Effects of Surface Displayed Targeting Ligand GE11 on Liposome

Subscriber access provided by NEW MEXICO STATE UNIV

Article

The Effects of Surface Displayed Targeting Ligand GE11 on Liposome Distribution and Extravasation in Tumor Hailing Tang, Xiaojing Chen, Mengjie Rui, Wenqiang Sun, Jian Chen, Jinliang Peng, and Yuhong Xu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp5001718 • Publication Date (Web): 02 Sep 2014 Downloaded from http://pubs.acs.org on September 8, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

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

Molecular Pharmaceutics

The Effects of Surface Displayed Targeting Ligand GE11 on Liposome Distribution and Extravasation in Tumor Hailing Tang†, Mengjie Rui‡, Xiaojing Chen†, Wenqiang Sun†, Jinliang Peng§，Jian Chen†, Yuhong Xu*†§

†School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, P. R. China;

‡School of Pharmacy, Jiangsu University, Zhenjiang 212013, P. R. China;

§Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, P.R. China

KEYWORDS: GE11; liposome; target delivery; doxorubicin; EGFR; extravasation

ACS Paragon Plus Environment

1


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

Page 2 of 27

ABSTRACT

Targeting ligands displayed on liposome surface had been used to mediate specific interactions and drug delivery to target cells. However, they also affect liposome distribution in vivo, as well as the tissue extravasation processes after IV injection. In this study, we incorporated an EGFR targeting peptide GE11 on liposome surfaces in addition to PEG at different densities, and evaluated their targeting properties and anti-tumor effects. We found that the densities of surface ligand and PEG were critical to target cell binding in vitro as well as pharmacokinetic profiles in vivo. The inclusion of GE11-PEG-DSPE and PEG-DSPE at 2% and 4% mol ratios in the liposome formulation mediated a rapid accumulation of liposomes within one hour after IV injection in the tumor tissues surrounding neovascular structures. This is in addition to the EPR effect which was most prominently described for surface PEG modified liposomes. Therefore, despite the fact that the distribution of liposomes into interior tumor tissues was still limited by diffusion, GE11 targeted doxorubicin loaded liposomes showed significantly better anti-tumor activity in tumor bearing mice as a result of the fast active-targeting efficiency. We anticipate these understandings can benefit further optimization of targeted drug delivery systems for improving efficacy in vivo.


2

Page 3 of 27

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


INTRODUCTION The discovery of critical molecular signatures of cancer cells has inspired the development of targeted therapeutics for cancer treatment1-3. Targeted therapies not only improve specificity and efficacy of the treatment, but also avoid toxic side effects 4 and spare physiological functions of normal tissues5. Great successes have been achieved in the application of antibodies and specific small molecule kinase inhibitors in cancer treatments6-9. Besides, there are growing interests in the development of smart nanomedical agents that could enable efficient delivery of drugs 10-12. Lipid based drug carriers including liposomes have already been demonstrated as one of the most promising nanomedical agents. Doxil and Daunosome have been put into market and showed significant effects for cancer patients13 . The lipid composition was found to be critical for the drug

delivery property14. For example, the incorporation of polyethylene glycol (PEG) conjugated lipids PEG-DSPE would help the liposomes to avoid rapid clearance by the reticuloendothelial system (RES)15, prolong blood circulation time, and improve drug accumulation in tumor tissues through the EPR effect 16. In addition, incorporation of tumor targeting ligands such as antibodies and peptides in the membrane were also beneficial by facilitating specific binding to cancer cells and mediate intracellular delivery of entrapped drug 12, 17-19. However, the exact mechanisms of targeting ligands on improving liposome distribution and extravasation in vivo after systemic administration were not fully understood. Goren et al. 20 were the first to point out that targeting ligands could only help in cell uptake but not liposome distribution in tumor tissues. But later studies reported contradictory results of drug concentration in tumor tissue17, 21-25. In their recent review, Lammers and colleagues26 attributed the discrepancies to the differences of liposome clearance rates, drug encapsulation stabilities, and


3


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

Page 4 of 27

tumor tissue heterogeneities among different active targeted liposome studies. Therefore it is difficult to optimize targeted liposome formulations because of numerous variables and large numbers of highly reproducible animal studies. Recently, noninvasive molecular imaging techniques have been explored to evaluate liposome formulations especially those active targeted liposomes 27, 28. Nevertheless, noninvasive imaging studies could only differentiate liposome distribution roughly down to the organ level, but not quantitatively on the cellular level29, 30. But ligand directed targeting properties rely on molecular interaction. Therefore in this study, we had attempted to offer a more in depth analysis of distribution and extravasation of targeting ligand directed liposome in xenograft tumor tissues. A

surprisingly fast ligand mediated liposome accumulation process in the neovascular regions was observed, resulting in the higher drug concentration, better pro-apoptotic activity, and more efficient tumor inhibition while the overall deep tissue penetration and uptake were still limited. These detailed findings are of great important for the future design and development of efficient targeted drug delivery systems. MATERIALS AND METHODS Materials Hydrogenated soybean phosphatidylcholine (HSPC) and 1, 2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](DSPE-PEG2000) were purchased from NOF Corporation (Tokyo, Japan). Cholesterol was from Avanti Polar Lipids (AL, USA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol) -2000] (ammonium salt) (DSPE-PEG2000-Mal) and GE11-cys(YHWYGYTPQNVIC) were custom made and supplied by Pharmaron Inc. (Beijing, China) with a purity of at least 95%.


4

Page 5 of 27

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


Doxorubicin Hydrochloride was from Shenzhen Wanle Pharmaceutical Co., Ltd (Shenzhen,China). HPLC grade methanol, tetrahydrofuran, isopropanol and trifluoroacetic acid (TFA) were all purchased from Tedia Inc. (OH, USA). Water was purified using a Millipore Milli-Q purification system (Bedford, MA). RhodamineB 1, 2-dihexadecanoyl-sn-glycero-3phosphoethanolamine (rhodamine-DHPE) and N-(fluorescein-5-thiocarbamoyl)-1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine (fluorescein-DHPE) were from Invitrogen Corporation (USA). Primary antibodies, rat anti-mouse CD105 were from eBioscience, Inc. (San Diego, USA), rabbit anti-mouse/human EGFR from Abcam (Cambridge, UK). The secondary antibody Alex488 donkey anti-rat or anti-rabbit IgG were from Invitrogen Corporation (USA). TdT-mediated dUTP Nick-End Labeling (TUNEL) KIT was from the manufacturer Beyotime, Inc. (Shanghai, China).The fluorescence dye 4, 6-diamidino-2-phenylindole (DAPI) was from Roche (USA). Normal Donkey Serum was obtained from Jackson, Inc. (USA). All other reagents were of chemical pure or analytical grade from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Preparation of GE11-conjugated Liposomes DSPE-PEG2000-Mal-GE11 (GE11-PEG-DSPE) was synthesized by conjugating GE11-Cys to the maleimide moiety of DSPE-PEG2000-Mal overnight under nitrogen at 10℃. Both DSPEPEG2000-Mal and GE11-Cys were confirmed by HPLC-ELSD/MS analysis to be at least 95% purity. The reactions were run using various molar ratios of DSPE-PEG-2000-Maleimide and GE11-Cys (1:0.2～1:3), followed by a purification step using the Amicon filter Ultracel PL-1 spun at 4,000×g for 30 min with deionized water washing for 3 times. The reaction yields and the purities of GE11-PEG-DSPE were examined by reverse phase HPLC (Agilent 1100)


5


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

Page 6 of 27

equipped with an evaporative light scattering detector (SEDEX 75, France). At DSPE-PEG2000Mal and GE11-Cys ratio of 1:2, the GE11-PEG-DSPE reached more than 90% purity and was used in the preparation of GE11 conjugated liposomes. Liposomes containing different amount of PEG and/or GE11 conjugated lipids were prepared using the thin film rehydration method. Specifically, HSPC, CHOL, PEG-DSPE and/or GE11PEG-DSPE were mixed in chloroform at various molar ratios and dried into a thin film in a rotary evaporator. The residual chloroform was removed under nitrogen flow. The thin film was rehydrated in PBS (pH7.4) with brief sonication, and the liposomes were extruded through a 100nm membrane. The size distribution and zeta potential of the liposomes were routinely determined using photon correlation spectroscopy (PCS) in a Zeta-sizer Nano-zs90 (Malvern Instruments, UK). The fluorescence labeled liposomes with similar formulations were made by incorporating 0.6% (molar percentage) of either fluorescein-DHPE or rhodamine-DHPE. For preparation of doxorubicin loaded liposomes, the thin lipid film was rehydrated using a solution containing 125mM (NH4)2SO4 and 20mM HEPES (pH 5.0). After size extrusion, the liposome solution was dialyzed against 1000ml HBS (NaCl 150mM, HEPES 20mM, pH7.5）for 2 h. Doxorubicin hydrochloride dissolved in HBS (pH7.5) was then added to the liposome solution and incubated at 60℃ for 1h for the remote loading of doxorubicin. The encapsulation efficiency was confirmed using Vis-UV spectroscopy. HPLC Quantification of Doxorubicin in Liposomes and Tissue Samples


6

Page 7 of 27

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


The amount of Doxorubicin encapsulated in liposomes was determined by HPLC. Daunorubicin was used as an internal standard. Specifically, liposomes were dissolved in 10% triton X solution, and then 20µl sample was injected into the Agilent 1200 HPLC system equipped with a Zorbax-C18 column (15×0.46cm). The initial elution phase consisted of the mixture of 57% methanol and 43% pH4.0 ammonium acetate buffer, changed to 83% methanol and 17% ammonium acetate in 8min, then a sharp increase of methanol ratio from 83% to 95% within 0.5 min, a 5.5 min elution at 95% methanol, and finally 10 min flushing of 57% methanol and 43% ammonium acetate buffer. The flow rate was maintained at 1ml/min and the column temperature at 25℃. The detection wavelength was set at 254 nm.

For determination of doxorubicin concentrations in blood and various tissues, samples were processed and analyzed according to the protocol developed in a previous study 31 with slight modifications. Briefly, each homogenate sample was centrifuged for 10min at 1,000 rpm and 100µl of the supernatant was collected to mix with 100µl of ammonium dihydrogen phosphate (NH4)H2PO4 (pH9.0), 160µl chloroform and 80µl 12.5µg/ml daunorubicin methanol standard solution. The mixture was then centrifuged for 15min at 5,000 rpm after 5min vortex. The separated chloroform layer was transferred to another EP tube and chloroform was removed in a low temperature vacuum drying oven (BIF-30, Shanghai Boxun, China). The residual was redissolved in 50µl methanol and centrifuged at 10,000rpm for 15min. The supernatant was directly injected into HPLC column to determine the doxorubicin content in tissues. Cancer Cell Lines and Xenograft Animal Models Human hepatoma cell line SMMC-7721, human non-small lung carcinoma cell line H1299 and murine breast cancer cell line 4T1 expressing EGFR, originally from Type Culture Collection of


7


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

Page 8 of 27

Chinese Academy of Sciences (CASTCC) were used in this study. The cells were cultured in RPMI1640 culture medium (GIBCO, USA) supplemented with 10% fetal bovine serum (PAA Laboratories, Australia) in a humidified incubator at 37 °C and 5% CO2 atmosphere. Male nude mice (SLAC Inc. Shanghai, China) at the age of 6～8 weeks were used in all studies. 2×106 cells in 100 mL of PBS were injected subcutaneously into the right flank of the mice to establish the SMMC7721 HCC xenograft models. After the tumor size reached 100～ 200 mm3, the mice were sacrificed and tumors were collected and cut into 1×1×1mm cubes. The small pieces of tumor were implanted in the right flank of nude mice. The protocols of animal studies were approved by the animal study committee of School of Pharmacy, Shanghai Jiao Tong University. FACS Analysis of Liposome Binding to Cancer Cells In Vitro H1299, SMMC7721 and 4T1 cells were seeded in 24-well culture plates and cultured in RPMI1640 medium containing 10% fetal bovine serum until they reached about 80% confluence. Fluorescein labeled liposomes containing different molar percentage of GE11-PEG-DSPE and/or PEG-DSPE were diluted in RPMI1640 and added into the culture wells at the dose of 2µM total lipids per well. The cells and liposomes were incubated at 37℃or 4℃for 0.5 hours or 4 hours. Afterwards, cells were washed three times with PBS (pH7.4) to remove unbound liposomes, and harvested separately by trypsin digestion. The binding efficiencies of the cells were analyzed using a BD FACS Calibur (Becton Dickinson, USA). Data from a total of 10,000 events were acquired for each sample and the mean fluorescence intensity of FITC in the cells was analyzed using the Flowjo software.


8

Page 9 of 27

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


Stability of Doxorubicin Loaded Liposomes In Vitro The stabilities of liposomes containing various ratios of GE11-PEG-DSPE and PEG-DSPE were examined by monitoring release of encapsulated doxorubicin in vitro. The release assay was carried out based on the protocol designed by Johnston et al32 in which ammonium chloride was added in the releasing buffer to accelerate the release process. Specifically, the liposomes were diluted 10 folds in release buffer (2mM ammonium chloride, 300mM sucrose, 20mM HEPES, 3mM EDTA, pH 7.4), and loaded into dialysis tubing (10K MW cut off) in a dissolution apparatus (TDTF, Tianjin, China) maintained at 45℃. The temperature was also chosen to accelerate the drug release process. Three aliquots of samples were collected at each time point (0min, 20min, 40 min, 1h, 2h, 4h, 8h) and assayed for doxorubicin concentration to estimate the drug release extent. Pharmacokinetic Properties Doxorubicin loaded liposomes containing various ratio of GE11-PEG-DSPE and PEG-DSPE were injected intravenously via tail vein in 250～300g SD rats at a doxorubicin drug dose of 2mg/kg. At specific time points (1min, 3min, 5min, 10min, 30min, 1h, 2h, 4h, 6h, 12h, 24h, 48h, 72h) after injection, 200µl blood was drawn from the venous plexus of the eyes and put into a 500µl EP tube containing 10µl heparin sodium. Doxorubicin concentrations were determined using the described HPLC assay. Pharmacokinetic parameters including T1/2 were calculated using the Kinetica software based a non-compartment model. Tissue Distribution of Liposomes in Tumor Bearing Mice


9


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

Page 10 of 27

Doxorubicin loaded liposomes containing various ratios of GE11-PEG-DSPE and PEG-DSPE were injected intravenously at a drug dose of 5 mg/kg into nude mice bearing SMMC7721 xenograft tumor. At 1h and 24h after injection, 3~5 mice per group were deeply anesthetized by injection of 200µl 4% chloral hydrate and terminated by exsanguination through cardiac puncture. After the blood vessels were completely flushed with about 40ml of PBS, the liver, heart, lung, kidney, and spleen were collected and weighted. Tissues were then individually homogenized in 2-fold volume of saline according to their weights, and the doxorubicin concentrations were determined. Immunohistochemistry Studies of Liposome Extravasation in Tumor Tissue Sections SMMC 7721 tumor-bearing nude mice were given IV injections of rhodamine-labeled liposomes at the dose of 80mg/kg. Mice were sacrificed at 1h or 24h after injection and tumors were collected after thorough perfusion using PBS. The tumor tissue was immediately embedded in OCT (Leika, German), quickly frozen in liquid nitrogen, and serial sectioned at the thickness of 14 um. The tissue sections were fixed by 4% paraformaldehyde for 15 min, washed with PBS for 3 times, and blocked with 5% donkey serum for 30min at 37℃. They were then stained using either CD105 antibody 33 for labeling proliferating endothelial cells (1:100 dilution, eBioscience), or EGFR antibody (1:100 dilution, Abcam) at 4℃ overnight. The secondary antibody Alex488 donkey anti-rat or anti-rabbit IgG (1:200 dilution, Invitrogen) were added and incubated at 37℃ for 30min. For colocalization of EGFR and blood vessels, primary antibodies were mixed and incubated with the same procedure. The secondary antibody Dylight 549 donkey anti-rat IgG (1:200 dilution, Invitrogen) were mixed with Alex488 donkey anti-rabbit IgG for the labeling. DAPI


10

Page 11 of 27

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


was used to stain the nucleus. After mounting with VECTOR (USA), the tissue section was observed using a confcoal fluorescence microscope (Leica TCS SP8，German).

Histology and Apoptotic Cell Analysis in Tumor Tissue Sections SMMC7721 xenograft mice were given IV injections of various doxorubicin loaded liposome formulations at a drug dose of 5mg/kg. Twenty-four hours after the injection, mice were sacrificed and tumors were collected and fixed in 10% formalin for 24 h and sectioned at a thickness of 14µm. The TdT-mediated dUTP Nick-End Labeling (TUNEL) staining was done as directed by the reagent supplier's protocol (Beyotime, Inc., Shanghai, China). DAPI mounting medium (Vector Laboratories, Inc., Burlingame, CA) was added for nucleus staining. The tissue sections were imaged using a confocal fluorescence microscope (Leica TCS SP8，German).

Tumor Growth Inhibition Study A tumor growth inhibition study was done on SMMC7721 HCC subcutaneous xenograft mouse models. When the tumor volumes reached about 100～200 mm3, the mice were randomly assigned into 5 treatment groups (n=7~8). Mice were intravenously injected PBS or different formulations with a drug dose of 5 mg/kg once a week for three weeks. Tumor size was measured every two days with a caliper across the perpendicular diameter. Tumor volume was calculated using the follow formula: V = π/6 × L × W , where V is the tumor volume, L is the longer perpendicular diameter, W is the shorter perpendicular diameter. Body weight of each mouse was recorded every other day. Humane sacrifice of mice was performed when tumors reached 20 mm in one dimension. Statistical Analysis


11


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

Page 12 of 27

Results are reported as mean±SD. Significancies were evaluated using the ANOVA analysis in the OriginPro 8.5.1software (Origin Lab Corporation, MA, USA). The results were considered statistically different when P

Effects of Surface Displayed Targeting Ligand GE11 on Liposome

Recommend Documents