Applied Microbiology and Biotechnology
Caleosin-assembled oil bodies as a potential delivery nanocarrier
--Manuscript Draft--Manuscript Number: AMAB-D-11-01454R1
Full Title: Caleosin-assembled oil bodies as a potential delivery nanocarrier
Article Type: Original Paper
Section/Category: Biotechnological products and process engineering Corresponding Author: Yun-Peng Chao
Feng Chia University
Taichung, TAIWAN, REPUBLIC OF CHINA Corresponding Author Secondary
Information:
Corresponding Author's Institution: Feng Chia University Corresponding Author's Secondary
Institution:
First Author: Chiang Chung-Jen, PhD
First Author Secondary Information:
All Authors: Chiang Chung-Jen, PhD
Lin Shen-Chuan Lin Li-Jen Chih-Jung Chen Yun-Peng Chao All Authors Secondary Information:
Abstract: Encapsulation of hydrophobic agents with nanocarriers is challenging. Therefore, we have sought to nanoscale artificial oil bodies (NOBs) as an alternative delivery carrier. To constitute NOBs, caleosin (Cal), a structural protein of plant seed oil bodies, was first fused with ZH2 (Cal-ZH2). ZH2 is a bivalent anti-HER2/neu affibody with a high affinity towards the HER2/neu receptor. After overproduction in Escherichia coli, insoluble Cal-ZH2 was isolated and used to assemble NOBs in one step.
Consequently, resulting NOBs had a zeta potential around -49 mV and ranged in size from 150 nm to 200 nm. Upon loading with a hydrophobic fluorescence dye, NOBs were found to be selectively internalized into HER2/neu-positive tumor cells. Further analyses showed that more than 90% cells were invaded by dye-loaded NOBs and the cargo dye was released in cells with time. In addition, the in vitro assay revealed the release of the dye from NOBs in a slow and prolong manner. Overall, these results indicate the potential of Cal-based NOBs as a delivery vehicle.
Response to Reviewers: Dear Professor Steinbuchel:
It is my pleasure to submit our revised manuscript (AMAB-D-11-01454) to Appl
Microbiol Biotechnol. An itemized list of changes addressing reviewers’ comments is as follows.
1.Fig. 5 (C) is changed to Fig. 5 (B).
2.The construction of the fusion protein, Cal fused with ZH2, was described under the Subsection “plasmid construction” in Materials and Methods. See line 14-22, P. 5. 3.As suggested, a scheme illustrating the structure of nanoscale artificial oil bodies (NOBs) was presented in Fig. 8.
4.The advantage of developed NOBs over other delivery systems was described in Discussion. See line 14-26, P. 12.
Best regards, Yun-Peng Chao Professor
Department of Chemical Engineering, Feng Chia Univeristy
October 25, 2011
Dear Professor Steinbuchel:
It is my pleasure to submit our revised manuscript (AMAB-D-11-01454) to Appl Microbiol Biotechnol. An itemized list of changes addressing reviewers’ comments is as follows.
1. Fig. 5 (C) is changed to Fig. 5 (B).
2. The construction of the fusion protein, Cal fused with ZH2, was described under the Subsection “plasmid construction” in Materials and Methods. See line 14-22, P. 5.
3. As suggested, a scheme illustrating the structure of nanoscale artificial oil bodies (NOBs) was presented in Fig. 8.
4. The advantage of developed NOBs over other delivery systems was described in Discussion. See line 14-26, P. 12.
I would love to acknowledge reviewers for their valuable comments. Your kind suggestions on our work are greatly appreciated as well.
Best regards, Yun-Peng Chao
Professor
Department of Chemical Engineering, Feng Chia Univeristy
October 25, 2011
Dear Professor Steinbuchel:
It is my pleasure to submit our revised manuscript (AMAB-D-11-01454) to Appl Microbiol Biotechnol. An itemized list of changes addressing reviewers’ comments is as follows.
1. Fig. 5 (C) is changed to Fig. 5 (B).
2. The construction of the fusion protein, Cal fused with ZH2, was described under the Subsection “plasmid construction” in Materials and Methods. See line 14-22, P. 5.
3. As suggested, a scheme illustrating the structure of nanoscale artificial oil bodies (NOBs) was presented in Fig. 8.
4. The advantage of developed NOBs over other delivery systems was described in Discussion. See line 14-26, P. 12.
I would love to acknowledge reviewers for their valuable comments. Your kind suggestions on our work are greatly appreciated as well.
Best regards, Yun-Peng Chao
Professor
Department of Chemical Engineering, Feng Chia Univeristy
Authors' Response to Reviewers' Comments
Caleosin-assembled oil bodies as a potential delivery nanocarrier
Chung-Jen Chiang1,*, S he n- C hu a n L in1, Li-Jen Lin2, Chih-Jung Chen1, and Yun-Peng Chao3,*
1
Department of Medical Laboratory Science and Biotechnology, China Medical University, 91 Hsue-Shih Road, Taichung 40402, Taiwan
2
School of Chinese Medicine, China Medical University, Taichung, Taiwan 3
Department of Chemical Engineering, Feng Chia University, 100 Wen Hwa Road, Taichung 40424, Taiwan
* Corresponding authors Chung-Jen Chiang
Phone: 886-4-22003366 ext. 7227; FAX: 886-4-22057414. E-mail address: [email protected]
Yun-Peng Chao
Phone: 886-4-24517250 ext. 3677; FAX: 886-4-24510890 E-mail address: [email protected]
*Manuscript
Click here to download Manuscript: Cal-ZH-AMB1.doc Click here to view linked References
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Abstract 1
Encapsulation of hydrophobic agents with nanocarriers is challenging. Therefore, we 2
have sought to nanoscale artificial oil bodies (NOBs) as an alternative delivery 3
carrier. To constitute NOBs, caleosin (Cal), a structural protein of plant seed oil 4
bodies, was first fused with ZH2 (Cal-ZH2). ZH2 is a bivalent anti-HER2/neu 5
affibody with a high affinity towards the HER2/neu receptor. After overproduction 6
in Escherichia coli, insoluble Cal-ZH2 was isolated and used to assemble NOBs in 7
one step. Consequently, resulting NOBs had a zeta potential around -49 mV and 8
ranged in size from 150 nm to 200 nm. Upon loading with a hydrophobic 9
fluorescence dye, NOBs were found to be selectively internalized into 10
HER2/neu–positive tumor cells. Further analyses showed that more than 90% cells 11
were invaded by dye-loaded NOBs and the cargo dye was released in cells with time. 12
In addition, the in vitro assay revealed the release of the dye from NOBs in a slow 13
and prolong manner. Overall, these results indicate the potential of Cal-based NOBs 14 as a delivery vehicle. 15 16 17 18 19 20
Keywords: artificial oil body, Caleosin, targeted delivery, HER2/neu 21 22 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Introduction 1
2
Plant seeds are rich in oil bodies (OBs) that provide the fuel for seedling growth 3
(Huang 1996; Napier et al. 1996). OBs consist of a triacylglycerol (TAG) matrix 4
surrounded by a monolayer of structural protein-bound phospholipids (PLs). Oleosin 5
(Ole) is recognized as the prominent structural protein associated with OBs (Frandsen 6
et al. 2001b). This protein has a central hydrophobic region along with two 7
hydrophilic domains. The former domain is embedded in TAG matrix while the latter 8
domains protrude outwards. The negative charge of these two terminal domains 9
provides OBs with the electronegative repulsion that prevents OBs from coalescence. 10
This in turn results in the stability of OBs in the form of separate entities both in vivo 11
and in vitro, and the size of OBs can reach 0.5-2 m in diameter on average (Tai et al. 12
2002). 13
OBs can be reconstituted in vitro to form artificial oil bodies (AOBs). This is 14
carried out by subjecting TAG, PLs, and structural proteins to sonication (Tzen et al. 15
1998; Tzen et al. 1992). Ole is the structural protein that has been primarily employed 16
for assembling AOBs (Peng et al. 2003). The resulting AOBs are comparable in size, 17
topology, and stability to those of isolated seed OBs (Tzen and Huang 1992). These 18
unique structural and topological features of Ole-based AOBs have found many 19
biotechnological applications, such as the scaffold-assisted refolding and purification 20
of recombinant proteins (Chiang et al. 2005; Chiang et al. 2007; Peng et al. 2004), 21
one-step immobilization of enzymes (Chiang et al. 2006), and cargo delivery (Chiang 22
et al. 2010; Chiang et al. 2011). 23
One promising approach to effectively combat cancers is to employ carriers to 24
selectively deliver drugs to tumor sites (Farokhzad and Langer 2009). This approach 25
relies on a drug-loaded carrier conjugated with a bioactive ligand that recognizes the 26
biomarker of tumor cells. Consequently, a sufficient dose of chemotherapeutic drugs 27
can reach cancerous loci, thereby minimizing the detrimental side-effect of drugs 28
toward normal cells (Cho et al. 2008). However, many potent anticancer 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
pharmaceuticals are poorly soluble. Oral or intravenous administration of 1
hydrophobic agents usually leads to low bioavailability of pharmaceuticals due to the 2
aggregate deposition at local sites (Fernandez et al. 2001; Lipinski et al. 2000). 3
Accordingly, various drug carrier systems have been proposed to address this issue, 4
including liposomes (Andresena et al. 2005), synthetic polymers (Zhang et al. 2008), 5
micelles (Torchilin 2005), and others (Cohen and Bernstein 1996). Nevertheless, 6
many technical difficulties need to be overcome before an effective formulation of 7
water-repelling agents is developed (Torchilin 2007). 8
In principle, an effective drug carrier must be small, biocompatible, and 9
biodegradable. AOBs, consisting of natural biomaterials, apparently hold these 10
characteristics. Recently, we have explored the feasibility of AOBs for targeted 11
delivery of hydrophobic agents (Chiang et al. 2010; Chiang et al. 2011). By tailoring 12
the ratio of Ole to oil, nanoscale AOBs (NOBs) could be obtained. Ole was fused with 13
a small bioactive domain, either the arginine-glycine-aspartate (RGD) motif or the 14
bivalent anti-HER2/neu affibody (denoted as ZH2). The resulting fusion genes were 15
overexpressed in Escherichia coli and the hybrid proteins were isolated for 16
constitution of NOBs. To illustrate, a hydrophobic dye was encapsulated into NOBs 17
that displayed RGD or ZH2 motif on the surface. Upon administration, the RGD- and 18
ZH2-displayed NOBs were selectively internalized into vintegrin- and 19
HER2/neu-positive tumor cells, respectively. The internalization efficiency could 20
reach 80% in both cases as revealed by the flow cytometry analysis. Moreover, NOBs 21
entered tumor cells via the endosomal entry pathway and disintegrated with time in 22
response to the low pH condition. The cargo dye was then released into cell cytoplasm. 23
Overall, the results indicate a new application of NOBs in the field of cancer 24
nanotechnology. 25
Caleosin (Cal) is another structural protein associated with seed OBs (Chen et al. 26
1999). The structure of Cal is very similar to that of Ole (Frandsen et al. 2001a). A 27
previous study found that AOBs reconstituted with Cal had a smaller size and 28
exhibited higher stability relative to those with Ole (Liu et al. 2009). Therefore, it was 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
intriguing to examine the feasibility of Cal-based NOBs for targeted delivery of 1
insoluble agents. To this end, Cal was fused with ZH2 (Cal-ZH2). A single domain of 2
ZH2 consists of 58 amino acid residues and exhibits a high binding affinity towards 3
the extracellular domain of HER2/neu (Orlova et al. 2006). After overproduction in E. 4
coli, Cal-ZH2 was isolated and used to assemble NOBs with the size ranging between 5
150 nm and 200 nm. By encapsulation with a hydrophobic dye, Cal-based NOBs were 6
shown to selectively penetrate HER2/neu-positive tumor cells in an effective way. 7
Moreover, the cargo dye was released from the pH-responsive nature of Cal-based 8
NOBs upon internalization. The result indicates the potential of Cal-based NOBs as a 9
delivery carrier. 10
11
Materials and methods 12
13
Plasmid construction 14
The ZH2 motif comprises two identical domains of ZHER2:342 (Orlova et al. 2006). It 15
was synthesized by Mission Biotech. Co. (Taiwan) and then subcloned into plasmid 16
pBluescriptSKII (Stratagene Co. USA) to obtain plasmid pBlue-ZH2 (Chiang et al. 17
2011). The ZH2 motif was recovered from plasmid pBlue-ZH2 with EcoRV-HindIII 18
digestion and incorporated into plasmid pET29a-Cal (Chen et al. 2004) to give 19
plasmid pJO1-Cal-ZH2. This plasmid contains the ZH2 domain fused to the C 20
terminus of Cal and the fusion gene is under the control of the T7 promoter. In 21
contrast, plasmid pET29a-Cal contained Cal alone. 22
23
Protein overproduction 24
E. coli strain BL21(DE3) was transformed with plasmid pJO1-Cal-ZH2 and 25
pET29a-Cal. These plasmid-bearing bacterial strains were grown in Luria-Bertani 26
(LB) medium (Miller 1972) at 37oC. To induce protein production, 100 M Isopropyl 27
β-D-1-thiogalactopyranoside (IPTG) was added to the culture medium. After 28
induction for 4 h, bacteria were harvested by centrifugation and resuspended in 1 mL 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
of 0.01 M sodium phosphate buffer (pH 7.5). Sulfate-polyacrylamide gel 1
electrophoresis (SDS-PAGE) was then conducted to analyze the proteins as previously 2
reported (Chiang et al. 2008). 3
4
Self-assembly of NOBs 5
NOBs were assembled essentially following the previous method (Chiang et al. 2005). 6
In brief, unless stated otherwise the assembly solution (1 mL) consisted of 100 g 7
olive oil, 150 g PLs, and 500 g Cal-ZH2 or Cal in 10 mM sodium phosphate buffer 8
(pH 7.5). The plant oils used here were soybean oil (Taiwan Sugar Co., Taiwan), 9
peanut oil (Leader Price Co., Taiwan), sesame oil (Taisun Co., Taiwan), olive oil 10
(Taisun Co., Taiwan), and mineral oil (Sigma, USA). The solution was then subjected 11
to sonication for 10 s with the amplitude set at 20%. Upon centrifugation, NOBs were 12
removed from the top of the solution. To encapsulate the cargo agent into NOBs, 1 g 13
yellow GGK dye (Widetex Co., Taiwan) was additionally added to the assembly 14
solution. 15
16
Morphology, size, and stability of NOBs 17
According to the previous report (Chiang et al. 2010), the morphology and size of 18
NOBs were analyzed with a light microscope (Nikon type E600, Japan) and a particle 19
size analyzer (Beckman Coulter, USA), respectively. The stability of NOBs was 20
determined by the turbidity test as reported previously (Chiang et al. 2010). In 21
addition, dye-loaded NOBs were analyzed by Atomic Force Microscopy (AFM) with 22
the NS4/D3100CL/Multi Mode (Digital Instrument, Germany). A drop of the 23
suspension containing NOBs was overlaid onto the mica surface at room temperature. 24
A cantilever was employed to scan the sample with a nominal force constant of 20 N 25 m-1. 26 27 Cell culture 28
Cell cultures were maintained following the reported protocol (Chiang et al. 2011). 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
The human tumor cell lines used here were MDA-MB-231 (ovarian), SKOV3 1
(ovarian), MCF7 (breast), and MCF7/Her18 (HER2-transfected stable cell line). In 2
essence, cell concentration was determined by a hemocytometer. For experiments, 3
cells were seeded into 24-well plates at a cell density of 1×105 cells per well. 4
5
Fluorescence microscopy, confocal Microscopy, and flow cytometry 6
Tumor cells were treated with dye-loaded NOBs and then prepared for further 7
analyses as reported previously (Chiang et al. 2011). The anti-HER2/neu antibody 8
(Santa Cruz Biotech., USA) was applied to cells. After washing, anti-mouse 9
IgG-TRIAC (Jackson ImmunoResearch Lab., USA) was administrated to stain cell 10
membranes. Meanwhile, cell nuclei were stained with diamidino-2-phenylindole 11
(DAPI) and rinsed with the sodium phosphate buffer. Both fluorescence microscopy 12
(Olympus IX71, Japan) and confocal microscopy (Leica TCS SP2 , Germany) were 13
used to analyze the cells. 14
In addition, tumor cells were seeded to 6-well plate at a cell density of 1×105 cells 15
per well. NOBs were then applied to cells for an indicated period of time. Followed 16
by washing, cells were trypsinized and harvested for analyses using a FACScanto 17
flow cytometer system (Becton Dickinson, USA). 18
19
In vitro assessment of dye release 20
The release profile of the yellow GGK dye from NOBs was determined following the 21
reported protocol (Chiang et al. 2011). In brief, Cal-ZH2-based NOBs (1 mL) were 22
placed in a dialysis bag (MW cut-off ranging 12,000-14,000 D) that was immersed in 23
the buffer at 37oC with constant stirring. At time intervals, aliquots of the solution 24
(100 L) were withdrawn and the content of dye was measured by a 25
spectrofluorometer (FP-6200, Jasco, Japan). The absorbance obtained was normalized 26
to that with the initial content of the dye. 27 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Results 1
2
Self-assembly of NOBs by Cal-ZH2 3
Production of Cal-ZH2 in E. coli was analyzed by SDS-PAGE (Fig. 1). Upon 4
induction by IPTG, the bacterial strain was able to overproduce Cal-ZH2 in an 5
insoluble form that was mainly present in the cell lysate (ppt-1). Self-assembly of 6
NOBs was carried out by subjecting the mixture, containing olive oil, PLs, and 7
isolated Cal-ZH2, to sonication. After centrifugation, NOBs sat on the top of mixture 8
solution while no Cal-ZH2 was left in the supernatant fraction (sup-2) and the cell 9
pellet part (ppt-2). NOBs were recovered and heated to liberate incorporated proteins. 10
Consequently, Cal-ZH2 was identified as the main associated protein (NOB). This 11
result indicates the strong association of Cal-ZH2 with oils. Similarly, Cal alone could 12
be overproduced in E. coli receiving IPTG induction. 13
14
Modulation of Cal-ZH2-assembled NOBs 15
According to our previous study (Chiang et al. 2010), NOBs with a smaller size 16
exhibited higher stability. Therefore, three factors affecting the size of NOBs were 17
investigated in a systematic way. NOBs were first assembled with various plant oils at 18
pH 7.5 by fixing the weight ratio of oil to Cla-ZH2 (O/P) at 1:1. As indicated in Fig. 19
2A, NOBs that were prepared from all types of oils except mineral oil exhibited a 20
relatively small and homogenous size. The stability assay for NOBs also showed a 21
similar trend (Fig. 3A). 22
At pH 7.5, NOBs were assembled using olive oil with various O/P ratios. Fig. 2B 23
and 3B show that lower O/P ratios gave a smaller size and higher stability of NOBs. 24
In particular, NOBs had a small and compact morphology when the O/P ratio was 25
lower than 1. 26
Finally, the effect of pH on NOB morphology was examined with the O/P ratio 27
set at 1:1. NOBs tended to have a larger size and lower stability at pH lower than 7 28
(Fig. 2C and 3C). This is in agreement with our previous findings (Chiang et al. 2011). 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
In contrast, NOBs had a smaller size and higher stability at an alkali condition. As 1
further analyzed, NOBs assumed a spherical shape with the size ranging 150-200 nm 2
by AFM (Fig. 4), and their zeta potential was estimated to be -49.1±2.3 mV. 3
4
Selective internalization of Cal-ZH2-assembled NOBs 5
The functionality of ZH2 displayed on Cal-based NOBs was further investigated. To 6
clearly illustrate, a hydrophobic fluorescence dye was encapsulated into NOBs with or 7
without ZH2. Upon administration of the fluorescent dye-loaded NOBs, strong 8
fluorescent signals could be detected in HER2/neu-postive cells (e.g., MCF7/Her18 9
and SKOV3) and the signal intensity increased in a NOB dose-dependent manner (Fig. 10
5A). In contrast, the signal was absent for HER2/neu-negative cells (e.g., MCF7 and 11
MDA-MB-231). Similarly, no fluorescence could be observed in any type of tumor 12
cells that were exposed to NOBs free of ZH2 (data not shown). Overall, the results 13
suggest the functional display of ZH2 via Cal onto the surface of NOBs. This in turn 14
leads to the specific association of functionalized NOBs with HER2/neu-positive 15
cells. 16
In addition, the localization of internalized NOBs was confirmed by confocal 17
microscopy. Fig. 5B shows that the fluorescent dye-loaded NOBs were located in the 18
cytoplasm of HER2/neu-positive cells. It clearly indicates the ability of functionalized 19
NOBs to target and penetrate HER2/neu-positive cells. 20
21
Internalization efficiency of Cal-ZH2-assembled NOBs 22
The internalization efficiency of Cal-ZH2-assembled NOBs was calculated as the 23
percentage of fluorescence-emitting cells in the whole cell population. After 24
administration of fluorescence dye-loaded NOBs for various time periods, tumor cells 25
were processed for analyses by flow cytometry. As depicted in Fig. 6A, internalization 26
efficiency of ZH2-displayed NOBs towards HER2/neu-positive cells generally 27
increased with the longer administration time. The maximum internalization 28
efficiency was more than 90% for SKOV3 and MCF7/Her18 cells upon 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
administration of functionalized NOBs for 60 and 120 min, respectively. A similar 1
observation could also be obtained with fluorescence microscopy (Fig. 6B). 2
3
Release of the cargo dye 4
As reported recently, Ole-based NOBs displaying ZH2 were internalized into tumor 5
cells via the endosomal entry pathway (Chiang et al. 2011). The cargo dye was 6
released as a result of the instability of Ole-based NOBs at an acidic condition. 7
Therefore, it promoted us to investigate the control-and-release feature of NOBs 8
assembled by Cal-ZH2. SKOV3 cells were then administrated with fluorescent 9
dye-loaded NOBs (25 g/mL) for 1 h. After washing, the fluorescent signals within 10
cells were monitored by confocal microscopy along the time course. As shown in Fig. 11
7A, the signals in the cells faded with time. After 5 days, the fluorescent images in the 12
cells were barely detectable. The result implies that the cargo dye is released from 13
Cal-based NOBs and gradually decays within the cells. 14
Furthermore, the dialysis analysis was conducted to analyze the in vitro release 15
profiles of the cargo dye from Cal-ZH2-assembled NOBs. Fig. 7B shows that an 16
initial burst release occurred at the first 6 h. The released cargo dye reached 55% at 17
pH 6.5 and 40% at pH7.5. After that, the dye was released in a stable and slow way. 18
This sustained and prolonged release curve resembles the typical profile as commonly 19
reported for many drugdelivery systems (Kim et al. 2007; Yang et al. 2002).
20 21
Discussion 22
23
Nanotechnology has transformed the medication method for treatment of diseases. 24
Apparently, targeted therapy is an emerging technology that receives the most 25
intensive study. In essence, the approach relies on a nanocarrier that delivers 26
therapeutic agents to target cells. However, one pressing challenge is to overcome the 27
technical difficulties in the robust formulation of hydrophobic drugs with nanocarriers. 28
In this study, we have sought to Cal-based NOBs as an alternative nanocarrier. 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Self-assembled NOBs comprise a central oil core that is enveloped by a monolayer of 1
Cal-bound lipid (Fig. 8). To make NOBs functional, the anti-HER2/neu motif (e.g., 2
ZH2) was displayed at their surface by linkage to the C-terminus of Cal. It is known 3
that HER2/neu belongs to the human epidermal growth factor receptor family (Hung 4
and Lau 1999). An abnormal and uncontrolled expression of HER2/neu can 5
eventually lead to the progression of many aggressive tumors (Citri and Yarden 2006). 6
As illustrated, Cal-based NOBs with surface display of ZH2 could specifically 7
penetrate HER2/neu-positive tumor cells (Fig. 5A and 5B). This indicates that fusion 8
of Cal with the bioactive motif provides a simple and feasible way for 9
functionalization of NOBs. 10
The translocation path of the HER2/neu receptor into cells was proposed 11
previously (Giri et al. 2005). The receptor enters cells by the endocytic internalization 12
and then interacts with importin, a transport protein. Escorted by the nuclear pore 13
protein, HER2/neu travels to cell nucleus. In agreement with this proposed pathway, 14
ZH2 on the surface of NOBs interacted with HER2/neu of tumor cells, leading to the 15
internalization of NOBs into cell endosomes (Chiang et al. 2011). This translocation 16
mechanism also resulted in the heterogeneous distribution of internalized NOBs in 17
cells (Fig. 5B). Moreover, the internalization behavior of Cal-based NOBs was 18
time-dependent (Fig. 6B), which is consistent with the invasion kinetics of 19
anti-HER2/neu affibody-conjugated materials as reported (Alexis et al. 2008). At the 20
acidic condition in cell endosomes (Ohkuma and Poole 1978; Yamashiro et al. 1983), 21
NOBs started to disintegrate with the release of the cargo dye (Fig. 7A). Based on the 22
in vitro assay, the release rate of cargo dyes from Cal-based NOBs is slower than that 23
from Ole-based NOBs (Fig. 7B). After 7 h at pH 6.5, there was 55% cargo dyes being 24
liberated for Cal-based NOBs whereas more than 90% was released for Ole-based 25
NOBs (Chiang et al. 2011). The result implies the potential of Cal-based NOBs for the 26
long-acting release system. 27
The size of Cal-based NOBs was tunable. As indicated in Fig. 2, the type of plant 28
oils could affect the morphology of NOBs. This result suggests that Cal-ZH2 likely 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
interacts differentially with various TAG compositions of oils. More Cal-ZH2 than oil 1
in the formulation could result in smaller and more stable NOBs. It indicates that 2
more terminal domains (bearing a negative charge) of Cal could contribute stronger 3
repulsion force to maintain the integrity of NOBs. However, under an acidic condition, 4
the electronegative repulsion force is likely negated as a result of neutralization by H+, 5
which leads to coalescence of NOBs. The size of NOBs assembled by Cal can be 6
tailored to reach less than 200 nm and is smaller than that with Ole (Chiang et al. 7
2011). In particular, the zeta potential of Cal-based NOBs (< -30 mV) was very high, 8
suggesting that they are stable (Lee et al. 2007). Moreover, Cal-based NOBs is 9
superior to Ole-based counterparts in terms of internalization efficiency (e.g., the 10
percentage of fluorescence-emitting cells and incubation time) (Chiang et al. 2011). 11
Regardless of the cell type, the uptake efficiency of Cal-based NOBs could reach 12
above 90% with incubation time less than 2 h (Fig. 6A). 13
As well recognized, liposomes and polymeric micelles are two primary 14
nanocarriers developed for a wide range of applications (Elbayoumi et al. 2007; 15
Shmeedaa et al. 2009; Sugarman et al. 1996). Liposomes have a lipid-enclosed 16
aqueous space that is not applicable for entrapment of water-repelling agents. 17
Polymeric micells contain a hydrophobic center surrounded by a hydrophilic shell 18
and are effective for encapsulation of hydrophobic cargos. However, both carriers 19
need to be conjugated with a functional ligand by a chemical modification before 20
they can selectively target tumor cells. In contrast, NOBs are oil droplets that 21
facilitate the entrapment of hydrophobic agents. They are biocompatible and can be 22
self-assembled in an easy and reproducible way. Simply fusing a bioactive motif 23
with Cal can result in functional NOBs that are highly invasive and featured with an 24
acid-triggered release nature. In conclusion, the advance of this novel approach may 25
open a new avenue in the filed of cancer nanotechnology. 26
27
Acknowledgement 28
We like to acknowledge Instrument Center of R&D Office at China Medical 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
University for technical assistance. This work was supported by National Science 1
Council of Taiwan (NSC 99-2313-B-039-003-MY3, NSC 2
98-2221-E-035-029-MY3), China Medical University (CMU100-S-29), and 3
Ministry of Economic Affairs (99-EC-17-A-10-S1-156). 4
5
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Figure legends:
Figure 1. SDS-PAGE analyses of Cal-ZH2 production in E. coli. E. coli strain bearing plasmid pJO1-Cal-ZH2 (left panel) or pET29a-Cal (right panel) was cultured and induced (+IPTG) for protein production. By centrifugation, proteins of plasmid pJO1-Cal-ZH2-bearing bacteria were fractioned into the soluble part (sup-1) and insoluble part (ppt-1). Insoluble Cal-ZH2 was used for assembly of NOBs with two fractions left after centrifugation, including the supernatant (sup-2) and precipitate (ppt-2). NOBs were recovered from the top of the solution and heated to release the protein (AOB).
Figure 2. Morphology of Cal-based NOBs at various conditions. Morphology of Cal-based NOBs was analyzed by light microcopy. (A) Assembly of Cal-based NOBs with various plant oils, including (1) mineral oil, (2) peanut oil, (3) soybean oil, (4) olive oil, and (5) sesame oil. (B) Assembly of Cal-based NOBs with various O/P ratios at (1) 10:1, (2) 2:1, (3) 1:1, (4) 1:5, and (5) 1:10. (C) Assembly of Cal-based NOBs at pH (1) 6.5, (2) 7.0, (3) 7.5, (4) 8.0, and (5) 9.0. The scale bar equals 2 m.
Figure 3. Stability of Cal-based NOBs at various conditions. The stability of NOBs was determined by the turbidity test as described. (a) The stability profile of NOBs assembled with various types of oils. (b) The stability profile of NOBs assembledat various O/P ratios. (c) The stability profile of NOBs assembled at various pHs.
Figure 4. Morphology of NOBs by AFM. NOBs were assembled with the O/P ratio at 1:5and at pH 7.5.
Figure 5. Selective internalization of NOBs. NOBs were assembled with Cal-ZH2 and encapsulated with 1 g/mL yellow GGK dye. The resulting NOBs (green) were added 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
to 1 x 105 cells to reach the indicated concentration (shown on the left) and incubated for 2 h. After repeated washing for 3 times, cells were processed for further analyses. (A) Analysis by fluorescence microscopy. Cell nuclei (blue) and the HER2/nue receptor (red) were stained with DAPI and anti-HER2/neu antibody, respectively. Individual images were taken and then merged as shown on the right. (B) Analysis by confocal laser scanning microscopy (CLSM). After treated with Cal-ZH2-based NOBs, SKOV3 cells were analyzed by CLSM. The insets show the two three-dimensional reconstruction sections, including X-Z (bottom) and Y-Z (right) sections.
Figure 6. Internalization efficiency of NOBs by flow cytometry and confocal microscopy. Refer to Fig. 5, cells were treated with Cal-based NOBs (green) at 25 g/mL. (A) Analysis by flow cytometry. Various cells were exposed to ZH2-displayed (Cal-ZH2) or ZH2-lacking NOBs (Cal) for an indicated time. All experiments were conducted in triplicate. (B) Analysis by fluorescence microscopy. Incubation time was shown on the left of each panel.
Figure 7. Release of the fluorescence dye carried by NOBs. (A) Analysis by confocal microscopy. SKOV3 cells were treated with Cal-ZH2-based NOBs that carried the hydrophobic dye. The fluorescence emitted by internalized NOBs in the cells was analyzed by confocal microscopy on (1) day 0, (2) day 1, (3) day 3, and (4) day 5. (B) In vitro dye release profile. NOBs were loaded with the dye (6.25 g/mL) and assessed for the dye release at pH 6.5 (●) or pH 7.5 (○) for 24 h. The experiment was conducted in triplicate.
Figure 8. A scheme illustrating the structure of Cal-ZH2-based NOBs. For clear illustration, a slice section of NOBs was removed to uncover the internal structure. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Like plant OBs, NOBs are mainly composed of a TAG matrix enclosed by a monolayer of Cal-bound PLs. The ZH2 motif linked to Cal is displayed at the surface of NOBs, consequently leading NOBs to targeting HER2/neu-expressing tumor cells. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Figure 1
Figure. 2 Figure
Figure 3A
Figure 3C
Figure 5A
Figure 6A
Figure 7A.
