LHD-Modified Mechanism-Based Liposome ... - ACS Publications

Subscriber access provided by - Access paid by the | UCSB Libraries

Article

LHD modified mechanism-based liposome co-encapsulation of mitoxantrone and prednisolone using novel lipid bilayer fusion for tissue-specific co-localization and synergistic anti-tumor effects Tingting Hu, Hua Cao, Chengli Yang, Lijing Zhang, Xiaohua Jiang, Xiang Gao, Fan Yang, Gu He, Xiangrong Song, Aiping Tong, Gang Guo, Changyang Gong, Rui Li, xiaoning zhang, Xinchun Wang, and Yu Zheng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 23 Feb 2016 Downloaded from http://pubs.acs.org on February 23, 2016

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.

ACS Applied Materials & Interfaces 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 61

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

ACS Applied Materials & Interfaces

LHD modified mechanismmechanism-based liposome coco-encapsulation of mitoxantrone and prednisolone using novel lipid bilayer fusion for tissuetissue-specific coco-localization and synergistic antianti-tumor effects Tingting Hua, Hua Caoa, Chengli Yangb, Lijing Zhanga, Xiaohua Jianga, Xiang Gaoa, Fan Yangc, Gu Hea, Xiangrong Songa, Aiping Tonga, Gang Guoa, Changyang Gonga, Rui Lia, Xiaoning Zhangd, Xinchun Wange, Yu Zhenga*

（#The first three authors contributed equally to the work.）

a, 17#, Section 3, Ren Min Nan Rd, Chengdu, Sichuan 610041, P.R.China. State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu,610041, P.R.China. Fax: +86 2885164060.Tel: +86 28 85503817 E-mail: [email protected] b, 201#, Dalian Road, Zunyi, Guizhou, 563000, China. School of Pharmacy, Zunyi Medical University, Zunyi, P.R. China. c, Department of Gynecology, West China Second University Hospital, Sichuan University, Chengdu,610041, P.R.China. d, 30#, Shuangqing Rd, Haidian Dist, Beijing, Laboratory of Pharmaceutics, School of Medicine, Tsinghua University, Beijing, 100084, P.R.China. e, NO. 221, North Fourth Rd, Shihezi, Xinjiang, School of Pharmacy, Shihezi

ACS Paragon Plus Environment


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

University, 832000, P.R. China. *E-mail:[email protected] (Y. Zheng).

Keywords: mitoxantrone, prednisolone sodium phosphate, co-encapsulation li posome, tissue-specific co-localization, synergistic anti-tumor effect

Abstract Co-encapsulation liposomes are of interest to researchers because they maximize the synergistic effect of loaded drugs. A combination regimen of mitoxantrone (MTO) and prednisolone (PLP) has been ideal for tumor therapy. MTO and PLP offer synergistic anti-tumor effects confirmed by several


Page 2 of 61

Page 3 of 61

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


experiments in this research. The deduced synergistic mechanism is regulation of Akt signaling pathway including the targets of p-Akt, p-GSK-3β, p-s6 ribosomal protein and p-AMPK by MTO reactivating PLP-induced apoptosis. The liposome fusion method is adopted to create co-encapsulation liposomes (PLP-MTO-YM). Then, low molecular weight heparin-sodium deoxycholate conjugate (LHD) is used as a targeting ligand to prove target binding

and

inhibition

of

angiogenesis.

LHD-modified

liposomes

(PLP-MTO-HM) have a high entrapment efficiency around 95% for both MTO and PLP. DSC results indicate that both drugs interacted with liposomes to prevent drug leak during liposome fusion. DiD-C6-HM dyes co-localize well to tumor tissue, and co-administration of DiD-HM and C6-CM do not achieve dye co-localization until 24 h after administration. In both CT26 and B16F10 mouse model, PLP-MTO-HM shows significantly higher tumor inhibition rate relative to the co-administration of MTO-HM and PLP-CM (p98%) was purchased from Biotium Inc. (Hayward, CA). Anti-CD31 primary antibody (ab28364) was a product of Abcam Trading (Shanghai) Company Ltd. (China); Caspase-8 primary antibody (AF705) and GAPDH primary antibody (AF5718) were all from R&D Systems, Inc. (USA)( Minneapolis, MN) and pAkt (ser473) primary antibody was purchased from Cell Signaling Technology, Inc. (USA)(Danvers, MA). All other reagents were of analytical reagent grade. 2.2 Cell line and animals Melanoma cells B16F10 and human prostate cancer PC-3 were cultured in DMEM supplemented with 10% (v/v) FBS, 100 IU/ml penicillin and 100 IU/ml



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

streptomycin at 37 °C in a humidified incubator containing 5% CO2. Mouse colon cancer CT26 and human prostate cancer DU145 cells were cultured in 1640 media supplemented with 10% (v/v) FBS, 100 IU/ml penicillin and 100 IU/ml streptomycin at 37 °C in a humidified incubator containing 5% CO2. Male C57BL/6 and Balb/c mice (6–8 weeks old, 18–22 g) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing, PR China). All animal experiments involved in the present study were approved by the Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China). 2.3 Study on synergistic effect of MTO and PLP 2.3.1 Combination index (CI) analysis of MTO and PLP in several cell lines Drug toxicity was evaluated by MTT assay. Briefly, B16F10, CT26, PC-3 and DU145 cells were seeded on 96-well plates 24 h before treatment. Free drug or the drug combination (various concentrations) was applied for 48 h. Non-treated cells were controls. Experiments were carried out in quadruplicate. MTT assay was used to measure cell viability. The percentage of cell viability relative to the negative control was calculated as an index of cytotoxicity. GraphPad Prism 5 was used to fit the curve of tumor inhibition and calculate IC50 values. Drug interactions were analyzed using a combination index (CI) method based on previous research.36 CI provides qualitative information about the drug interaction. The calculation formula of CI is as follows: CI =

C A, X IC X , A

+

C B, X IC X , B


Page 8 of 61

Page 9 of 61

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


CA,X and CB,X are the concentrations of drug A and drug B used in combination to achieve x% inhibition rate. ICX,A and ICX,B are single agent concentrations required to achieve the same inhibition rate. A CI of less than 1 indicates synergy. 2.3.2 Caspase-8 activation induced by MTO and PLP Post-treated cells (MTO or PLP alone or co-administration) were harvested and washed in PBS. Pellets were resuspended in RIPA cytosolic lysis buffer (Beyotime, China) supplemented with protease inhibitor cocktail (Biotool, China) on ice. Cells lysis solutions were centrifuged at 14,000 rpm at 4 °C. The resulting supernatant was collected as the cytosolic fraction. Western blot was used to quantify expression of caspase-8 after drug treatment. Primary antibody against caspase-8 was purchased from R&D Systems, Inc. (city, state). 2.3.3 MTO’s effect on pAkt MTO-treated cells were harvested and washed in PBS. Pellets were resuspended in RIPA cytosolic lysis buffer (Beyotime, China) supplemented with protease inhibitor cocktail and phosphatase inhibitor (Biotool, China) on ice. Cells lysis solutions were centrifuged at 14,000 rpm at 4 °C. The resulting supernatant was collected as the cytosolic fraction. Western blot quantified expression of pAkt after drug treatment. Primary antibody against pAkt (Ser473) was purchased from Cell Signaling Technology, Inc. (Beverly, MA). 2.3.4 MTO’s effect on Akt signaling pathway



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

Cells lysis solutions were prepared as described is section 2.3.3. And the cell lysis was used to treat pathScan Akt signaling antibody array kit following the protocol which was purchased from Cell Signaling Technology, Inc. (Beverly, MA). 2.4 Preparation of drug-loaded liposomes 2.4.1 Preparation of MTO loaded liposomes (MTO-YM) An ammonium sulfate gradient method was used to prepare MTO liposomes as described previously.16, 37 The composition of lipid membrane was DOTAP:HSPC:CHOL:DSPE-PEG (15:40:40:5 molar ratio). For neutral MTO liposomes, the formulation was HSPC:CHOL:DSPE-PEG (55:40:5, molar ratio), and prepared as above to yield MTO-CM. After MTO loading, unentrapped MTO was removed by Sephadex G-25. Liposomal MTO was measured by UV-Vis spectrophotometry (λ610 nm , PE, Massachusetts，USA). The encapsulation efficiency (EE) and drug loading efficiency (DL) were calculated according to the following equations. Entrapment efficiency (%) = Ce/Ct×100%。 Drug Loading amount in the liposomes/total lipid amount in the liposomes) × 100% Ct = initial drug added Ce = drug encapsulated in liposomes Blank YM was prepared as described above without MTO. DiD, a lipophilic near infrared fluorescent membrane dye, was used to


Page 10 of 61

Page 11 of 61

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


label liposomes. According to preliminary studies, leakage of DiD from liposomes 24 h after preparation was negligible. DiD-loaded liposomes were prepared as described above with little modification. DiD was dissolved in anhydrous ethanol with lipids to form a thin film. Encapsulated DiD was measured in liposomes by deducting unloaded DiD in the supernatant from the amount of drug added initially (DiD-YM: fluorescent liposome). 2.4.2 Preparation of PLP-loaded liposomes (PLP-CM) A traditional thin film-hydration method was used to prepare PLP liposomes. The composition of lipid membrane was HSPC:CHOL:DSPE-PEG (55:40:5 molar ratio). EE and DL were calculated according to the equations mentioned previously. Coumarin-6 (C6), a lipophilic fluorescent membrane dye, was used to label liposomes. According to preliminary studies, leakage of C6 from liposomes 24 h after preparation was negligible. C6-loaded liposomes were prepared as described above with some modification.C6 was dissolved in anhydrous ethanol with lipids to form a thin film. The fluorescent liposome (C6-CM) was assessed for C6 by deducting unloaded C6 in the supernatant from the amount of drug added initially. 2.4.3 Preparation of liposomes co-encapsulated with MTO and PLP (PLP-MTO-YM) First, MTO-loaded cationic liposomes and PLP-loaded liposomes were prepared as described above.38-39 Then, each oppositely charged, individually



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

loaded liposome was fused. MTO and PLP liposomes with equal lipid concentrations were mixed and incubated at 65 oC for several hours to achieve complete fusion. Classic TbCl3/DPA assay was used to study the fusion ratio and the resultant PLP-MTO-YM was stored at 4 oC before use. DiD-C6-YM was prepared as described above after liposome fusion of DiD-YM and C6-CM. A classical three terbium chloride (TbCl3)/dipicolinic acid (DPA) assay was used to investigate liposome fusion ratios. Briefly, DPA and TbCl3 were separately encapsulated in cationic and anionic liposomes, respectively. Upon liposome fusion, TbCl3 and DPA formed complexes [Tb(DPA)3]3- and fluorescence increased by four orders of magnitude. Fluorescence was measured at λex 277 nm and λem 545 nm. TbCl3-loaded and DPA-loaded liposome mixtures dissolved in sodium cholate were set as positive controls. The fusion ratio was the percentage of sample fluorescence relative to positive control. TbCl3 and DPA alone had no emission fluorescence and each was separately encapsulated in the aqueous phase of each liposome. After lipid bilayer full fusion, liposomes contents mixed and yielded fluorescent complexes [Tb(DPA)3]3-. 2.5. Preparation and characterization of LHD and LHD-modified blank YM (HM) 2.5.1 Synthesis of N-deoxycholylethylenediamine (DOCA-NH2) and LHD DOCA-NH2 was synthesized according to published method.40 LHD7 was


Page 12 of 61

Page 13 of 61

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


synthesized according to published method. The degree of substitution (DS) of DOCA to LMWH was calculated by deducting the remaining carboxylic groups in LHD conjugates from the total carboxylic groups in LMWH, which was measured with titration as previously described.41 2.5.2 Preparation and characterization of HM and blank HM First, FITC-labeled LHD7 was synthesized according to published methods.42 Then, the cationic liposome was prepared as described above (without MTO) and was mixed with different concentrations of FITC-LHD at 25 oC

for 30 min to obtain LHD-LP. To separate free LHD from LHD-LP, agarose

gel electrophoresis was performed to characterize the extent to which LHD was adsorbed onto the liposomes. After electrophoresis, migration of FITC-LHD7 was visualized using a Gel Doc System (Bio-Rad, Hercules, CA). All liposomes were stored at 4 °C prior to use. LHD was adsorbed onto blank YM to yield HM (blank). 2.5.3 LHD and HM Zebrafish neovascularization inhibition Tg (flk1: EGFP) transgenic zebrafish were bred and maintained routinely at 28 oC with a 14/10 h on/off light cycle. Healthy embryos at 15 h post fertilization (hpf) were transferred into 24-well plates (N = 6 embryos per well). Then, fresh fish water (1 ml) containing various concentrations of LHD-LP were immediately added into each well. 2.5.4 In vivo Imaging of DiD-labeled HM in Balb/c Mice DiD loaded liposomes were administered to mice (0.5 mg/kg, iv) and 6 h



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

later, mice were anaesthetized. Fluorescent imaging of the heart, liver, spleen, lung, kidney and of tumors was sequentially obtained under an in vivo fluorescent imaging system (Quick View 3000 Bio-Real, Austria; λex 474 nm; λem 525 nm). 2.5.5 Binding of FITC-LHD7 to tumor cells in vitro CT26 and B16F10 cells were seeded on 24-well plates 24 h before treatment. FITC-LHD-containing media was incubated with the cells for 0.5 h at 4 oC. Cell fluorescence was visualized under a fluorescent microscope (OLYMPUS BX43, Tokyo, Japan) after two washes in PBS. 2.6 Characterization of liposomes 2.6.1 Particle size and zeta potential Particle size distribution and zeta potential of the liposomes were measured using a Dynamic Light Scattering Detector (Zetasizer, Nano-ZS, Malvern, UK). 2.6.2 Transmission electron microscopy (TEM) 2.6.2.1 Visualization of liposomes All liposome preparations (PLP-CM, MTO-YM, PLP-MTO-YM and PLP-MTO-HM1:15) were negatively stained with 2% phosphotungstic acid before visualization with TEM (H-600, Hitachi, Japan). 2.6.2.2 Visualization of liposomes during fusion Liposome fusion is dynamic and gradually reaches equilibrium. According to the TbCl3/DPA assay, fusion of MTO and PLP liposomes equilibrated ~2 h


Page 14 of 61

Page 15 of 61

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


after mixing. Thus, liposomes were sampled and visualized with TEM at different time points during the fusion process. Samples were negatively stained with 2% phosphotungstic acid before visualization with TEM (Tecnai G2 F20 S-Twin, FEI, Hillsboro, Oregon). 2.6.3 Differential scanning calorimetry measurements (DSC) To determine phase transition temperatures of liposomes, weighed lyophilized liposomes were placed in an aluminum pan and sealed. Samples were thermally scanned from -10 to 400 °C with 10 °C /min incremental increases in temperature with differential scanning calorimetry (TA Instruments, New Castle, DE). 2.6.4 In vitro release of MTO and PLP from liposomes Liposomes were enclosed in a dialysis bag (MW cut off 8,000 Da) in PBS (10 mM, pH 5, 6.8 and 7.4) containing 0.02% sodium azide at 37 oC, under horizontal shaking at 100 rpm. Drug released into the media was sampled at 1, 3, 4, 5, 8, 12, 24, 96, 120, and 144 h and measured as described previously. 2.6.5. MTT Assay and cytotoxicity Liposomal cytotoxicity in CT26 and B10F10 cells was measured with MTT assay. Cells were seeded in 96-well plates (2 × 103 cells/well) 24 h before treatment. Then liposomes with various drug concentrations were added for 24 h. The MTT assay was performed per the instructions on the kit (20 μl MTT solution; 5 mg/ml was added to media and incubated for 4 h). Formazan crystals formed were dissolved in 150 μL DMSO per well. Absorbance was



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

read (570 nm) on a plate reader. Percentage of cell viability relative to the negative control was calculated to assess the cytotoxicity. GraphPad Prism 5 was used to fit the curve of tumor inhibition and calculate IC50 values. 2.6.6 Stability of liposomes in PBS The stability of PLP-MTO-HM was observed by measuring particle size and drug EE. Several batches of samples were stored in 10 mM PBS at 4 °C. At various time points, particle sizes and EE were measured as depicted previously. 2.6.7 Cellular uptake of PLP-MTO-HM in cell lines B16F10 and CT26 cells were seeded in 24-well plates. After removal of culture medium 24 h after plating, cells were incubated with PLP-MTO-HM for 0.5, 1, 2, and 4 h at 37 °C. Nuclei were stained with DAPI and cellular uptake and subcellular localization of liposomes was observed under high-content screening (HCS) and laser scanning confocal microscopy (Olympus FV1200, Japan). 2.6.8 Surface plasmon resonance (SPR) analysis Biacore T100 (GE Healthcare, Waukesha, WI) was used for SPR analysis. VEGF165A was immobilized (1200 resonance units, RU) on the sensor chip using an amide-coupling method. PLP-MTO-YM and PLP-MTO-HM with various weight ratios of LHD were prepared (20–200 μg/ml in PBS buffer containing 150 mM NaCl), which was also used as a running buffer (flow rate 10 μl/min). The contact time was 240 sec followed by a minimum of a 600 sec


Page 16 of 61

Page 17 of 61

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


delay before regeneration with 3 mM NaOH of the sensor chip surface after each analysis. Binding of liposomes to the sensor chip surface changed the refractive index on chip surface leading to a shift in resonance angle that was proportional to the mass change on the chip. The change in resonance angle is expressed as RU in the sample flow cell, minus the change of RU in the blank flow cell, and this represents the extent of liposome adsorption to the immobilized proteins (∆RU). Each sample was performed at least 6 times. 2.7 Safety assessment of blank HM 2.7.1 Hemolytic experiments Healthy erythrocytes were collected, washed, and resuspended in NS (2% final concentration). Empty liposomes were added to the erythrocyte suspension (final concentrations 1.6, 3.2, 4.8, 6.4, and 8 mg/ml) for 3 h at 37 °C. Erythrocytes treated with purified water and NS were as positive and negative controls, respectively. Hemoglobin release in the supernatant was measured by UV spectrophotometry (545 nm). The mixtures were centrifuged at 1,500 rpm for 10 min before analysis and the percent of hemolyzed cells were calculated (see below) and less than 5% hemolysis was interpreted as hemocompatibile. Hemolysis (%)＝(Absample －Ab

negative control)/

(Ab

positive control －Ab negative

control)×100%

2.7.2 H&E staining of main organs To study liposome toxicity, H&E stained sections of Balb/c mouse tissues



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 18 of 61

were visualized after animals were treated with blank liposomes (100-400 mg/kg, iv tail vein) and histopathological alterations were observed and imaged under light microscopy (OLYMPUS BX43, Tokyo, Japan). 2.8 In vivo imaging of CT26-tumor bearing mice after liposome treatment To establish a CT26 Balb/c mouse model, 5×106 CT26 cells (in 100 μL) were carefully inoculated into animal right limbs (sc). Ten days after cell implantation, mice (N=3/group) were treated with fluorescently labeled liposomes. DiD, a lipophilic near infrared fluorescent membrane dye, was used to label liposomes. DiD-loaded liposomes were prepared as previously described. DiD-C6-HM and DiD-YM and C6-CM were given to animals (0.25 μg/g DiD; 0.4 μg/g C6, iv). At 6 h post-injection, mice were anaesthetized and fluorescent imaging of the whole animal and heart, liver, spleen, lung, kidney and tumors was performed with in vivo fluorescent imaging (Quick View 3000 Bio-Real, Austria; λex 615 nm; λem 747 nm for DiD; λex490 nm and λem 520 nm for C6). Data were analyzed with molecular imaging software and fluorescent signal intensities were expressed as average area values. DiD-HM was administered to CT26-tumor bearing mice and mice were sacrificed

at

various

intervals

after

treatment.

Tumor

tissue

was

immunolabeled with CD31 (primary antibody) and Alexa Fluor 488 (secondary antibody). Co-localization of HM and tumor blood vessels were assessed. 2.9. Tumor inhibition in vivo 2.9.1 Single- versus multiple-dose therapy


Page 19 of 61

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


To study antitumor activity of liposomes in xenograft CT26-tumor bearing mice, animals were divided into 10 groups (N = 8) normal saline (NS, i), free MTO (FM, ii), MTO-CM (iii), MTO-YM (iv), MTO-HM with a LHD to MTO-YM weight ratio of1:60 (MTO-HM1:60, v), MTO-HM1:30 (vi), MTO-HM1:15 (vii), PLP-MTO-YM

(viii),

co-administration

of

PLP-CM

and

MTO-HM1:15

(PLP-CM+MTO-HM1:15, ix) and PLP-MTO-HM1:15 (x) when tumor volumes reached ~100 mm3. Single-dose therapy was either free MTO or liposomal MTO (6 mg/kg, iv) or liposomal PLP (10 mg/kg, iv). All drugs were administered 7 days after tumor inoculation. Multiple-dose therapy was given on day 7, 10, and 13 after tumor inoculation (same dose, same route as single therapy). Controls received equal volumes of NS. Tumor length and width and animal weights were measured every two days. Tumor volume was determined as follows: volume (mm3) ＝ (length × width2)/2. Mice were sacrificed on day 11 after single-dose therapy and on day 16 after multiple-dose therapy. Organs were harvested and frozen for future use. 2.9.2 Liposomes and B16F10 tumor growth inhibition To establish the B16F10 C57L/6 mouse model, 5 × 106 B16F10 cells in 100 μL were inoculated into mouse right limbs (sc). When tumor volume reached ~100 mm3, mice were divided into seven groups (N = 6) NS (i), FM (ii), MTO-CM (iii), MTO-YM (iv), MTO-HM1:15 (v), PLP-CM+MTO-HM1:15(vi) and PLP-MTO-YM (vii). On experiment day 7, free MTO or liposomal MTO was administered once (6 mg/kg, iv). Liposomal PLP was given once as well (10



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

mg/kg, iv). Controls were given saline in the same volume via the same route. Tumor length and width and animal weight were measured every two days and tumor volume was measured. Mice were sacrificed on day 11 and heart, liver, spleen, lung, kidney and tumor tissues were excised and snap-frozen for later use. 2.9.3 Histological analysis Immunohistochemical analysis of tumor microvessel formation of frozen tumor sections was performed with rabbit anti-mouse CD31 (Abcam, city, MA) as the primary antibody. Apoptosis was measured via TUNEL assay. All samples were visualized under fluorescent microscopy (IX73, Olympus, Japan).

3.Results and Discussion

3.1 Synergistic effect of MTO and PLP


Page 20 of 61

Page 21 of 61

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


3.1.1 Combination index (CI) of MTO and PLP in several cell lines CI to evaluate synergism between the two drugs was calculated in four cell lines: CT26, B16/F10, HUVEC, PC-3 and DU145 cells (Table S1). PLP (200 μ g/ml) and various concentrations of MTO were measured and CI gradually decreased as MTO increased, suggesting synergism. CI values were low overall, so the drug interactions were likely similarly synergistic in all cell lines studied. 3.1.2 MTO and PLP cooperate to induce caspase-8 activation Tumor cell susceptibility to MTO and PLP is likely due to apoptosis,36, 43 so we measured caspase-8 activation in the caspase cascade to confirm this. Caspase-8 activation was assessed by the appearance of the p41/43 cleavage fragment. In all cell lines tested, both MTO and PLP activated caspase-8 and MTO combined with PLP enhanced single drug-induced processing of caspase-8 into active cleavage fragments (Figure 2a-d). This synergism was different with respect to cell line tested, and these data agreed with CI results. Thus, MTO and PLP are synergistic via apoptotic induction. 3.1.3 MTO synergize with PLP via regulation of Akt signaling pathway Glucocorticoid resistance is a major cause of therapeutic failure in cancer therapy. Recently, researchers confirmed that Akt activation and additional mechanisms downstream of Akt can block glucocorticoid-induced apoptosis and

induce

resistance

to

glucocorticoid

therapy.44

pAkt

impairs

glucocorticoid-induced gene expression by direct phosphorylation of the



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

NR3C1

glucocorticoid

receptor

at

position

S134m

Page 22 of 61

thereby

blocking

glucocorticoid-induced NR3C1 nuclear translocation.45 Therefore, the Akt pathway has been recognized as a therapeutic target for reversing primary glucocorticoid resistance.45-46 To investigate mechanisms behind synergistic induction of apoptosis between MTO and PLP, we studied the influence of MTO on pAkt (Figure 2e). pAkt inhibition varied among different cells and at various time points and MTO depleted pAkt in all four tumor cell lines used. Thus, pAkt depletion and reactivation of PLP-induced apoptosis may explain MTO and PLP synergism. However, additional mechanisms downstream of Akt may also promote glucocorticoid resistance.47-48 Akt signaling antibody array kit verified the inhibition of MTO on pAkt (Figure 2f). The target map of the Akt Signaling Antibody Array Kit was shown in Figure S1. In addition to pAkt, the targets in Akt signaling pathway influenced by MTO included GSK-3α（Ser21）and 3β (Ser9) (CT26, PC-3 and DU145),

S6

ribosomal

protein （ Ser235/236 ） (CT26

and

PC-3),

PRAS40(Thr246) (PC-3 and DU145), PTEN(Ser390)(PC-3) and AMPKα （Thr172）(DU-145). Expression of all the targets was attenuated after the treatment of MTO. Phosphorylation of the multifunctional kinases GSK-3α and GSK-3β at Ser21 and Ser9, respectively, by Akt inhibits their activity and promotes cell survival. So, attenuation of phosphorylated GSK-3α and GSK-3 β by MTO would weaken the promoting survival effect. On the other hand, there are several reports about the influence of GSK on phosphorylation of the


Page 23 of 61

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


glucocorticoid receptor through ubiquitin-proteasome-mediated pathway. The S6 ribosomal protein is found downstream of p70 S6 kinase and its phosphorylation at Ser235/236 reflects mTOR pathway activation, which is the core regulator in the mTOR/p70S6K/glycolysis signaling pathway. And the inhibition of this pathway would reverse the GC resistance. Phosphorylation of PTEN at residues ser390 leads to loss of phosphatase activity and tumor suppressor function. So, abolishment of phosphorylation of PTEN by MTO would benefit for tumor inhibition. AMPK is an energy sensor that is activated by phosphorylation at Thr172 in response to elevated AMP levels. AMPK can inhibit glucocorticoid-induced apoptosis. Inhibition of AMPK by MTO might contribute to PLP-induced apoptosis. Phosphorylation of PRAS40 was positively correlated with activation of Akt/PRAS40/mTOR signaling pathway, suggesting p-PRAS40 as a potential therapeutic target for various cancers. Above all, the targets of MTO in Akt signaling pathway including GSK-3β, S6 ribosomal protein and AMPK might be related to glucocorticoid resistance, which need further investigation. Generally speaking, the number of targets influenced by MTO was more on PC-3 and DU145 relative to that on CT26 and B16/F10, which was probably responsible for the better synergistic effect of MTO and PLP on prostate cancer in clinic. 3.2Preparation of drug-loaded liposomes 3.2.1 Impact of drug dose on entrapment efficiency of MTO-YM and PLP-CM



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

Increasing MTO from 5 to 10% increased entrapment efficiency approached 100%, and loading efficiency increased linearly (Figure s2). MTO at 15% decreased entrapment efficiency, suggesting gradual saturation of drug loading. PLP behaved the same way. 3.2.2 Liposome fusion for liposome co-encapsulation of MTO and PLP Prior to fusion, we used the passively-loading method to encapsulate the two drugs into liposomes, and hydration media containing both drugs was used in the hydration process. However, precipitates appeared immediately after drug mixing. MTO is a strong acid-weak base salt, and PLP is an alkali salt and together form an insoluble salt. Also, passive loading was used to encapsulate PLP and remote loading was used to load MTO.49 PLP containing ammonium sulfate solution (pH 4) was used to hydrate the thin lipid film but the entrapment efficiency of PLP in acidic medium was low (Table S3). Thus, the special physical and chemical properties of both drugs did not permit existing methods for liposome preparation and fusion was attempted. As described in Figure 1, MTO-loaded cationic and PLP-loaded anionic liposomes were prepared separately. MTO-YM was fused with PLP-CM via heating. 3.3 Preparation and Characterization of LHD and LHD-modified liposomes (LHD-LP) 3.3.1 Synthesis and characterization of LHD LHD synthesis is depicted in Figure s2. The characteristic proton peaks of DOCA in CD3OD included 0.59 ppm (s, 3H, 18-CH3), 0.86 ppm (s,3H,19-CH3),


Page 24 of 61

Page 25 of 61

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


0.91 ppm (d, 3H, 21-CH3) and 1.03–2.50 (25H, steroidal H), 2.81 (s,4H,CH2CH2), 3.78 ppm (m, 1H, 3-H), 4.06 ppm (br,s,1H,12-H). Proton peaks of LHD in D2O/CD3OD included 8.04 ppm of an amide bond. These data confirmed synthesis of LHD Toluidine blue colorimetric assay50 confirmed the DOCA substitution degree was ~7. 3.3.2 LHD7 and LHD7-LP and inhibition of Zebrafish neovascularization Transgenic zebrafish eggs are fertilized in vitro and became transparent after fertilization. Microscope imaging was simple and permitted the transgenic zebrafish to be used for angiogenesis study.51 LMWH cause 50% anti-angiogenesis at 2.5 mg/ml. For free LHD, inhibition of angiogenesis was 50% at 1 mg/ml (Figure 3a). Modification of LMWH with sodium deoxycholate not only reduced its anticoagulant effect but also enhanced anti-angiogenicity In previous studies,52 other traditional methods have been used to evaluate anti-angiogenicity of LHD.35, 53 Here, a transgenic zebrafish model was used to estimate anti-angiogenicity of LHD and we confirmed data in vivo. 3.3.3 Preparation and characterization of LHD7 modified liposome (blank HM) Negatively charged LHD7 might migrate from anode to cathode like free pDNA on agarose gel electrophoresis which was performed to study the adsorption on LHD7 by cationic liposomes. Adsorption was probably driven by electrostatic adherence between counterparts with opposite charges and hydrophobic association between deoxycholate and the lipid bilayer. When the weight ratio of LHD to cationic lipids was 1:5, much unbound LHD7 was



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

observed to migrate to the cathode. When the weight ratio was 1:10, LHD7 was almost absorbed completely. When the weight ratio was greater than or equal to 1:15, LHD was completely absorbed by cationic lipids (Figure 3b). Thus, weight ratios of 1:15, 1:30 and 1:60 were used for further study. Corresponding liposomes are HM1:15, HM-LP1;30 and HM1:60. LHD-LP particle size increased at temperatures greater than 37 oC and 2 h was confirmed to be sufficient for adsorption saturation (Figure 3c). As the same as the LHD, LHD-modified liposome also exhibited anti-angiogenicity (Figure 3d). Research shows that a near-infrared dye DiD-labeled liposome had similar biodistribution as radiolabeled liposomes.54 So, an noninvasive optical animal imaging method would be useful for studying in vivo behavior of nano-scaled vectors. In ex vivo organs 6 h post-injection, greater fluorescent intensity represented the most DiD-liposome accumulation. Liposomes with more LHD modification (1:15) were superior to other liposomes (1:30 and 1:60) and circulated longer (Figure 3e and 3f). LHD modification decreased the surface charge of liposomes from ~+36 mV to +24 mV, which may have decreased protein adsorption during circulation. 3.3.4 Binding of FITC-LHD7 to its receptors in vitro VEGF proteins are members of the PDGF/VEGF growth factor family and are active in angiogenesis, vasculogenesis, and endothelial cell growth. They can induce endothelial cell proliferation, promote cell migration, inhibit apoptosis and induce permeabilization of blood vessels. VEGFs bind to


Page 26 of 61

Page 27 of 61

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


FLT1/VEGFR1 and KDR/VEGFR2 receptors, heparan sulfate and heparin. Some are secreted proteins and some are cell-associated.55 Binding of FITC-LHD to secreted VEGF associated with cancer cell surfaces were studied and we observed that FITC-LHD bound to CT26 and B16/F10 cells (Figure s3). Thus, liposome modification with LHD might increase cellular uptake of liposomes and increase tumor targeting in vivo. 3.4 Characterization of liposomes 3.4.1 Particle size and zeta potential The particle size, PDI and zeta potential of the liposomes in purified water is shown in Table. S. All liposomes were between 100 and 120 nm with uniform distribution (PDI ≈0.2), and LHD modification slightly increased particle size ~10 nm. Phospholipid type influences liposome fusion. Reports about molecular dynamic simulation of liposome fusion suggest that main contribution to free energy barriers can be overcome during fusion by removal of water between two vehicles approaching each other.56 In purified water, liposomes made from HSPC/CHOL/PEG-DSPE were negatively charged, and liposomes made from DOTAP/CHOL/PEG-DSPE were positive. Generally, drug loading would affect the net charge value to some extent, but would not change the charge property. Both liposomes had high net charge, and this facilitated closeness. Also, barrier crossing was affected by lipid tail splaying, which was correlated to lipid fusogenicity. DOTAP had high fusogenicity and the double bond in the lipid end facilitated contact between the lipid tail and



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

external water molecules. Comparing MTO-YM, PLP-CM and PLP-MTO-YM, we might conclude that liposome fusion would not affect particle size distribution uniformity (Table S3). In contrast, it slightly increased uniformity. Smaller vesicles had larger curvature effects, which made it easier for a single splayed lipid to form a bridge between the two outer leaflets and promote additional lipid mixing and eventually fuse. So, the fusion time for small vesicles was substantially shortened but these times gradually extended with greater particle size.57 This accounted for increased uniformity. Of note, both MTO and PLP DiD not leak during liposome fusion.58 According to the TbCl3/DPA assay, liposome fusion was complete and aqueous contents both liposomes mixed. Thus, we interpreted this to mean that both drugs remained inside the fused liposomes. 3.4.2 Liposome visualization during fusion with TEM Liposome fusion was observed under high resolution electron microscopy and before fusion, oppositely charged liposomes were drawn together via electrostatic attraction and fused, and this fusion as driven further by charge until the liposome was large (Figure 4a ,b). Under low resolution electron microscopy, all liposomes including PLP-CM, MTO-YM and PLP-MTO-YM were spherical and uniformly distributed (Figure. 4c). After absorption of LHD, a homogeneous distributed shell covered on the outer liposome surface (Figure 4c). 3.4.3 Cellular uptake of liposomes in CT26 and B16/F10 cells


Page 28 of 61

Page 29 of 61

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


PLP-MTO-HM uptake at various time was studied in CT26 and B16/F10 cells (Figure s4) and it was rapid. During the incubation time (0.5 to 2 h), red fluorescence (MTO) accumulated and then stabilized. Liposome incubation with cells at 4 h DiD not increase fluorescence. Under confocal microscopy (Figure 4d), most of MTO was in the nuclei by 2 h, suggesting that PLP-MTO-HM was an efficient vector for intracellular delivery of MTO. Most pAkt is located in nuclei so MTO may be effective for decreasing nuclear pAkt. 3.4.4 Cytotoxicity evaluation by MTT assay. IC50 values of liposomes were calculated by MTT assay (See Table S5). In CT26 cells, IC50 of YM decreased relative to that of CM but this was not related to toxicity. According to previous work, YM increased anti-tumor effects via enhanced cellular uptake and lysosome escape.59 Modifying LHD and combining it with PLP decreased the IC50 of MTO-HM1:15 and MYO-PLP-HM and this occurred in both CT26 and B16/F10 cells. 3.4.5 Differential scanning calorimetry measurements (DSC) DSC curves of all liposomes are shown in Figure 5a. The main phase transition peak of all liposomes was around 50 oC. Main characteristic peaks of drugs were between 100–150 oC. In the DSC curve of the co-encapsulation liposome, drug peaks disappeared. In contrast, characteristic drug peaks were visible in the mechanically mixed sample of drug and blank liposomes. From previous research, this result suggested an interaction between drug and the liposome lipid bilayer and explained why during liposome fusion, drug leakage



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

may have occurred due to interactions between the lipid bilayer and drug.60 3.4.6 In vitro release of drug from liposomes MTO was released slowly from liposomes in release media of various pHs (Figure 5b). Cumulative release was ~20% by 144 h at pH 5 and this observation agreed with published data.61-62 PLP was released rapidly from liposomes and cumulative release approached 100% at 120 h (pH 7.4), at 96 h for pH 6.8 and at 48 h for pH 5. MTO-loaded liposomes were made using an ammonium sulfate gradient method, and MTO formed precipitates with sulfate ion at the internal aqueous phase. So, the low solubility of MTO at the internal aqueous phase did not create an effective concentration gradient between the internal and outer aqueous phases, which consequently failed to drive the MTO molecule from the phospholipid bilayer under an osmotic pressure gradient. 3.4.7 Liposome stability in PBS A phospholipid bilayer may form pores and consequently disintegrate due to phospholipid oxidation, so liposome stability was studied. We observed that liposomes in PBS at 4 oC may be stable for at least 28 d according to particle size, entrapment efficiency and PDI screening factors (Figure 5c). High liposome stability was ascribed to the introduction of oxidation-resistant HSPCs and repulsion forces among positively charged particles. 3.4.8 Surface plasmon resonance (SPR) analysis SPR is useful for studying binding events. Because both PLP-MTO-YM


Page 30 of 61

Page 31 of 61

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


and PLP-MTO-HM are positively charged and may be attracted by the negatively charged VEGF165-coated dextran surface, a high concentration sodium chloride was included in the running buffer to reduce unspecific binding. Protein, small molecule antigens, and liposomes were not absolutely uniform spheres and this might affect the measured kinetic association constant and qualitative comparison was the main purpose for use of SPR. Data suggested that ∆RU of PLP-MTO-HM increased with increased liposome concentration (20–200 μ g/ml). ∆RU of PLP-MTO-YM also increased with liposome concentration, but there was a difference between ∆RU of PLP-MTO-HM and that of PLP-MTO-YM (p 0.05). Thus, low dose PLP-CM (10 mg/kg) offered almost no tumor inhibition in the CT26 mouse model. Previously, liposomal prednisolone phosphate (PLP-CM) was reported to inhibit up to 90%


Page 34 of 61

Page 35 of 61

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


of tumors in several mouse models including CT26 and B16/F10. Here, PLP-CM DiD not have these effects in the B16/F10 mouse model (Table S8) and we attributed this to dose and treatment frequency. Treatment began when were 100 mm3 and in previous studies, treatment began when tumors were palpable (~20 mm3) and a higher dose (20 mg/kg) was used which offered a significant anti-immune effect as evidenced by total white blood cell (TWBC) counts dropping to 16% of baseline and staying low for four days. We estimated that PLP-CM at 10 mg/kg was sufficient for synergism with MTO while avoiding side effects. Tumor inhibition of MTO-HM (1:15) was better than that of MTO-YM, confirming the contribution of LHD modification (Table S8). As doses were repeated, the advantages between PLP-CM+MTO-HM and PLP-MTO-HM were apparent (p < 0.01) suggesting that co-encapsulation of liposomes with both PLP and MTO was better than single therapy for tumor inhibition in a CT26 model. 3.7.2 Inhibition effect of the liposomes on B16F10 tumor growth NS and PLP group tumors grew rapidly (See Figure 9a-b and Table S9) on 9th day after inoculation and other treatment groups had different degrees of tumor inhibition. Table S9 depicts the treatment groups and outcomes. PLP-CM (10 mg/kg) used here was likely too low, as previously mentioned (Table S9). Cationic liposome YM inhibited up to 57% of tumors, and LHD modification increased this to 68.9%. Thus, YM and HM15 might be promising



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

liposomal

formulations

for

MTO.

Page 36 of 61

Therapeutic

efficacy

of

PLP-CM+MTO-HM1:15 was not distinctly enhanced relative to that of HM1:15, suggesting that two-drug formulations in this experiment were not synergistic and likely ascribed to poor co-localization of both drugs after co-administration therapy as previously mentioned. PLP-CM+MTO-HM15 and PLP-MTO-HM had different inhibitory effects (p < 0.05), suggesting that loading MTO and PLP in the same vector may maximize synergism and be better than simple co-administration therapy. In the CT26 tumor model, PLP-MTO-HM outperformed PLP-CM+MTO-HM15 in a B16/F10 tumor model (p < 0.05), suggesting that loading both drugs on the same vector for synergism was superior to simple co-administrations of separate drug formulations. 3.7.3 Weight change Mouse weight over time after tumor inoculation indicated that the free-MTO group lost weight relative to the other treatment groups and this indicated low or acceptable toxicity (Figure 8c and Figure 9c). 3.7.4 Histological analysis Fluorescent semi-quantitative of immunolabeled blood vessels (CD31) confirmed that all the treatment groups experienced tumor anti-angiogenesis (Figure 8d and Figure 9d). In the CT26 mouse model, the angiogenic density in each

group

sorted

in

ascending

is:

PLP-MTO-HM1:15

LHD-Modified Mechanism-Based Liposome ... - ACS Publications

Recommend Documents