Edible Ginger-derived Nano-lipids Loaded with Doxorubicin as a Novel Drug-delivery Approach for Colon Cancer Therapy

ACCEPTED ARTICLE PREVIEW

Accepted Article Preview: Published ahead of advance online publication Edible Ginger-derived Nano-lipids Loaded with Doxorubicin as a Novel Drug-delivery Approach for Colon Cancer Therapy

Mingzhen Zhang, Bo Xiao, Huan Wang, Moon Kwon Han, Zhan Zhang, Emilie Viennois, Changlong Xu and Didier Merlin

t p ri

Cite this article as: Mingzhen Zhang, Bo Xiao, Huan Wang, Moon Kwon Han, Zhan Zhang, Emilie Viennois, Changlong Xu and Didier Merlin, Edible Ginger-derived Nano-lipids Loaded with Doxorubicin as a Novel Drug-delivery Approach for Colon Cancer Therapy, Molecular Therapy accepted article preview online 05 August 2016; doi:10.1038/mt.2016.159

c us

an m

This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

d e t

p e c

c A

Received 17 April 2016 ; accepted 28 July 2016 ; Accepted article preview online 05 August 2016

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Edible Ginger-derived Nano-lipids Loaded with Doxorubicin as a Novel Drug-delivery Approach for Colon Cancer Therapy Mingzhen Zhang1, 2*, Bo Xiao1, 2, 5, Huan Wang4, Moon Kwon Han1, 2, Zhan Zhang1, 2, Emilie Viennois1, 2, Changlong Xu1, 2, 6 and Didier Merlin1, 2, 3

Affiliations: 1

Institute for Biomedical Sciences, 2 Center for Diagnostics and Therapeutics, Georgia State

University, Atlanta, GA 30303, USA

t p ri

3

Veterans Affairs Medical Center, Decatur, GA, USA

4

Center for Molecular and Translational Medicine, Georgia State University, Atlanta, GA 30303,

c us

USA 5

Institute for Clean Energy and Advanced Materials, Faculty for Materials and Energy,

an m

Southwest University, Chongqing, 400715, P. R. China 6

The 2nd Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University,

Wenzhou, 325027, P. R. China.

d e t

*Author for correspondence Tel.: +1 (404) 413 3597

p e c

Fax: +1 (404) 413 3580 Email: [email protected]

c A

Keywords: ginger derived nanoparticles, edible ginger derived lipids, doxorubicin, colon cancer, chemotherapy

Abstract

The use of nanotechnology for drug delivery has shown great promise for improving cancer treatment. However, potential toxicity, hazardous environmental effects, issues with large-scale production and potential excessive costs are challenges that confront their further clinical applications. Here, we describe a nanovector made from ginger-derived lipids that can serve as a delivery platform for the therapeutic agent doxorubicin (Dox) to treat colon cancer. We created nanoparticles from ginger and reassembled their lipids into ginger-derived nanovectors (GDNVs). A subsequent characterization showed that GDNVs were efficiently taken up by colon cancer cells. Viability and apoptosis assays and ECIS technology revealed that GDNVs exhibited excellent biocompatibility up to 200 M; by contrast, cationic liposomes at the same concentrations

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

decreased cell proliferation and increased apoptosis. GDNVs were capable of loading Dox with high efficiency and showed a better pH-dependent drug-release profile than commercially available liposomal-Dox. Modified GDNVs conjugated with the targeting ligand folic acid (FAGDNVs) mediated targeted delivery of Dox to Colon-26 tumors in vivo and enhanced the chemotherapeutic inhibition of tumor growth compared with free drug. Current experiments explore the feasibility of producing nature-derived nanoparticles that are effective as a treatment vehicle while potentially attenuating the issues related to traditional synthetic nanoparticles.

Introduction Colorectal cancer is the third-most common neoplasm and the second-leading cause of cancer-

t p ri

related mortality for men and women worldwide.1 The incidence of colorectal cancer has increased over the last few years with about one million new cases being diagnosed annually.2-4

c us

Chemotherapy remains the most common therapeutic strategy available for colon cancer patients.5 However, current chemotherapeutic strategies cannot distinguish cancerous and healthy

an m

cells, which leading to poor therapeutic effect on tumors and severe toxic side effects on healthy cells. Therefore, a strategy that could simultaneously maintain (or improve) drug therapeutic efficacy while decreasing its toxicity would represent a major development in chemotherapy for

d e t

advanced colon cancer.

p e c

Drug delivery systems using nanotechnology have shown great promise for improving cancer treatment.6,7 Such delivery vehicles have enhanced permeability and retention (EPR) effect that

c A

allow drugs to reach tumors more passively through leaky vasculatures surrounding the mass.8-10 However, this passive approach has limits in that passive targeting depends on the degree of tumor vascularization and angiogenesis.11-13 Moreover, the high interstitial fluid pressure of solid tumors works against the successful uptake and homogenous distribution of drugs in the tumor.14 As an alternative strategy, modifying nanovehicles to include targeting ligands, such as antibodies,15 proteins16 or aptamers, 17 would allow active targeting through binding to cognate receptors overexpressed by cancer cells or angiogenic endothelial cells. In this approach, targeting ligands attached to the surface of nanovehicles may act as homing devices, improving the selective delivery of drug to specific tissues and cells.18 To date, approximately 150 nanotechnology-based anticancer therapeutics have advanced to various stages of development, some of which are widely used in the clinic. 19 Doxorubicin (Dox) encapsulated in liposomes (Doxil) and paclitaxel encapsulated in protein-based nanoparticles (Abraxane) represent two such successful applications. 20 The types of nanoparticles currently used in research for cancer drug delivery

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

include dendrimers,21 liposomes,22 polymeric nanoparticles,23 micelles,24 protein nanoparticles,25 ceramic nanoparticles,26 viral nanoparticles,27 metallic nanoparticles,28 and carbon nanotubes.29 Although there is some evidence supporting the use of these synthetic nanoparticles as delivery vehicles for therapeutic agents, including siRNA, proteins and pharmaceutical agents,30,31 the potential in vivo toxicity of some synthetic nanoparticles and issues associated with their largescale economical production still present obstacles to their clinical application.32 Unlike artificially synthesized nanoparticles, naturally released nano-sized particles derived from different types of mammalian cells and exosome-like nanoparticles from edible plants play an important role in intercellular communication.33-36 Research has shown that nano-sized particles released from mammalian cells can be utilized for encapsulating drugs and siRNAs to treat diseases in mouse

t p ri

disease models without obvious side effects.36 Although a nanoparticles-based approach is promising, production of large quantities of mammalian cell-derived nanoparticles and evaluation

c us

of their potential biohazards still pose a challenge. Recently, Zhang et al reported edible grapefruit derived lipid could be employed as vectors to deliver chemotherapeutic agents, RNA

an m

and DNA vectors and proteins to cells, suggesting development of edible plant derived vector for therapy has many potential advantages over current commercial available vectors. 32,33,35,37,38

d e t

In this study, we isolated nanoparticles in large quantities from Zingiber officinale, the edible ginger, and characterized ginger-derived nanovectors (GDNVs). We found that GDNVs were

p e c

efficiently taken up by colon cancer cells, and the modification with targeting ligand folic acid (FA), achieved active specific targeting to Colon-26 tumors in vivo (Figure 1). More importantly,

c A

GDNVs, made from ginger lipids, showed excellent biocompatibility. This study will expand current concepts of drug-delivery systems and lay the foundation for a less toxic drug-delivery approach.

Results Fabrication and characterization of GDNVs Using standard techniques reported in the literature,39,40 we isolated large amounts of nanoparticles from ginger extract (48.5

4.8 mg/kg ginger). Particles from an initial sucrose

density gradient purification step were separated by ultracentrifugation (Figure 2a) and identified as nanoparticles based on transmission electron microscopy (TEM) (Figure 2b) and atomic force microscopy (AFM) (Figure 2c) examination. These data suggest that ginger can serve as a natural source for the large-scale production of ginger-derived nanoparticles.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Lipid profile analysis revealed that ginger-derived nanoparticles were enriched in phosphatidic acid (PA) (~47% of total lipids), digalactosyldiacylglycerol (DGDG) (~15% of total lipids) and monogalactosyldiacyglycerol (MGDG) (~27% of total lipids) (Figure 2d). Research recently showed that phosphatidic acid (PA) has a function to control membrane fission and fusion, because of its small negatively charged headgroup very close to the acylchain region of the bilayer, its high affinity for divalent cations, and its propensity to form intermolecular hydrogen bonds.41,42 Digalactosyldiacylglycerol (DGDG) and monogalactosyldiacyglycerol (MGDG) are important glycolipids, It has been shown that glycolipids can stabilize phospholipid liposomes during freeze-thawing freeze-drying, making glycolipids interesting potential stabilizers for liposome-encapsulated drugs.43 To determine whether lipids from ginger-derived nanoparticles

t p ri

could be assembled into nano-sized particles (GDNVs) to serve as therapeutic delivery vector, we employed a standard method based on hydration of lipid films similar to that used for fabrication of liposomes.

44

c us

TEM images of purified nanoparticles showed that GDNVs were generally

spherical in shape (Figure 2e-g), and with an average hydrodynamic diameter (as determined by

an m

dynamic light scattering) of approximately 188.5 nm, with a lower polydispersity (Figure 2h). These data were also confirmed by AFM (Figure 2i and j), which showed that GDNVs were highly dispersed with a uniform size distribution. In addition, we confirmed that the critical

d e t

concentration of ginger-derived lipid nanoparticles for GDNV formation was about 5 M (Figure

p e c

S1a), and further found that GDNVs with a much more stable size distribution could be assembled by increasing the concentration of total lipids, determined as described previously,33 up to 256 M.

c A

We believe that this finding is very important for selecting the concentration of ginger-derived lipid nanoparticles needed to generate GDNVs with the desired size. Moreover, GDNVs were very stable when stored at 4ºC for up to 25 days, based on the analysis of their zeta potential and size distribution (Figure S1b–d), a property that is very important for delivery in therapeutic applications. Collectively, these results suggest that lipids derived from edible ginger can be reassembled into stable nano-sized particles and produced in large scale. GDNVs are taken up by colon cancer cells and do not affect cell viability Efficient cellular uptake and cytotoxicity are major requirements for the therapeutic efficacy of nanoparticles. 6 To evaluate the potential use of GDNVs as vectors to deliver therapeutic agents to colon cancer, we first evaluated the cellular uptake of GDNVs by colon cancer cells. As shown in Figure 3a, after incubation with cells for 4 h, GDNVs labeled with DiL (red dots) were internalized by both Colon-26 cells and HT-29 cells with high efficiency (87.5%

2.5% and 75.8%

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

0.8%,

ACCEPTED ARTICLE PREVIEW

respectively) (Figure 3b). A confocal microscopy examination of cells incubated with free DiL revealed a different pattern of DiL staining compared with DiL-labeled GDNVs (Figure S2). These data suggest that DiL signals were derived from GDNV-positive cells, not from the contamination by free DiL. Together, these results indicate that GDNVs made from ginger-derived lipids are taken up by the tested colon cancer cell lines at physiological temperatures (37 °C). To further investigate the pathway(s) of GDNVs internalization, Colon-26 cells were first co-incubated with specific endocytosis inhibitors (amiloride, indomethacin, chlorpromazine and cytochalasin D), as shown in Figure 3c,d, the uptake of DiL-GDNVs were significantly inhibited by cytochalasin D (***, p＜ ＜0.001), an inhibitor of actin polymerization required for phagocytosis (comprises a series of events, starting with the binding and recognition of particles by cell surface receptors, followed

t p ri

by the formation of actin-rich membrane extensions around the particle).45 Amiloride (an inhibitor of macropinocytosis),46 indomethacin (an inhibitor of caveolae-medicated endocytosis),47

c us

and chlorpromazine (an inhibitor of clathrin-mediated endocytosis) 48 have slight effect on the uptake of DiL-GDNVs. We also observed the same results in HT-29 cells (Figure S3), suggesting

an m

that GDNVs were most possibly internalized via phagocytosis pathway. Next, we sought to determine whether GDNVs were toxic to Colon-26 and HT-29 cells. In these

d e t

experiments, the cationic liposome, DC-Chol/DOPE (30/70, w/w), which has been used extensively for the delivery of therapeutic reagents, 49 were used as a standard control (zeta potential: 42 mV

p e c

and the Z-average: 106.1 nm in our measurement). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) assays, which quantitatively measure cell viability, revealed that

c A

GDNVs at concentrations up to 200 M had less of an effect on the viability of Colon-26 cells, HT29 cells and Caco2-BBE cells than did DC-Chol/DOPE cationic liposomes following co-incubation for 24 or 48 h (Figure S4a-f). As previously reported, MTT assays are not suitable for real-time analysis of cellular transformation.50 As an alternative, we employed electric cell-substrate impedance-sensing (ECIS) technology, an automated, real-time analytical tool for measuring cell proliferation, cytotoxicity, apoptosis, and attachment.6 As shown in Figure S4g, after cells had attached to the electrode surface to form a confluent monolayer, they were incubated with GDNVs (200 M), cationic liposomes (200 M), or phosphate-buffered saline (PBS; control). Caco2-BBE monolayers treated with GDNVs yielded a resistance curve similar to that of PBS-treated cells, whereas the resistance of monolayers treated with liposomes gradually decreased. This shows that liposomes significantly reduced the resistance of Caco2-BBE cell monolayers, an observation consistent with MTT assay results. Together, these results show that GDNVs have less of an effect

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

on cell proliferation than cationic liposomes at equivalent lipid concentrations. We also evaluated the cytotoxicity of GNDVs by quantifying cell apoptosis using an annexin V-FITC/ Propidium Iodide assay (annexin V-FITC/PI). Four distinct phenotypes are distinguishable using this approach: 1) viable cells, lower right quadrant; 2) early apoptotic cells, lower right quadrant; 3) necrotic cells, upper left quadrant; and 4) apoptotic cells-upper right quadrant. These assays revealed that incubation of cells with GDNVs at concentrations up to 200 M exerted no significant apoptotic effect on Colon-26 or HT-29 cells, whereas cationic liposomes at concentrations greater than 20 M (for Colon-26) and 100 M (for HT-29) induced significant apoptotic effects (Figure S5 and Figure S6). Collectively, these results indicate that GDNVs are nontoxic in vitro.

t p ri

Next, we explored the potential in vivo cytotoxic effects of GDNVs. As shown in Figure S7, intravenous injection of mice with GDNVs or cationic liposomes did no change serum levels of

c us

pro-inflammatory cytokines significantly such as interleukin (IL)-6, IL-1β or tumor necrosis factor (TNF-α). Histological analyses of hematoxylin and eosin (H&E)-stained organ slices showed

an m

no noticeable evidence of organ damage in either GDNVs or DC-Chol/DOPE cationic liposome treatment groups. Hepatocytes in the liver appeared normal; no myocardial fibrillary loss or vacuolation was observed in the heart; no pulmonary fibrosis was detected in lung samples; and

d e t

no necrosis was observed in any of the histological samples analyzed (Figure S8). Moreover, as

p e c

shown in Figure S9, hemolysis of nanoparticles in blood—a serious limitation for nanoparticle use in in vivo application—was not observed for GDNVs or cationic liposomes at concentrations up to

c A

200 M lipids; Triton X-100, used a positive control, achieved 100% hemolysis. These results suggest that GDNVs would be nontoxic towards erythrocytes after intravenous administration. Collectively, these findings indicate that GDNVs made from ginger-derived lipid nanoparticles can be taken up effectively by colon cancer cells and are nontoxic both in vitro and in vivo, and thus can be used as vectors to deliver therapeutic agents for colon cancer therapy.

GDNVs can be effectively loaded with Dox (Dox-GDNVs) Next, we sought to determine whether GDNVs could load Dox, which was used as a representative chemotherapeutic drug. Dox has a slightly positive charge, and most of ginger- derived lipid components have negative charge, with the help of sonication, ginger-derived lipids can form bilayer and Dox can be encapsulated into ginger lipids by electrostatic interaction. The size of Dox-GDNVs nanoparticles can be further decreased by AVESTIN liposomes extruder with

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

polycarbonate membrane. As shown in Figure 4a and Figure S10, GDNVs are capable of loading with Dox, achieving a loading efficiency up to 95.9%

0.26% at a ginger lipid concentration of

100 M. Representative TEM images showed that Dox-GDNVs were nearly spherical in shape (Figure 4b). The size distribution and zeta potential of Dox-GDNVs were about 188 nm and -15.5 mV, respectively (Figure 4 c and d), similar to GDNVs alone—Values that are very important for in vivo application to enhance blood circulation time and tumor accumulation probability. In addition, Dox-GDNVs were very stable at 4°C (up to 25 days) and did not lose their ability to load Dox (Figure 4e). The ideal drug-delivery system requires increased release after reaching its specified location. Extracellular microenvironment of tumor is acidic (pH 6.5–6.9), which can be employed to increase drug release. To test Dox-GDNVs release performance in a biological model,

t p ri

they were dispersed in buffer solutions (PBS) at different pH values. As shown in Figure S11a, Dox-GDNVs were stable without an increase of Dox release until they were in a pH value lower

c us

than 6.5, in contrast, a pH value as low as 5.5 did not result in an increased releasing of Dox from commercially available liposomes (Figure S11b), suggesting Dox-GDNVs were able to release

an m

loaded drug more rapidly in acidic pH close to the tumor microenvironment than commercially available liposomes. This improved pH-dependent drug-release profile of Dox-GDNVs could decrease the severe side effects of Dox, such as leucopenia, thrombocytopenia, anemia and

d e t

gastrointestinal toxicities, caused by intravenous administration of chemotherapeutic drugs. In

p e c

summary, these results show that GDNVs made from ginger-derived lipid can successfully encapsulate the chemotherapeutic drug, Dox, with a high loading efficiency and exhibit a pH-

c A

dependent release profile, indicating that GDNVs could be an effective delivery vector for chemotherapeutic drug delivery.

Dox-GDNVs exert apoptotic effects In vitro antitumor activity was first evaluated by assaying apoptosis. To quantitatively compare the apoptotic effects of free Dox and Dox-GDNVs, we treated Colon-26 and HT-29 cells with three different concentrations of each preparation (3.25, 6.5 and 13 M) for 8 h, and then quantified apoptosis by flow cytometry using an annexin V-FITC/PI apoptosis assay detection kit. As shown in Figure 5, very few necrotic or apoptotic cells were detected in control Colon-26 cells (untreated group) (Figure 5a). In contrast, both free Dox and Dox-GDNVs induced a concentrationdependent decrease in the percentage of viable cells. In the free Dox group (Figure 5b,d), the percentage of viable cells was 79.5% at 3.25 M Dox, 63.7% at 6.5 M Dox and 23.3% at 13 M

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Dox. For Dox-GDNVs, the percentages of viable cells at the corresponding Dox concentrations were 82.2%, 70.9% and 64.8% (Figure 5c,d). The lower percentage of viable cells following treatment with free Dox compared with that observed following treatment with Dox-GDNVs suggests that free Dox rapidly penetrates cells through passive diffusion across the plasma membrane to directly exert its antitumor effects in the nucleus. Dox-GDNVs showed a timedependent release profile (Figure S11a), which delayed the release of Dox from GDNVs and produced a sustained cytotoxic action. We also obtained similar results in HT-29 cells (Figure S12). ECIS technology was also employed for real-time measurement of apoptosis. Caco2-BBE cells attached to the electrode surface to form a confluent monolayer with a resistance of approximately

t p ri

13 kOhms. These cells were then incubated with Dox-GDNVs or free Dox, as shown in Figure 5e. Untreated Caco2-BBE monolayers exhibited a steady resistance curve, whereas those treated with

c us

Dox-GDNVs showed a concentration-dependent decrease in resistance: the higher the DoxGDNVs concentration, the sharper the decrease in resistance. Consistent with the results of

an m

annexin V-FITC/PI apoptosis assays, noted above, a lower concentration of free Dox (3.25 M) induced a significant reduction in the resistance of the Caco2-BBE monolayer. Dox-GDNVs–induced apoptosis was further confirmed by staining for the apoptosis marker,

d e t

cleaved (activated) caspase-3/7. These qualitative analyses revealed apoptotic cells with bright

p e c

green nuclei in both Colon-26 and HT-29 following treatment with Dox-GDNVs; cells without activated caspase-3/7 exhibited a minimal fluorescence signal (Figure S13). Colon-26 cells and HT-

c A

29 cells showed good cell confluence, indicating that after co-incubation with Dox-GDNVs (6.5 M) for 4h, cells were in the state of early apoptosis.

FA-GDNVs loaded Dox (Dox-FA-GDNVs) are effective in targeting tumors and reducing their volume To evaluate the tumor-targeting ability and antitumor capacity of Dox-FA-GDNVs in vivo (Figure S14), biodistribution of DiR-labeled GDNVs and FA-GDNVs were investigated by noninvasive in vivo imaging following intravenous administration. DiR has an emission in the near-infrared window, and has been widely used for noninvasive in vivo imaging. Here, we did not employ fluorescence of Dox to investigate the biodistribution, because comparing with DiR, the emission of Dox is in the visible light window and its optical penetration depth is too small to noninvasively image in vivo. As shown in Figure S15, DiR fluorescence could be observed in different organs, including the heart, liver, spleen, lung, kidney and tumor. Liver showed the highest DiR fluorescence both in GDNV group and FA-GDNV group when compared with other organs, a

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

probable contribution to the detoxification of liver. On the other hand, DGDG, a component of ginger-derived lipids, can be recognized by asialoglycoprotein receptors (ASGPR) which are expressed in hepatocytes, leading to an enhanced liver uptake by our prepared GDNVs or FAGDNVs nanoparticles. Importantly, we found that the fluorescence intensity of DiR-labeled FAGDNVs in tumors was higher (~2.8-fold) than that of GDNVs, confirming that FA-GDNVs are capable of targeting tumors, probably through active (FA-FRs interaction) mechanisms. On the other hand, the accumulation of FA-GDNVs, determined by DiR fluorescence intensity, was significantly reduced in spleen and slightly reduced in liver compared with GDNVs, suggesting that the use of FA-GDNVs could decrease the systemic toxicity of drugs to normal tissue/organs while extending the circulation time of the drug in blood and thereby increase its efficacy against

t p ri

tumors.

Next, the stability of circulating FA-GDNVs in blood was investigated by intravenous

c us

administration of DiR-labeled FA-GDNVs into mice bearing Colon-26 tumors. These experiments showed that FA-GDNVs were still stable and detectable up to 48 h after intravenous injection

an m

(Figure 6a), an observation consistent with our in vitro results. This longer residence time provides a longer time window for FA-GDNVs to accumulate at the tumor site. Thus, the longer nanoparticles remain in the circulation, the greater their opportunity to penetrate tumor tissues.

d e t

To investigate the antitumor effects of Dox-FA-GDNVs in vivo, we randomly assigned BALB/c nu/nu female mice bearing Colon-26 subcutaneous xenografts into four groups: saline, free Dox,

p e c

FA-GDNVs, and Dox-FA-GDNVs. We then treated mice according to the protocol illustrated in Figure 6b. Tumors grew progressively and rapidly in mice treated with FA-GDNVs or saline

c A

(Figure 6c), although body weight did not change significantly (Figure 6d). By contrast, Dox-FAGDNVs dramatically inhibited tumor growth, as shown in Figure 6e and f; free Dox also reduced tumor volume, albeit to a much lesser extent. Consistent with these decreases in tumor volume, tumor weight in mice treated with Dox-FA-GDNVs was the lowest of all treatment groups (Figure 6g). These results demonstrate that GDNVs with active-targeting FA ligands are able to further enhance the antitumor effects of Dox. To further confirm this improved therapeutic efficacy, we performed a histological analysis of tumor sections at the end of the experiment. H&E-stained images shown in Figure 6h reveal that treatment with Dox-FA-GDNVs resulted in a significant decrease in the number of cancerous cells compared with saline, free Dox, and FA-GDNVs groups. The antitumor efficacy of Dox-FA-GDNVs against implanted Colon-26 cells was also evaluated by immunohistochemical detection of the cellular proliferation marker, Ki67, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assays. As shown in Figure 7, tumors from

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

mice treated with Dox-FA-GDNVs exhibited a significant decrease in the proportion of Ki67positive cells compared with tumors from mice treated with free Dox. In tumors from free-Dox– treated mice, the average percentage of Ki67-positive cells was 64.67%, whereas the corresponding value for Dox-FA-GDNVs-treated tumors was 10%. TUNEL assays showed that TUNEL-positive (green) apoptotic cells were barely detectable in tumors from mice treated with saline or FA-GDNVs (4.7% and 3.3%, respectively). Apoptotic cells were readily detectable in tumors from mice treated with free DOX (18%) and were prominent in tumors from Dox-FAGDNVs-treated mice, where they constituted 43.3% of all cancer cells (Figure 7). These results confirm that Dox-FA-GDNVs exert greater antitumor efficacy than free Dox. H&E-stained sections further revealed that, compared with the saline-treated control group, none

t p ri

of the treatment groups showed noticeable signs of tissue or cellular damages in the heart, liver, spleen, lung or kidney, such as myocardial fibrillar loss and vacuolation in heart tissues, edema

c us

and ballooning degeneration of hepatocytes, increased numbers of granulocytes in spleen, tubular vacuolization and tubular dilation with hemorrhagic areas in the kidney, and increased alveolar

an m

wall thickness and cellular infiltration in lung tissue (Figure 8). Collectively, these findings indicate that FA-GDNVs can be used as a drug-delivery platform with the potential to decrease the toxicity and adverse effects of free drug.

d e t

Discussion

p e c

Synthetic nanoparticles have the capacity to serve as delivery vehicles for therapeutics, such as siRNA, proteins, and pharmaceutical drugs. However, the potential toxicity of these artificial

c A

nanoparticles and issues surrounding their large-scale, economic production that have limited their applications must first be addressed.32 We speculated that nanoparticles from natural sources, such as edible plants, might address these limitations because such plants are consumed regularly, have no known potential toxicity to humans, and can be produced in large quantities. Recently, Zhang et al reported edible grapefruit derived lipid could be employed as vectors to deliver chemotherapeutic agents, microRNA, DNA vectors and proteins for cancer treatment and inflammation and achieved good results. 32,33,35,37,38 Their excellent work encourages us to explore new edible plants whether they can be employed as similar delivery vehicle for cancer prevention and therapy, and it is necessary to further development edible plant derived vector for therapy. In this study, we identified that edible ginger can release nanoparticles, and their lipids can serve as a platform for therapeutic drug (Dox) delivery with excellent biocompatibility, we demonstrated

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

that GDNVs were better delivery vehicle than commercial available ones, including high efficiency in packaging, stability, pH dependent releasing, and targeting delivery in vivo. In cancer therapy, accurate targeting of tumor tissue is a key determinant of successful therapy. To achieve this, we modified GDNVs with folic acid (FA), a ligand for high-affinity folate receptors (FRs), which are expressed at elevated levels on many tumors and in almost negligible amounts on non-tumor cells.35 As is the case for liposomes, FA can be incorporated into the lipid bilayer during GDNVs fabrication by mixing this lipophilic FR ligand with ginger lipid components. Either GDNVs phospholipids or cholesterol can be used as the lipophilic anchor for FA. 32, 35, 51,52 In summary, our results show that GDNVs made of edible ginger-derived lipids could shift the

t p ri

current paradigm of drug delivery away from artificially synthesized nanoparticles toward the use of nature-derived nanovectors from edible plants. Because they are nontoxic and can be produced

c us

in large scale, nanovectors derived from edible plants could represent one of the safest therapeutic delivery platforms. Notable in this context, our results demonstrated that GDNVs loaded with Dox

an m

successfully inhibited tumor growth in a Colon-26 xenograft tumor model. This study will expand current perspectives on drug-delivery systems and could lay the foundation for a less toxic drugdelivery approach.

d e t

Materials and Methods

p e c

Chemicals. The fluorescent lipophilic dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocar bocyanine perchlorate (DiL), and 1,1'-dioctadecyl-3,3',3'- tetramethylindotricarbocyanine iodide

c A

(DiR), were purchased from Promokine (Heidelberg, Germany); DC-Chol/DOPE blend was purchased from Avanti Polar Lipids (Alabaster, AL, USA); Ascorbic acid and phalloidin-FITC were purchased from Sigma (St. Louis, MO, USA). Doxorubicin (D-4000) was purchased from LC Laboratories (Woburn, MA, USA). Duoset enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, USA). CellEvent™ Caspase-3/7 Green Detection Reagent, Vybrant® MTT cell proliferation assay kit and Annexin V-FITC/propidium iodide (PI) apoptosis detection kit were obtained from Molecular Probes (Eugene, OR, USA). Ginger derived nanoparticles isolation and purification. For isolation of Ginger derived nanoparticles, ginger or Zingiber officinale (Family, Zingiberaceae; Order, Zingiberales; Superorder, Lilianae; Subclass, magnoliidae; Class, Equisetopsida) was purchased from a local farmers’ market. Ginger was ground in a blender to obtain juice. Then the juice was centrifuged at 3000 g for 20 min, 10 000 g for 40 min to remove the large fiber of ginger with Type 45Ti rotor. The supernatant was

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

then ultracentrifuged at 150 000 g for 2 h, the pellet was suspended in PBS with the help of sonicator. Then the suspension was transferred to a sucrose gradient (8 %, 30 %, 45 % and 60 % (g/v)) and ultracentrifuged at 150 000 g for another 2 h. The bands between the layer of 8/30 % and 30/45% were harvested and noted as ginger derived nanoparticles according to the literature.40 Lipidomic analysis of ginger derived nanoparticles. For lipidomic analysis, lipids from ginger derived nanoparticles were submitted to the Lipidomics Research Center, Kansas State University (Manhattan, KS, USA) for analysis. Briefly, the lipid composition was determined using a triple quadrupole mass spectrometer (Applied Biosystems Q-TRAP; Applied Biosystems, Foster City, CA, USA) as described in an online protocol (http://www.k-

t p ri

state.edu/lipid/lipidomics/profiling.html). Data for each lipid molecular species were presented as mol % of the total lipids analyzed.

c us

Phosphorus quantification. Phosphate in ginger derived nanovectors (GDNVs) was quantified by using a standard phosphorus solution (0.65 mM, Sigma). First, different amounts of phosphate

an m

standard solutions (0, 6.5, 13, 26, 52, 104 nmol) and GDNVs samples were added in glass test tubes, then 30 L 10% Mg(NO3)2.6H2O (prepared in 95% alcohol) was added, and the mixture were heated by shaking the tubes over the strong flame of burden burner until the brown fumes

d e t

disappeared. After cooling, 300 L HCl (prepared in 0.5 N) was added to each tube and allowed to

p e c

heat in a boiling water bath for 15 min to hydrolyze any pyrophosphate to phosphate. After the tube has cooled, 0.1 mL 10 % ascorbic acid (prepared in ddH2O) and 0.6 mL 0.42 % ammonium

c A

molybdate.4H2O (prepared in 1 N H2SO4) were added in sequence. Finally, the mix solutions were incubated at 45 ºC for 20 min and read the absorbance at OD820. Preparation of ginger derived nanovectors (GDNVs). Firstly, total lipids were extracted using the Bligh and Dyer method. Briefly, 6 ml of methyl alcohol/chloroform with a volume ratio of 2:1 (v/v) was added to 1.6 ml of ginger derived nanoparticles (1mg/ml) in PBS and shake well. Chloroform (2 ml) and ddH2O (2 ml) were added sequentially and vortexed. The mixture was centrifuged at 2,000 r.p.m. for 10 min at room temperature in glass tubes to separate the mixture into two phases (aqueous phase and organic phase). For collection of the organic phase, a glass pipette was inserted through the aqueous phase with gentle positive pressure and the bottom phase (organic phase) was aspirated and dispensed into fresh glass tubes. Then the samples were washed once with a small volume KCl (1 M, 0.5 ml) and once with a small volume of water (0.5 ml). Finally, the organic phase samples were dried by heating (60 ºC) under nitrogen. For GDNVs fabrication,

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

residual chloroform was removed using a vacuum pump for 15 min and the dried lipids was immediately suspended in 500 L Hepes buffer (20 mM, sigma, pH 7.4). After a bath sonication for 5 min, an equal volume of buffer was added and sonicated for another 5 min. Finally, the solution was passed through AVESTIN liposomes extruder for 20 times (Ottawa, ON, Canada) with 200 nm polycarbonate membrane. Fabrication of Dox-GDNVs and Dox-FA-GDNVs. Doxorubicin (200 g) or/and folic acid (50 l, 1.5 mg/ml) in DMSO were mixed with total lipids in chloroform and dried under nitrogen to obtain a thin lipids-complex film. Then an identical protocol as described above was used for making DoxGDNVs and Dox-FA-GDNVs. Characterization-size, zeta potential, AFM and TEM. The size and zeta potential of liposomes, and

t p ri

lipid nanovector were measured by dynamic light scattering using a Zetasizer nano ZS (Malvern, Southborough, MA). The AFM images were taken by a SPA 400 AFM (Seiko Instruments Inc,

c us

Chiba, Japan). For TEM imaging, a drop of sample were deposited onto the surface of formvarcoated copper grids, then add 1% uranyl acetate for 15s, the samples were left to dry at room temperature.

an m

Lipid nanovector labeling. To label the lipid nanovector, stain solutions (DiL, or DiR) in DMSO (500 µM) were prepared first, then 1 ml lipid nanovector in Hepes (the concentration of lipid is

d e t

200 M) mixed with stain solution (20 l) at room temperature for 30 min (the mol ratio of lipid

p e c

/dye is 20:1). Finally, the mix solutions were centrifuged to remove the uncombined free dye. Loading efficiency. To evaluate the loading efficiency of Dox by GDNVs, particles were prepared

c A

by adding 200 g of doxorubicin into the ginger lipid film, after sonicated as the method described above, Dox-GDNVs nanovector were centrifuged at 100,000 g for 30 min. The supernatant was collected and the residual doxorubicin was measured using the microplate reader at the wavelength of 497 nm. The loading efficiency was calculated as follows: Loading efficiency= (Total Dox-free Dox)/Total Dox

100 %

Dox release profile from GDNVs and Liposomes. To test the in vitro release of Dox from DoxGDNVs and Dox-Liposomes, Dox-GDNVs and Dox-Liposomes with 100 g doxorubicin were suspended in 1 mL Hepes and added into the slide-A-Lyzer Mini dialysis devices (20K MWCO, 2 mL) at different pH buffer (5.0, 5.5, 6.0, 6.5, 7.0, 7.5). Devices were placed at 37 ºC with shaking slowly. At the predetermined time points (0, 2, 4, 6, 20, 24 and 48 h), the absorbance of suspensions were measured at 497 nm.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Cell culture. HT-29, Caco-2BBE, and colon 26 cells were cultured to confluence in 75-cm2 flasks at 37°C in a humidified atmosphere containing 5% CO2. HT-29 Cell were cultured in McCoy’s 5A medium, Caco-2BBE cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM), and colon 26 cells were culture in RPMI 1640 medium (Life Technologies, NY, USA), which were supplemented with penicillin (100 U/ml), streptomycin (100 U/ml), and heat-inactivated fetal bovine serum (10 %) (Atlanta Biologicals, GA, USA). Mice. Athymic BALB/c nu/nu mice, C57BL/6 and FVB/NJ mice (6-8 wk old) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed under specific pathogen-free conditions. All the experiments involving mice were approved by the institutional animal care and use committee (IACUC) of Georgia State University (Atlanta, GA, USA).

t p ri

Fluorescence imaging in vitro and in vivo. For confocal imaging of cellular uptake of GDNVs in vitro, Colon-26 and HT-29 cells with the concentration of 1 105/well were seeded on eight-chamber

c us

slides (Tissue-Tek, Sakura, USA) and cultured overnight at 37 ºC. Then cells were cultured with fresh culture media in the presence of DiL labeled GDNVs (50 l of 100 M/well). After 4 h

an m

incubation, the cells were fixed with 4 % paraformaldehyde (PFA) for 10 min, and then dehydrated with acetone at -20°C for 5 min. After blocking with 1% bovine serum albumen (BSA) in PBS for 30 min, 100 l of phalloidin-FITC (1:40 dilution) was added and the mixture was

d e t

incubated for an additional 30 min. Finally, cells were coverslip-mounted with mounting medium

p e c

containing 4-, 6-diamidino-2-phenylindole (DAPI, H-1500; Vector Laboratories, Burlingame, CA, USA). Cells were observed and imaged using a Zeiss LSM 700 confocal microscope with Zen 2014 software version 9.1.

c A

To evaluate the potential endocytic pathways involved in GDNVs uptaken, endocytosis inhibitors (amiloride 34mg/ml, indomethacin 18mg/ml, chlorpromazine 4.5mg/ml and cytochalasin D 2.5 mg/ml) were first co-incubated with Colon-26 cells or HT-29 cells for 1h, then DiL labeled GDNVs nanovectors were added for another 4h incubation at 37 ºC. Followed, cells were fixed for fluorescence imaging or quantification by flow cytometry. To evaluate the stability of circulating FA-GDNVs in mice, DiR dye-labeled FA-GDNVs (200 nmol) were injected into mice via tail vein. Blood was drawn into BD Microtainer MAP tube with K2EDTA (1 mg) at various time points (1 h, 3 h, 6 h, 24 h, 48 h) after injection. Then the intensity of DiR signals from equal volume blood samples were measured using IVIS series pre-clinical in vivo imaging systems. To determine whether the circulating FA-GDNVs in mice are free or not with blood cells, DiR dye-labeled FA-GDNVs were injected into mice, whole blood from mice was

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

collected at various time points (1 h, 3 h, 6 h, 24 h, 48 h) after injection. Blood cells and plasma were separated by centrifuging at 8000 g for 8 min. Then the intensity of DiR signals from equal volume from blood cells and plasma were measured using IVIS series pre-clinical in vivo imaging systems. For the biodistribution of GDNVs or FA-GDNVs in tumor-bearing mice, mice were i.v. administrated DiR-labeled GDNVs and FA-GDNVs (200 nmol), Imaging of tumors and different organs were done at 48 h after the injection. The intensity of DiR signal from different samples was then measured using IVIS series pre-clinical in vivo imaging systems (PerKinElmer, Massachusetts). Hemolysis assay. Fresh mouse blood was collected in BD Microtainer MAP tube with K2EDTA (1

t p ri

mg). Then red blood cells (RBC) were separated, purified and diluted to 1

108 for the hemolysis

according to the literature.53 Different concentrations of GDNVs and liposomes (5, 10, 20, 50, 100

c us

and 200 M) were incubated with RBC at 37 ºC for 2 h. The absorbance of the supernatants from each group was measured using a microplate reader at 540 nm. The group which treated by

an m

Ttiton-100 (1 %) was used as a positive control, and the release rate of hemoglobin for this group was set as 100 %. The hemolysis percentage was calculated as follows:

d e t

Hemolysis percentage = (O.D.540 of samples)/ O.D.540 of positive control

100 %

Cytotoxicity assay. For in vitro toxicity comparison of GDNVs with DC-Chol/DOPE liposomes,

ep

MTT assay was used to analysis the cell viability of Colon-26 cells and HT-29 cells. Briefly, Colon-

c c A

26 and HT-29 cells were seeded in 96-well plates at a density of 1

104 cells/well and incubated

overnight. Cells were then incubated with different amounts of GDNVs and liposomes (5, 10, 20, 50, 100 and 200 M) in PBS for 24 h and 48 h. After the GDNVs or Liposomes-containing medium was removed and cells were thoroughly rinsed once with PBS. Cells were then incubated with 20 L of MTT (5 mg/ml) at 37°C for 4 h until a purple precipitate was visible. Thereafter, the media were discarded and 50 l dimethyl sulfoxide (DMSO) was added to each well prior to spectrophotometric measurements at 570 nm. Untreated cells were used as a negative control. Cell-attached assays were performed to investigate the real-time cytotoxicity using electrical impedance sensing (ECIS) technology (Applied BioPhysics, Troy, NY, UAS), which is based on AC impedance measurements using weak and noninvasive AC signals. The attachment and spread of cells on the electrode surface change the impedance in such a way that morphological information about attached cells can be inferred. The measurement system consists of an 8-well culture dish (ECIS 8W1E plate) with the surface treated for cell culture. The bottom of each well contains a

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

small, active electrode and a large counter electrode. A lock-in amplifier with an internal oscillator is used to switch among the different wells, and a personal computer controls the measurement and stores the data. For experiment, Caco2-BBE cells at a density of 2×105/well were seeded in the plate. Once cells reached confluence, control (PBS) GDNVs (200 M), Liposomes (200 M), DoxGDNVs (equivalent 3.25, 6.5 and 13 M Dox) or free Dox (3.25, 6.5 and 13 M Dox) were added to the wells. Basal resistance measurements were performed using the ideal frequency for Caco2BBE cells, 500Hz, and a voltage of 1V. For evaluation of toxicity of GDNVs and DC-Chol/DOPE Liposomes (30/70, w/w) in vivo, mice were injected (i.v.) with GDNVs or Liposomes (200 M) once/day for 1 and 7 days. 24 h after the last cycle injection, serum were collected for proinflammatory cytokines quantification (TNF-α,

t p ri

IL-1β and IL-6) using Duoset enzyme-linked immunosorbent assay kits from R&D Systems (Minneapolis, MN, USA). Body and different organ weights were measured at the same time.

c us

H&E staining was performed on paraffin-embedded heart, liver, spleen, lung and kidney sections using the standard method.

an m

Cell apoptosis study. The annexin V-FITC/propidium iodide apoptosis assay was used to quantify cell apoptosis and death in vitro. Colon-26 and HT-29 cells with the cell concentration of 5 105/well were seeded in 12-well culture plates and cultured in the presence of GDNVs or Dox-

d e t

GDNVs (equivalent 3.25, 6.5 and 13 M Dox) or free Dox (3.25, 6.5 and 13 M Dox) for

p e c

predetermined time. At the end of cultured, cells were washed twice with cold PBS ad then suspended cells in annexin V binding buffer at a cell concentration of 1 106 cells/mL. Then 100 l

c A

of cells suspension were transferred to a 5 ml culture tube. Followed, 5 l annexin V-FITC and 5 l PI were added and incubated at RT for 15 min in the dark. Finally, 400 l of annexin V binding buffer were added to the tube for analysis. At the same time, unstained cells and cells stained with FITC or PI were also prepared. Healthy cells were double negative in annexin V and PI staining, and early apoptotic cells were positive for annexin V but negative for PI staining, and necrotic cells were positive only for the PI while late apoptotic cells were double positive. Excitation wave was set at 488 nm and the emitted green fluorescence of annexin V and red fluorescence of PI were collected using 525 and 575 nm band pass filters, respectively. Triplicate samples were analyzed for each experiment. For caspase-3/7 detection, Colon-26 and HT-29 cells were first treated with Dox-GDNVs for predetermined time (4h) with the concentration of 6.5 M. Then detection reagent (5 M) were added and incubated for 30 min at 37 °C. Cells were then followed by fixation with 3.7 %

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

formaldehyde for 15 min at RT. Finally, mounted with ProLong Gold reagent and cured for 24 h at RT in the dark before imaging. Apoptotic cells with activated caspase-3/7 show bright green nuclei, while cells without activated caspase-3/7 exhibit minimal fluorescence signal. The excitation/emission maxima for the CellEvent™ Caspase-3/7 Green Detection Reagent are 502/530. Colon-26 subcutaneous xenograft model. Xenograft tumor growth models were used to demonstrate Dox-FA-GDNVs mediated targeted delivery of chemotherapy drug to tumors versus free doxorubicin (Free Dox). To grow subcutaneous tumor xenografts, female athymic BLAB/c nu/nu mice were implanted with 2× 106 cells (Colon-26 cells) in Matrigel (BD Biosciences, 1:1 dilution) subcutaneously in the right flank. When tumors reached approximately 100 mm3 in volume, the

t p ri

mice were randomly assigned to four different treatment groups: 1) Saline; 2) Free Dox (100 g/dose); 3) FA-GDNVs and 4) Dox-FA-GDNVs (100 g/dose). Mice were treated every 4 days for

c us

20 days. The tumor volumes and body weights of the mice were measured and recorded. Tumor volume was calculated as follows: volume =1/2LW2, where L is the long diameter and W is the

an m

short diameter of a tumor. Animals were euthanized when the signals of sickness, such as breathing problems, failure to eat and drink, lethargy or abnormal posture, were observed. To conduct histological analysis, tumors and the major organs (heart, liver, spleen, lung and kidney)

d e t

were fixed for 2 days in 10 % buffered formalin solution and embedded in paraffin. After

p e c

deparaffinization, the tissue sections (5 m) were stained with hematoxylin/eosin (H&E). Images were acquired using an Olympus equipped with a Hamamatsu Digital Camera DP-26. The

c A

apoptosis of tumor cells was determined using 4,6-diamidino-2-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assays, with a commercial apoptosis detection kit according to the manufacturer's standard protocols (Roche Diagnostics, Indianapolis, IN, USA). Images were acquired using an Olympus equipped with a Hamamatsu Digital Camera ORCA-03G. Three fields of each section after Ki67＋ and TUNEL staining were chosen randomly for counting the proliferative and apoptosis cells. Statistical analysis. One-way and two-way analyses of variance (ANOVA) and t-tests were used to determine statistical significance (*p<0.05, **p<0.01, ***p<0.0001).

Acknowledgments This work was supported by the National Institutes of Health of Diabetes and Digestive and Kidney (RO1-DK-071594 to D.M).

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

References 1.

Twelves, C, Wong, A, Nowacki, MP, Abt, M, Burris, H, Carrato, A, et al. (2005). Capecitabine as adjuvant treatment for stage III colon cancer. The New England Journal of Medicine 352: 26962704.

2.

Sung, JJY, Lau, JY, Goh, KL, Leung, WK, and Colorecta, APWG (2005). Increasing incidence of colorectal cancer in Asia: implications for screening. The Lancet Oncology 6: 871-876.

3.

Yeo, H, Niland, J, Milne, D, ter Veer, A, Bekaii-Saab, T, Farma, JM, et al. (2015). Incidence of Minimally Invasive Colorectal Cancer Surgery at National Comprehensive Cancer Network Centers. Journal of the National Cancer Institute 107：dju362

4.

Arnold, M, Sierra, MS, Laversanne, M, Soerjomataram, I, Jemal, A, and Bray, F (2016). Global

t p ri

patterns and trends in colorectal cancer incidence and mortality. Gut: gutjnl-2015-310912. 5.

de Gramont, A, Van Cutsem, E, Schmoll, HJ, Tabernero, J, Clarke, S, Moore, MJ, et al. (2012).

c us

Bevacizumab plus oxaliplatin-based chemotherapy as adjuvant treatment for colon cancer (AVANT): a phase 3 randomised controlled trial. The Lancet Oncology 13: 1225-1233. 6.

an m

Xiao, B, Zhang, MZ, Viennois, E, Zhang, YC, Wei, N, Baker, MT, et al. (2015). Inhibition of MDR1 gene expression and enhancing cellular uptake for effective colon cancer treatment using

d e t

dual-surface-functionalized nanoparticles. Biomaterials 48: 147-160. 7.

Zhang, MZ, Yu, Y, Yu, RN, Wan, M, Zhang, RY, and Zhao, YD (2013). Tracking the Down-

p e c

Regulation of Folate Receptor-alpha in Cancer Cells through Target Specific Delivery of Quantum Dots Coupled with Antisense Oligonucleotide and Targeted Peptide. Small 9: 4183-4193. 8.

c A

Liu, X, Situ, A, Kang, Y, Villabroza, KR, Liao, Y, Chang, CH, et al. (2016). Irinotecan Delivery by Lipid-Coated Mesoporous Silica Nanoparticles Shows Improved Efficacy and Safety over Liposomes for Pancreatic Cancer. ACS nano 10: 2702-2715.

9.

Kunjachan, S, Detappe, A, Kumar, R, Ireland, T, Cameron, L, Biancur, DE, et al. (2015). Nanoparticle Mediated Tumor Vascular Disruption: A Novel Strategy in Radiation Therapy. Nano Letters 15: 7488-7496.

10. Hansen, AE, Petersen, AL, Henriksen, JR, Boerresen, B, Rasmussen, P, Elema, DR, et al. (2015). Positron Emission Tomography Based Elucidation of the Enhanced Permeability and Retention Effect in Dogs with Cancer Using Copper-64 Liposomes. ACS Nano 9: 6985-6995. 11. Danhier, F, Feron, O, and Preat, V (2010). To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. Journal of Control Release 148: 135-146.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

12. Singh, R, Norret, M, House, MJ, Galabura, Y, Bradshaw, M, Ho, DW, et al. (2016). DoseDependent Therapeutic Distinction between Active and Passive Targeting Revealed Using Transferrin-Coated PGMA Nanoparticles. Small 12: 351-359. 13. Tiash, S, and Chowdhury, E (2016). Passive targeting of cyclophosphamide-loaded carbonate apatite nanoparticles to liver impedes breast tumor growth in a syngeneic model. Current pharmaceutical design. 14. Mohammadi, M, and Chen, P (2015). Effect of microvascular distribution and its density on interstitial fluid pressure in solid tumors: A computational model. Microvascular research 101: 2632. 15. Yang, E, Qian, WP, Cao, ZH, Wang, LY, Bozeman, EN, Ward, C, et al. (2015). Theranostic

t p ri

Nanoparticles Carrying Doxorubicin Attenuate Targeting Ligand Specific Antibody Responses Following Systemic Delivery. Theranostics 5: 43-61.

c us

16. Caracciolo, G (2015). Liposome-protein corona in a physiological environment: Challenges and opportunities for targeted delivery of nanomedicines. Nanomedicine: Nanotechnology, Biology and Medicine 11: 543-557.

an m

17. Wu, X, Chen, J, Wu, M, and Zhao, JXJ (2015). Aptamers: Active Targeting Ligands for Cancer Diagnosis and Therapy. Theranostics 5: 322-344.

d e t

18. Fahmy, TM, Fong, PM, Goyal, A, and Saltzman, WM (2005). Targeted for drug delivery. Materials Today 8: 18-26.

p e c

19. Li, F, Zhao, X, Wang, H, Zhao, RF, Ji, TJ, Ren, H, et al. (2015). Multiple Layer-by-Layer LipidPolymer Hybrid Nanoparticles for Improved FOLFIRINOX Chemotherapy in Pancreatic Tumor

c A

Models. Advanced Functional Materials 25: 788-798. 20. Pattni, BS, Chupin, VV, and Torchilin, VP (2015). New Developments in Liposomal Drug Delivery. Chemical Reviews 115: 10938-10966. 21. Mignani, S, El Kazzouli, S, Bousmina, M, and Majoral, JP (2013). Expand classical drug administration ways by emerging routes using dendrimer drug delivery systems: A concise overview. Advanced Drug Delivery Reviews 65: 1316-1330. 22. Allen, TM, and Cullis, PR (2013). Liposomal drug delivery systems: From concept to clinical applications. Advanced Drug Delivery Reviews 65: 36-48. 23. Cheng, R, Meng, FH, Deng, C, Klok, HA, and Zhong, ZY (2013). Dual and multi-stimuli responsive polymeric nanoparticles for programmed site-specific drug delivery. Biomaterials 34: 3647-3657. 24. Wu, H, Zhu, L, and Torchilin, VP (2013). pH-sensitive poly(histidine)-PEG/DSPE-PEG co-polymer

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

micelles for cytosolic drug delivery. Biomaterials 34: 1213-1222. 25. Lohcharoenkal, W, Wang, LY, Chen, YC, and Rojanasakul, Y (2014). Protein Nanoparticles as Drug Delivery Carriers for Cancer Therapy. Biomedical Research International 2014:180549. 26. Bose, S, and Tarafder, S (2012). Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: A review. Acta Biomaterialia 8: 1401-1421. 27. Steinmetz, NF (2013). Viral Nanoparticles in Drug Delivery and Imaging. Molecular Pharmaceutics 10: 1-2. 28. Probst, CE, Zrazhevskiy, P, Bagalkot, V, and Gao, XH (2013). Quantum dots as a platform for nanoparticle drug delivery vehicle design. Advanced Drug Delivery Reviews 65: 703-718. 29. Meng, LJ, Zhang, XK, Lu, QH, Fei, ZF, and Dyson, PJ (2012). Single walled carbon nanotubes as

t p ri

drug delivery vehicles: Targeting doxorubicin to tumors. Biomaterials 33: 1689-1698. 30. Cho, K, Wang, X, Nie, S, and Shin, DM (2008). Therapeutic nanoparticles for drug delivery in

c us

cancer. Clinical cancer research 14: 1310-1316.

31. Laroui, H, Viennois, E, Xiao, B, Canup, BSB, Geem, D, Denning, TL, et al. (2014). Fab'-bearing

an m

siRNA TNF alpha-loaded nanoparticles targeted to colonic macrophages offer an effective therapy for experimental colitis. Journal of Controlled Release 186: 41-53.

32. Wang, QL, Zhuang, XY, Mu, JY, Deng, ZB, Jiang, H, Xiang, XY, et al. (2013). Delivery of

d e t

therapeutic agents by nanoparticles made of grapefruit-derived lipids. Nature Communications 4:1687.

p e c

33. Wang, QL, Ren, Y, Mu, JY, Egilmez, NK, Zhuang, XY, Deng, ZB, et al. (2015). GrapefruitDerived Nanovectors Use an Activated Leukocyte Trafficking Pathway to Deliver Therapeutic

c A

Agents to Inflammatory Tumor Sites. Cancer Research 75: 2520-2529. 34. Zhang, M, Viennois, E, Xu, C, and Merlin, D (2016). Plant derived edible nanoparticles as a new therapeutic approach against diseases. Tissue Barriers: e1134415. 35. Zhuang, XY, Teng, Y, Samykutty, A, Mu, JY, Deng, ZB, Zhang, LF, et al. (2016). Grapefruitderived Nanovectors Delivering Therapeutic miR17 Through an Intranasal Route Inhibit Brain Tumor Progression. Molecular Therapy 24: 96-105. 36. El-Andaloussi, S, Lee, Y, Lakhal-Littleton, S, Li, JH, Seow, Y, Gardiner, C, et al. (2012). Exosomemediated delivery of siRNA in vitro and in vivo. Nature Protocols 7: 2112-2126. 37. Zhuang, X, Deng, Z-B, Mu, J, Zhang, L, Yan, J, Miller, D, et al. (2015). Ginger-derived nanoparticles protect against alcohol-induced liver damage. Journal of Extracellular Vesicles 4:28713.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

38. Teng, Y, Mu, J, Hu, X, Samykutty, A, Zhuang, X, Deng, Z, et al. (2016). Grapefruit-derived nanovectors deliver miR-18a for treatment of liver metastasis of colon cancer by induction of M1 macrophages. Oncotarget 7: 25683-25697. 39. Sun, DM, Zhuang, XY, Xiang, XY, Liu, YL, Zhang, SY, Liu, CR, et al. (2010). A Novel Nanoparticle Drug Delivery System: The Anti-inflammatory Activity of Curcumin Is Enhanced When Encapsulated in Exosomes. Molecular Therapy 18: 1606-1614. 40. Mu, JY, Zhuang, XY, Wang, QL, Jiang, H, Deng, ZB, Wang, BM, et al. (2014). Interspecies communication between plant and mouse gut host cells through edible plant derived exosome-like nanoparticles. Molecular Nutrition & Food Research 58: 1561-1573. 41. Yang, J-S, Gad, H, Lee, SY, Mironov, A, Zhang, L, Beznoussenko, GV, et al. (2008). A role for

t p ri

phosphatidic acid in COPI vesicle fission yields insights into Golgi maintenance. Nature cell biology 10: 1146-1153.

c us

42. Kooijman, EE, Chupin, V, de Kruijff, B, and Burger, KN (2003). Modulation of membrane curvature by phosphatidic acid and lysophosphatidic acid. Traffic 4: 162-174.

an m

43. Popova, AV, and Hincha, DK (2003). Intermolecular interactions in dry and rehydrated pure and mixed bilayers of phosphatidylcholine and digalactosyldiacylglycerol: a Fourier transform infrared spectroscopy study. Biophysical journal 85: 1682-1690.

d e t

44. Wei, T, Liu, J, Ma, HL, Cheng, Q, Huang, YY, Zhao, J, et al. (2013). Functionalized Nanoscale Micelles Improve Drug Delivery for Cancer Therapy in Vitro and in Vivo. Nano Letters 13: 2528-

p e c

2534.

45. Herre, J, Marshall, AS, Caron, E, Edwards, AD, Williams, DL, Schweighoffer, E, et al. (2004).

c A

Dectin-1 uses novel mechanisms for yeast phagocytosis in macrophages. Blood 104: 4038-4045. 46. Koivusalo, M, Welch, C, Hayashi, H, Scott, CC, Kim, M, Alexander, T, et al. (2010). Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. The Journal of cell biology 188: 547-563. 47. Chang, J, Jallouli, Y, Kroubi, M, Yuan, X-b, Feng, W, Kang, C-s, et al. (2009). Characterization of endocytosis of transferrin-coated PLGA nanoparticles by the blood–brain barrier. International Journal of Pharmaceutics 379: 285-292. 48. Boucrot, E, Ferreira, AP, Almeida-Souza, L, Debard, S, Vallis, Y, Howard, G, et al. (2015). Endophilin marks and controls a clathrin-independent endocytic pathway. Nature 517: 460-465. 49. Yang, SY, Zheng, Y, Chen, JY, Zhang, QY, Zhao, D, Han, DE, et al. (2013). Comprehensive study of cationic liposomes composed of DC-Chol and cholesterol with different mole ratios for gene transfection. Colloid Surface B 101: 6-13.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

50. Zhang, MZ, Yu, RN, Chen, J, Ma, ZY, and Zhao, YD (2012). Targeted quantum dots fluorescence probes functionalized with aptamer and peptide for transferrin receptor on tumor cells. Nanotechnology 23. 51. Zhao, XBB, Muthusamy, N, Byrd, JC, and Lee, RJ (2010). Cholesterol as a Bilayer Anchor for PEGylation and Targeting Ligand in Folate-Receptor-Targeted Liposomes (2007). Journal of Pharmaceutical Sciences 99: 4753-4753. 52. Dauty, E, Remy, JS, Zuber, G, and Behr, JP (2002). Intracellular delivery of nanometric DNA particles via the folate receptor. Bioconjugate Chemistry 13: 831-839. 53. Huang, C, Jin, HL, Qian, Y, Qi, SH, Luo, HM, Luo, QM, et al. (2013). Hybrid Melittin Cytolytic Peptide-Driven Ultrasmall Lipid Nanoparticles Block Melanoma Growth in Vivo. ACS Nano 7:

t p ri

5791-5800.

c us

d e t

an m

p e c

c A

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Figure legends Figure 1 Preparation of DOX-FA-GDNVs nanovectors from ginger derived lipids and schematic diagram of the targeted anti-tumor effect in vivo. (a) Ginger derived nanoparticles (NPs) were isolated and purified from edible ginger by ultracentrifugation (150, 000g) and sucrose density gradient (8%/30%/45%/60%), the lipids isolated from ginger derived nanoparticles were reassembled in GDNVs, simultaneously, GDNVs could be modified with targeting ligand folic acid mediating targeted delivery of chemotherapy drug (Dox). (b) The schematic diagram showed that i.v. injection of DOX-FA-GDNVs could achieve targeted delivery of chemotherapy drug to tumors through blood vessel.

t p ri

Figure 2 Characterization of GDNVs prepared from ginger-derived lipid. (a) Ginger juice was purified by sucrose density gradient (8%/30%/45%/60%) under ultracentrifugation, band from

c us

the interface of 30%/45% (as marked in red rectangle) was harvest and note as ginger derived NPs according to literature for further use. (b) Ginger derived NPs harvest from sucrose density

an m

gradient (30%/45%) were characterized by transmission electron microscopy (TEM), the scale bar indicates 100 nm. (c) Ginger derived NPs harvest from sucrose density gradient (30%/45%) were also characterized by atomic force microscopy (AFM), the scale bar indicates 1 m. (d) Pie

d e t

chart of lipid profile of ginger derivered NPs was indicated in the percentage of total lipids. The

p e c

lipid composition of ginger derivered NPs was determined by using a triple quadrupole mass spectrometer. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; LPG,

c A

lysophosphatidylglycerol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidic acid, (n=5). (e) GDNVs were purified from the sucrose gradient as marked in red rectangle. (f) Purified GDNVs were characterized by TEM, the scale bar indicates 500 nm. (g) High-resolution TEM image of GDNVs, the scale bar indicates 100 nm. (h) Particle size of GDNVs were measured by measured by dynamic light scattering (DLS) using a Zetesizer Nano ZS. (i-j) Purified GDNVs were characterized by AFM, the scale bar indicates 1 m.

Figure 3 Evaluation the uptaken efficiency of GDNVs by Colon-26 and HT-29 cancer cells and potential endocytosis pathway. (a) Confocal images of Dil-labeled GDNVs taken up by Colon-26 cells and HT-29 cells. Cells were incubated with DiL--labeled GDNVs for 4 h (red channel) and then labeled with phalloidin-FITC (green channel) and DAPI (blue channel) to display the

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

distribution of F-actin and nucleus. The scale bar indicates 20 m. (b) Quantitative flow cytometry analysis of DiL-labeled GDNVs taken up by Colon-26 cells and HT-29 cells. (c) Potential endocytosis pathway utilized by GDNVs to enter Colon-26 cells. Endocytosis inhibitors (amiloride 34mg/ml, indomethacin 18mg/ml, chlorpromazine 4.5mg/ml and cytochalasin D 2.5 mg/ml) were first co-incubated with Colon-26 cells for 1 h, and then DiL labeled GDNVs nanovectors were added for 4 h incubation at 37 ºC. Followed, cells were fixed for fluorescence imaging. Scale bar indicates 20 m. (d) Uptaken efficiency was quantified by flow cytometry (n=5).

Figure 4 GDNVs can be used as a drug carrier and load therapeutic agent-Dox efficiency. (a) Loading efficiency of Dox in GDNVs was measured. 200 g of Dox solution was added into the

t p ri

ginger lipid film (the lipid concentrations of 5, 10, 20, 50, 100 and 200 M were tested), DoxGDNVs nanovector were centrifuged at 100,000 g for 30 min. The supernatant was collected and

c us

the residual doxorubicin was measured using the microplate reader at the wavelength of 497 nm. (b) Morphology of Dox-GDNVs was observed by TEM. (c) Particles size of Dox-GDNVs was

an m

measured by dynamic light scattering (DLS) using a Zetesizer Nano ZS. (d) Zeta potential of DoxGDNVs was measured by Zetesizer Nano ZS. (e) The stability of Dox-GDNVs was evaluated. Dox-

d e t

GDNVs were suspended in buffer (pH 7.4) and stored for 25 days at 4 ºC. The stability of DoxGDNVs was indicated by size and zete potential change. Data are Mean S.E.M. of three

p e c

independent experiments.

c A

Figure 5 Evaluation the apoptosis in Colon-26 cells induced by free Dox and Dox-GDNVs using Annexin V-FITC/PI staining and ECIS technology. (a) Control group. (b) Free Dox group. (c) Dox-GDNVs group. Cells were treated with three different Dox concentrations (3.25, 6.5 and 13 M) for 8 h, and the apoptosis were evaluated by FACS using annexin V-FITC/PI staining. (d) Cell viability in each concentration were quantified and compared (n=3, **＜ ＜0.01, ***＜ ＜0.001). (e) Apoptosis of Caco2-BBE cells manolayers induced by Dox-GDNVs and free Dox was evaluated by real-time ECIS technology.

Figure 6. Anticancer effect of Dox-FA-GDNVs was evaluated in vivo using Colon-26 xenograft mouse model. (a) Stability of circulating GDNVs by observing the fluorescence of DiR. DiRlabeled GDNVs were i.v. injected into mice and the whole blood from mice was collected at different time points (1, 3, 6, 24 and 48 h) after i.v. injection. The DiR signals from whole blood,

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

blood cells and blood plasma were measured and imaged. The images are representative of three times independent experiments (n=3). (b) Treatment protocol used in present study. Female athymic BLAB/c nu/nu mice were implanted with Colon-26 cells subcutaneously in the right flank at day 0, from day 8, mice at each group were treated every 4 days for 3 times. At the end of the experiments, mice were sacrificed and the anticancer effects in each group were evaluated and compared (n=5). (c) Tumor growth profiles in different treatment groups (saline, free Dox, FAGDNVs and Dox-FA-GDNVs). Arrows indicate the injection time (8, 12, and 16 day after i.v. injection); each point represents the mean

SEM (n = 5). (d) Body weight changes in different

treatment groups. Mouse body weight was normalized to that at the time of injection; each point represents the mean

SEM (n = 5). (e) Xenografts from each group were imaged and compared at

t p ri

the end of the experiments (n = 5). (f) Tumor volumes at the end of the experiments were compared (n = 5). (g) Tumor weights at the end of the experiment were compared (n = 5). (h)

c us

Tumor tissues were removed, fixed and sectioned. H&E-staining of tumor tissues from each group were used to evaluate the anti-tumor effects. Scale bar: 50 m. *p < 0.05, **p < 0.01.

an m

Figure 7 Effect of inhibiting cell proliferation and stimulation of apoptosis by Dox in Colon-26 xenografts was enhanced by Dox-FA-GDNVs. (a) Immunohistochemical analysis of Ki67 and

d e t

TUNEL staining of apoptosis cells in Colon-26 xenograft tissues. (b) Rate of cell proliferation in

ep

Colon-26 xenograft tissues from different treatment groups. (c) Frequency of apoptosis in Colon-

c c A

26 xenograft tissues from different treatment groups. Results are presented as means

SEM

(n = 5). ***p < 0.001. Scale bar: 50 m.

Figure 8 Histological analysis were performed to evaluate toxicity of Dox-FA-GDNVs. H&Estained sections of major organs obtained from tumor-bearing mice treated with saline, free Dox, FA-GDNVs or Dox-FA-GDNVs obtained at the end of the experiment. Scale bar: 50 m.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Figure 1

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Figure 2

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Figure 3

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Figure 4

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Figure 5

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Figure 6

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Figure 7

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Figure 8

© 2016 The American Society of Gene & Cell Therapy. All rights reserved