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Modulating Drug Release Rate from Partially Silica-Coated Bicellar Nanodisc by Incorporating PEGylated Phospholipid Li Lin, Xiaoyou Wang, Xiaoda Li, Yongbo Yang, Xiuli Yue, Qiang Zhang, and Zhifei Dai Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00508 • Publication Date (Web): 10 Oct 2016 Downloaded from http://pubs.acs.org on October 14, 2016

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Bioconjugate Chemistry

Modulating Drug Release Rate from Partially SilicaCoated Bicellar Nanodisc by Incorporating PEGylated Phospholipid Li Lin1, Xiaoyou Wang3, Xiaoda Li1, Yongbo Yang1, Xiuli Yue2*, Qiang Zhang3, Zhifei Dai3*

1

School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, PR

China 2

School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin

150080, PR China 3

Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery System, College

of Engineering, School of Pharmaceutical Sciences, Peking University, Beijing 100871, China Corresponding author: Xiuli Yue*, Email: [email protected] Zhifei Dai*, Email: [email protected]

KEYWORDS: Nanodisc, Silica, bicelles, doxorubicin, Drug release, Cancer

ABSTRACT: This article reports an effective method to regulate hydrophobic drug release rate from partially silica-coated bicellar nanodisc generated from proamphiphilic organoalkoxysilane

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and dihexanoyl phosphatidylcholine by introducing different molar percentages of 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000

(DSPE-PEG2000)

into

planar

bilayers of hybrid bicelles. It was found that the drug release rate increased with increasing the molar percentages of DSPE-PEG2000, and 57.38%, 69.21%, 78.69%, 81.64% and 82.23% of hydrophobic doxorubicin was released within 120 h from the nanodics incorporating with 0%, 2.5%, 5%, 10% and 20% DSPE-PEG2000, respectively. Compared with the non-PEGylated nanodisc and free doxorubicin, the PEGylated nanodiscs showed good biocompatibility, high cellular uptake and adhesion, as well as high local drug accumulation. In addition, both in vitro and in vivo results demonstrated significantly improved antitumor efficacy of the PEGylated nanodisc than its control groups. Thus, the PEGylated nanodisc with partial silica coating offers a facile and efficient strategy of drug delivery for chemotherapy with improved patient acceptance and compliance.

Introduction Chemotherapy remains one of the most widely used means for cancer treatment 1, 2. Yet, patients undergoing chemotherapy often suffer from serious side effects of anticancer drugs of nanocarriers, such as polymeric nanoparticles

5-8

, liposomes

9-11

3, 4

. A range

, have all been fabricated to

enhance the therapeutic efficiency of anticancer drugs by facilitating local drug accumulation via enhanced permeability and retention (EPR) effect

12

, developing drug bioavailability, and

prolonging systemic circulation13. Nevertheless, irrespective of their design, mere small part of the drug nanocarriers can enter tumor in vivo and some may reach normal tissue 14-16. Hence, we have pressing need to develop novel nanocarriers to control the drug release from nanocarriers specifically in tumor with minimal distribution in healthy tissues.


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Many studies have shown that the particle shape could affect the circulation time, cellular uptake, biodistribution, cytotoxicity and therapy efficacy

17-25

. It was found that the disc-like

nanoparticles displayed preferential cellular uptake and higher microvascular adhesion than spherical nanoparticles

17-19, 26, 27

. The discoidal bicelles, or bilayered micelles, consist of long-

chain phospholipid and short-chain phospholipid or detergent

28-35

. As a model biological

membrane system, the bicelles have been successfully used in studying the structure of membrane proteins

36-38

and function of membrane associated proteins

39, 40

. Also, hydrophobic

drugs can be loaded into the hydrophobic domain of nanodisc for controlled drug release 35, 41, 42. However, the biomedical applications of phospholipid bicelles have been largely limited due to their instability43, 44. The interactions between bicellar nanodiscs and plasma proteins may result in destabilization of the bicelles and drug leakage during circulation. In order to overcome the general problems associated with current phospholipid bicelles, Kichuchi et al recently developed a new type of organic-inorganic hybrid bicelles (HBs) with partial silica coating 45, 46. The silica-like surface may protect the inner lipid bilayer, hence offer the hybrid bicelles significantly higher stability than conventional phospholipid bicelles

47-52

.

Therefore, such hybrid bicelles may show great potential use as a nanocarrier for a range of hydrophobic guest species. Modulating drug release rate from nanocarriers enable us to shield drug from premature elimination, and to lead drug to the desired action site while minimizing drug exposure elsewhere in the body. The aim of this study was to propose the encapsulation of hydrophobic drugs with partially silica-coated nanodisc and modulation of drug release rate by introducing different molar ratios of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 (DSPEPEG2000)

53, 54

into planar bilayers of bicelles. An anticancer drug, hydrophobic doxorubicin


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(DOX) was selected as a model drug. The doxorubicin loaded hybrid bicelles (DOX@HBs) were generated from cerasome-forming lipid (CFL) and dihexanoyl phosphatidylcholine (DHPC) and DSPE-PEG2000 at the designated molar ratio by using conventional Bangham method in combination with sol-gel reaction and self-assembly process (Fig. 1). The PEGylation by incorporating DSPE-PEG2000 could protect the silica-coated bicellar nanodisc from agglomeration and macrophage capture, reduce protein absorption, and consequently prolong the blood circulating time55,

56

. The influence of the nanodisc composition on the morphology,

encapsulation efficiency and drug release behavior were investigated. Furthermore, the biocompatibility, tissue distribution and anti-tumor efficacy of DOX@HBs were also examined in vitro and in vivo.

Fig. 1 Schematic illustration of the formation of the PEGylated bicellar nanodisc with partial silica coating for modulating the drug release rate of hydrophobic doxorubicin.


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RESULTS AND DISCUSSION Preparation and Characterization DOX@HBs. It was reported that the addition of an appropriate amount of DSPE-PEG2000 could stabilize the nanodisc41,

52, 57

. The PEGylated

bicellar nanodiscs with partial coating were generated from proamphiphilic organoalkoxysilane and dihexanoyl phosphatidylcholine by introducing DSPE-PEG2000 at the designated molar percentages of 0%, 2.5%, 5%, 10% and 20%. The polyorganosiloxane coating on the lipid bilayer membrane was formed by in situ sol-gel processes (Si-OCH2CH3 + H2O CH3CH2OH followed by 2Si-OH

Si-OH +

Si-O-Si + H2O). The crosslinked network was proved by

Fourier transform infrared (FT-IR) spectroscopy (Fig. S1). The HBs exhibited significant peaks at 1095.90 cm-1 and 3319.49cm-1 corresponding to stretching vibration of Si-O-Si and O-H, respectively, indicating the existence of silica-coating of bicellar nanodisc.

Fig. 2 TEM images of drug free hybrid bicelles doped with different molar ratios of DSPE-PEG2000, (a) 0%; (b) 2.5%; (c) 5%; (d) 10%; (e) 20%; and DOX@HBs doped with 5% DSPE-PEG2000 (f). Red arrows represent edge-on bicelles.


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The dynamic laser scattering (DLS) measurements showed that the hydrodynamic particle size of the non-PEGylated HBs was about 61.9 ± 3.8 nm, a little larger than the HBs doped with 5% DSPE-PEG2000 (56.2 ± 3.2 nm) (Table 1). Similar phenomenon was also observed in the literature

58

. As seen in TEM images (Fig. 2), the nanodisc shape was observed in all samples

showing ellipsoidal (face-on) and rod-like (edge-on, red arrows) shape, which was consistent with the atomic force microscopy (AFM) images (Fig. S2). Compared with the non-PEGylated nanodiscs (Fig. 2a), the rod-like shape were more obvious for the PEGylated nanodiscs (Fig. 2be). In table 1, TEM measurements showed that the particle size was 54.06 ± 8.74, 53.13 ± 9.64, 47.82 ± 9.59, 45.32 ± 5.98 and 54.25 ± 8.70 for hybrid bicelles doped with 0%, 2.5%, 5%, 10% and 20% DSPE-PEG2000, smaller than the hydrodynamic size measured by DLS measurements. The thickness of the bicellar nanodiscs was evaluated to be about 6 nm. As shown in Fig. 2f, after loading DOX into the HBs doped with 5% DSPE-PEG2000, the obtained DOX@HBs still kept the discoidal shape and the diameter had no apparent change. Table1 The particle size, encapsulation efficiency (EE) and drug-loading content (DLC) of bicelles doped with different molar ratios of DSPE-PEG2000

Molar ratio of DSPEPEG2000(%)

0

2.5

5

10

20

Size (nm) by DLS

61.90±3.75

63.52±4.12

56.21±3.25

56.06±0.65

60.42±2.13

Size (nm) by TEM

54.06 ±8.74

53.13±9.64

47.82±9.59

45.32±5.98

54.25±8.70

EE%

66.03±1.31

75.02±1.20

85.27±1.02

84.02±0.52

84.73±1.05

DLC%

2.24±0.05

2.12±0.10

2.35±0.05

2.13±0.16

2.01±0.01

The drug-loading content (DLC) and encapsulation efficiency (EE) of HBs were evaluated by using the traditional spectrophotometry method. The molar ratio of the doped DSPE-


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PEG2000 had effect on the DLC% and EE% (Table 1). The EE% and DLC%s were about 66.03 ± 1.31% and 2.24 ± 0.05% for the non-PEGylated HBs, respectively. By incorporating 5% DSPE-PEG2000 into HBs, the EE% and DLC% increased to 85.27 ± 1.02% and 2.35 ± 0.16%. However, no significant change was seen in the EE% and DLC% when the molar percentage of DSPE-PEG2000 increased to 10% or 20%. Release behavior and mathematical model of DOX@HBs. The cumulative release of DOX from HBs was performed in vitro over an experimental time period of 120 h. As shown in Fig. 3 a, free DOX exhibited an initial burst release and nearly 97.4% of DOX were released within 24 h. In contrast, the HBs exhibited slower release rate attributed to the higher density of siloxane networks formed by siloxane bonds. Nevertheless, the length of siloxane bond was much shorter than the diameter of the cross-section of the alkyl-chain segment of the hybrid amphiphiles. This effect may have induced the formation of pores which were sufficiently large to allow leakage of DOX

59

. The release rate of DOX from HBs increased with increasing the molar percentage of

DSPE-PEG2000. The total amount of released DOX was up to about 53.30 ±3.08%, 63.81 ± 2.90%, 68.97 ±0.37%, 65.52 ±4.5% and 68.02 ±2.36% within 24h, and 57.38% ± 1.18, 69.21% ± 0.92, 78.69% ± 0.14, 81.64% ± 0.99 and 82.23% ± 1.61 within 120 h for hybrid bicelles doped with 0%, 2.5%, 5%, 10% and 20% DSPE-PEG2000, respectively. A little initial burst release of DOX was seen probably due to the drug adsorption on the nanodisc surface. The increased release rates and amounts of DOX from the HBs are probably because of the enhanced membrane permeability due to the incorporation of DSPE-PEG2000. Since DSPE-PEG2000 molecules are more mobile than cerasome-forming lipid and not crosslinkable, the PEGylated HBs possess greater membrane fluidity than non-PEGylated HBs. As a result, the membrane fluidity of HBs and their permeability to DOX can be regulated by varying lipid membrane


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composition. It opens a new way to modulate the release profiles of hydrophobic drugs from HBs.

Fig. 3 Cumulative release of DOX from hybrid bicelles doped with different molar percentages of DSPEPEG2000 (a) (**p

Modulating Drug Release Rate from Partially Silica ... - ACS Publications

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