG2 liver cancer cells to assess the in vitro anticancer activities (Fig. 3). The in vitro anticancer efficiency of both 9-NC and 9-NC loaded micelles was dose-dependent. The IC50s (the concentration of 9-NC that killed 50% of cells) of free 9-NC, 9-NC loaded mPEG–CIN and mPEG–DCIN micelles were 0.3, 0.04 and 0.02 mg/mL, respectively. The inhibition effect of mPEG–DCIN micelles was the best. The results demonstrated that the HepG2 cells were more sensitive to 9-NC loaded mPEG–DCIN micelles, the anticancer activity of 9-NC was enhanced after encapsulated in mPEG–DCIN micelles.

micelles was lower than that of free 9-NC and 9-NC loaded mPEG–CIN micelles. The mPEG–DCIN micelle is a promising carrier for 9-NC delivery.

4. Conclusions Polymeric micelles with small cinnamic acid molecule as lipophilic moiety were synthesized for 9-Nitro-20(S)-camptothecin delivery. The drug loaded micelles were monodisperse and the mean diameter was less than 100 nm. The in vitro anticancer assessment revealed that the IC50 of 9-NC loaded mPEG–DCIN

Acknowledgments This research work was supported by Ministry of Science and Technology of China (No. 2011CB606206), National Science Foundation of China (Nos. 51222304, 31170921, 51133004 and 50830105), Ministry of Education of China (NCET-10-0564).

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.matlet.2013.01.093.

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