Myristic Acid Modified DA7R Peptide for Whole ... - ACS Publications

Subscriber access provided by Uppsala universitetsbibliotek

Biological and Medical Applications of Materials and Interfaces

Myristic Acid Modified DA7R Peptide for WholeProcess Glioma Targeted Drug Delivery Man Ying, Songli Wang, Mingfei Zhang, Ruifeng Wang, Hangchang Zhu, Huitong Ruan, Danni Ran, Zhilan Chai, Xiaoyi Wang, and Weiyue Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05235 • Publication Date (Web): 23 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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 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 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.

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 25 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

Myristic Acid Modified DA7R Peptide for Whole-Process Glioma Targeted Drug Delivery Man Ying,†,# Songli Wang,†,# Mingfei Zhang,† Ruifeng Wang,† Hangchang Zhu,† Huitong Ruan,† Danni Ran,† Zhilan Chai,† Xiaoyi Wang,† Weiyue Lu*,†, ‡ †

Department of Pharmaceutics, School of Pharmacy, Fudan University, and Key Laboratory of

Smart Drug Delivery (Fudan University), Ministry of Education, Shanghai 201203, & State Key Laboratory of Medical Neurobiology, and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China. ‡

Minhang Branch, Zhongshan Hospital and Institute of Fudan-Minghang Acadimic Health System,

Minghang Hospital, Fudan University, Shanghai 201199, China Keywords: glioma, MC-DA7R, liposomes, BBB, whole-process glioma targeted drug delivery ABSTRACT Clinical treatment of aggressive glioma has been a great challenge, mainly due to the complexity of glioma microenvironment and the existence of blood-brain tumor barrier (BBTB)/blood-brain barrier (BBB), which severely hamper the effective accumulation of most therapeutic agents in glioma region. Additionally, vasculogenic mimicry (VM), angiogenesis and glioma stem cells (GSC) in malignant glioma also lead to the failure of clinical therapy. To address the aforementioned issues, a whole-process glioma targeted drug delivery strategy was proposed. DA7R peptide has effective BBTB penetrating and notable glioma, angiogenesis and VM targeting abilities. Herein, we designed a myristic acid modified DA7R ligand (MC-DA7R), which combines tumor-homing DA7R with BBB-penetrable myristic acid. MC-DA7R was then immobilized to PEGylated liposomes (MC-DA7R-LS) to form a whole-process glioma targeting system. MC-DA7R-LS exhibited exceptional internalization in glioma, tumor neovascular and brain capillary endothelial cells. Enhanced BBTB and BBB traversing efficiency was also observed on MC-DA7R-LS. Ex vivo imaging on brain tumors also demonstrated the feasibility of MC-DA7R-LS in intracranial glioma homing, while immunofluorescence studies demonstrated its GSC and angiogenesis homing. Furthermore, doxorubicin (DOX)-loaded MC-DA7R-LS accomplished remarkable therapeutic outcome, as a result of a synergistic improvement on glioma microenvironment. Our study 1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 25

highlights the potential of MC-modified DA7R peptide as a great candidate for whole-process glioma targeted drug delivery.

1. INTRODUCTION Glioblastoma multiforme, the most common and lethal primary central nervous system tumor, occurs at an annual incidence of 5.3 per 100,000 people.1,2 Even with extensive therapies being performed (i.e. surgical resection, radiation therapy and chemotherapy, etc.), their clinical outcome is somehow very limited.3 Less than 5.1% of the patients could survive 5 years after diagnosis with 15 months median survival time.4,5 This unsatisfactory prognosis is mainly due to the complexity of glioma

microenvironment.6

Tight

blood-brain

barrier

(BBB)

rigorously

inhibits

most

chemotherapeutic agents from infiltrating into glioma cells, therefore impeding the success of chemotherapy.7,8 Moreover, blood-brain tumor barrier (BBTB) of glioma hampers the accumulation of therapeutic paradigms from systemic administration, as the resemblance to blood-tumor barrier of peripheral tumors.9 The high angiogenicity of glioma could also be responsible for glioma recurrence and metastasis.10,11 In addition, aggressive glioma cells could form vasculogenic mimicry (VM) as a part of vasculature without the assistance of endothelial cells, contributing to the failure of treatment.12-14 Last but not least, tumorigenic glioma stem cells (GSC) would also cause drug resistance and glioma recurrence.15,16 The distinct benefits of drug delivery systems with active targeting feature have been verified in improving the therapeutic outcome of glioma treatment.17 Incorporation of targeting ligands, including peptides, small molecules, antibodies, and antibody fragments, significantly improves the delivery efficiency and reduces the off-target scene. To date, several ligand-containing targeted delivery systems have been reported for the treatment of glioma.18-26 In the previous study, a glioma-homing heptapeptide DA7R (DRDPDPDLDWDTDA) was designed as an effective ligand of both neuropilin-1 (NRP-1) and vascular endothelial growth factor receptor 2 (VEGFR2), obtaining the muti-targeting to VM, angiogenesis and glioma cells.27,28 In another study, multifunctional glioma targeted drug delivery was achieved by dual modification of DA7R and a brain-targeted ligand DCDX to liposomes.29 However, to best of our knowledge, so far delivery system with whole-process glioma-targeting capability has not been reported yet. Based on the characteristics of glioma microenvironment, herein we propose a whole-process glioma targeted delivery strategy, 2


Page 3 of 25 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


which could simultaneously target angiogenesis and VM, overcome BBTB and BBB, then target GSC and glioma cells. Myristic acid (MC), a saturated fatty acid with high hydrophobicity, acts as a lipid anchor to phospholipid bilayer of cell membranes. Therefore, by incorporating MC into delivery systems, it could enhance their cellular uptakes to a great extent.30 Moreover, it has been demonstrated that MC is an efficient brain targeting ligand which could benefit delivery systems with significant brain accumulation.31 In the previous reports, MC was conjugated to a polyarginine peptide (MC-R7) to improve its uptake efficiency, as BBB-penetrable MC-R7 was used for in vivo neuroimaging.32,33 Inspired by these findings, we postulate that MC modification could also empower other tumor-homing peptides with BBB penetration feature, which is the key for whole-process glioma targeting. Herein, we modified MC with DA7R peptide by a sensitive coupling reaction. To verify our hypothesis, we first investigated both in vitro and in vivo glioma homing efficiency of synthesized MC-DA7R. Furthermore, we designed MC-DA7R modified PEGylated liposomes (MC-DA7R-LS) and evaluated its glioma targeting efficiency (Figure 1). Finally, the therapeutic outcome of MC-DA7R-LS was investigated by the intracranial U87 tumor-bearing mice while encapsulating doxorubicin (DOX) as the model drug.

2. EXPERIMENTAL SECTION 2.1. Materials. Peptide DA7R were synthesized by GL Biochem (China). GL Biochem (China) provided the Boc-protected D-amino acids. Myristoyl chloride was provided by Sigma-Aldrich (USA). Fanbo Biochemicals (China) supplied the Fluorescein-5-maleimide. Mal-PEG3400-DSPE and mPEG2000-DSPE were provided by Laysan Bio Co. (USA). Hydrogenated soy phosphatidylcholine (HSPC) was obtained from A.V.T. Pharmaceutical (China). Anti-CD31 and anti-CD133 antibody were from Abcam. DAPI was provided by Roche (Switzerland). LysoTracker Red DND-99 and DiR dye were supplied by Invitrogen (USA). FAM (5-carboxyfluorescein) was got from Sigma-Aldrich (USA). Doxorubicin hydrochloride (DOX) was obtained from Haizheng Co. (China). Cholesterol and other reagents were provided by Sinopharm Chemical Reagent Co. LTD. (China). U87 cells were provided by ATCC (Manassas, VA) and human umbilical vein endothelial cells (HUVEC) were supplied by Shanghai Institute of Cell Biology. Cells were cultured in Dulbecco’s 3



Page 4 of 25

Modified Eagle Medium (DMEM) containing 10% FBS (Gibco) at 37 °C with 5% CO2. Male ICR mice of 25-30 g weight as well as BALB/c nude mice of 4-6 weeks age were supplied by BK laboratory animal Co. LTD (China) and housed under specific pathogen free conditions. 2.2. Synthesis of MC-DA7R and Functional Material. Solid phase peptide synthesis was applied to synthesize MC-DA7R by firstly linking the deprotected resin with Boc-protected D-amino acids as the sequence of DRDPDPDLDWDTDADC. Then, myristoyl chloride was coupled to the peptide by reaction between acyl chloride group of MC and amino group of arginine. After that, trifluoroactic acid (TFA) was used to remove all the Boc groups in the sequence. MC-DA7R was cleaved from resin using HF and purified by preparative C18 reverse-phase HPLC. After purification, MC-DA7R was marked by Fluorescein-5-maleimide through maleimide-sulfhydryl covalently conjugation. All products were confirmed by ESI-MS and HPLC. The functional material, MC-DA7R-PEG3400-DSPE was formed by covalent conjugation between cysteine and maleimide. According to the previous method, Mal-PEG3400-DSPE was dissolved in Dimethylformamide at first, then this organic solution was slightly dropped into peptide phosphate buffered solution. Excessive ligand was eliminated through dialysis (MWCO 3.5 kDa) against distilled water. Then the homogenized and lyophilized material was ascertained by 1H-NMR. 2.3. Preparation and Characterization of Liposomes. All liposomal formulations, including liposomes with MC-DA7R-PEG3400-DSPE decoration (MC-DA7R-LS),

D

A7R-PEG3400-DSPE

decoration (DA7R-LS), MC-PEG3400-DSPE decoration (MC-LS) and plain liposomes without targeting material (LS) were constructed by thin-film hydration and extrusion method.34 In brief, a mixture

of

cholesterol/HSPC/mPEG2000-DSPE

(molar

ratio:

45/50/5)

or

cholesterol/HSPC/mPEG2000-DSPE/functional materials (molar ratio: 45/50/4/1) was dissolved in CHCl3 solution. Then a thin film was formed after evaporation. To prepare DiR labeled liposomes, DiR dye was added to the film and hydrated using normal saline solution. FAM solution was used to hydrate the thin film for the construction of FAM-loaded liposomes. After hydration, Avanti Mini Extruder was used to homogenize the mixture by extruding across a series of polycarbonate membranes with the pore size ranging from 200 nm to 50 nm. DOX encapsulated liposomes were constructed using an ammonium sulfate gradient method with high loading efficiency as reported.35 A fluorescence spectrophotometer was used to detect the concentrations of DOX, DiR and FAM. A dynamic light scattering (DLS) zetasizer (Malvern) was applied to record the size distribution and 4


Page 5 of 25 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


zeta potential of each formulation. 2.4. Cellular Uptake of Peptides and Liposomes. Primary brain capillary endothelial cells (BCEC), HUVEC and U87 cells were incubated with Fluorescein-labeled ligands or FAM-loaded liposomes for 4 h. In all cases, the dye concentrations were 5 µM in DMEM with 10% FBS. The nuclei of cells were labeled by DAPI staining. Cellular internalization was captured by a laser scanning confocal microscope (TCS SP5, Leica, Germany). Flow cytometry (FACSAria, BD, USA) was also conducted using suspended cells and fluorescence-positive cells were counted for quantitative analysis. To investigate the subcellular location of ligands, BCEC, U87 cells and HUVEC were incubated with 5 µM Fluorescein marked MC-DA7R, DA7R and MC for 1 h, followed by incubation of 50 nM LysoTracker for 30 min. Cells were gently washed and stained with DAPI for imaging by a confocal microscope. 2.5. In Vitro BBB and BBTB Penetration. In vitro BBTB and BBB models were built up in accordance with precious studies.36,37 20 µM FAM-loaded liposomal formulations dispersed in culture media were slightly added into the upper chambers. The solution in the basal chamber was collected at each predetermined time point. A fluorescence microplate reader was utilized for the measurement of fluorescence intensity. 2.6. Targeting Property on BBB or BBTB/U87 Tumor Spheroids Co-culture Model. Tumor spheroids were built up by plating U87 cells onto agarose-coated 48-well plates with a density of 3000 per well and kept steady for a week. Tumor spheroids were transferred to the lower compartment of BBTB or BBB model. Then 20 µM FAM-loaded liposomes were added into the upper inserts for 4 h incubation at 37 °C. Then tumor spheroids were gently rinsed with PBS and visualized by a confocal laser microscope. 2.7. In Vivo Brain and Glioma Targeting Property. DiR-encapsulated liposomes were prepared for in vivo targeting measurement. For brain targeting evaluation, different liposomal formulations were intravenously injected to ICR mice. After 1 h or 4 h, mice were sacrificed and brains were obtained for ex vivo imaging (IVIS Spectrum, Caliper, USA). To investigate glioma targeting property of liposomes, intracranial U87 glioblastoma bearing nude mice model was established in accordance with previous method.38 Thereafter at day 6 or day 15, 100 µL DiR-loaded liposomes were intravenously administrated to the model mice. 4 h post injection, mice were sacrificed and whole brains were obtained for ex vivo imaging For both ex vivo imaging experiments, the mean 5



fluorescence intensity was quantified and analyzed. 2.8. Immunofluorescence Analysis. To localize the molecules and liposomes in intracranial glioma, immunofluorescence assay was carried out using intracranial U87 xenograft glioblastoma bearing nude mice model. Mice were intravenously treated with various FAM-encapsulated liposomes or Fluorescein-labeled peptides. Mice were sacrificed after 4h for liposomal formulations and 1 h for peptides. The obtained whole brains were fixed with 4% paraformaldehyde solution overnight at least. After dehydration using gradient sucrose, samples were frozen within O.C.T. and then cut into 10 µm slices. The slides were firstly blocked with bovine serum albumin for 1 h and then incubated with anti-CD31 antibody or anti-CD133 antibody overnight. The slides were visualized using a confocal laser microscope after staining with secondary antibody and DAPI. 2.9. Pharmacokinetics Property. The pharmacokinetic properties of various DOX-encapsulated liposomal formulations were measured using ICR mice intravenously treated with a single dose of 5 mg/kg. Blood was sampled from the retro-orbital sinus at each predetermined time point at 3, 30 min and 1, 2, 4, 8, 24, 48 h post injection. 30 µL blood samples were diluted in 70 µL distilled water for fluorescent measurement at 480/590 nm by a fluorescence microplate reader. The DOX-free blood sample was used as blank control. The pharmacokinetic results were directly calculated using DAS2.0 software. 2.10. Cytotoxicity Study. MTT assay was performed to assess the toxicity of different DOX-encapsulated liposomal formulations on U87 cells and HUVEC. In brief, cells were seeded in 96-well plates and cultured overnight. Then various formulations with a series of DOX concentrations were added into cells for 4 h incubation. After that, the media were replaced by DOX-free DMEM containing 10% FBS. After 3 days culture, the absorbance at 490 nm was measured using a plate reader while using untreated cells as the control. 2.11. In Vivo Antitumor Study. Male nude mice bearing U87 intracranial glioblastoma were utilized to evaluate in vivo antitumor efficacy. The model mice were randomly separated into 6 groups (n = 11). Mice were intravenously administrated with free DOX, MC-DA7R-LS/DOX, D

A7R-LS/DOX, MC-LS/DOX, LS/DOX and saline with DOX dose of 2 mg/kg at 6, 9, 12, 15, 18

days after implantation. Survival of mice was monitored and Kaplan-Meier survival curves were plotted. At last administration, one mouse of each group was randomly selected for immunohistochemical study. In brief, the brains of sacrificed mice were excised, formalin-fixed and 6


Page 6 of 25

Page 7 of 25 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


parrffin-embedded and sectioned into 5µm slices. Microvessels (include VM and angiogenesis) in glioma were detected by CD31/PAS dual staining.39 TUNEL assay was further performed to estimate the apoptosis in glioma regions.40 CD133 staining was also performed to detect the tumor stem cells. Software Image pro plus 6 was used to count the positive cells in three different equal-sized fields, which were randomly selected. 2.12. Statistical Analysis. All data were presented as mean ± SD. One-way ANOVA was carried out to accomplish the comparison between different groups by using software GraphPad prism 6.0. p < 0.05 was considered as statistically significant.

3. RESULTS AND DISCUSSION It has been reported that novel multifunctional ligand was formed by simple conjugation of two peptide ligands.41 For instance, CK sequence-containing SHH peptide and K237 peptide were fused together to facilitate the targeting to glioma cells, angiogenesis and VM.42 Stapling strategy was also used to modify RGD peptide, and the formed sRGD peptide could simultaneously cross BBB and BBTB.24 In another study, a Y-shape ligand was generated by linking cyclic RGD with p-hydroxybenzoic acid (pHA) through a short spacer. As a result, similar BBB and BBTB escape effects were observed.18 In this study, we extrapolated ligand modification strategy to whole-process glioma active targeting and drug delivery. Heptapeptide DA7R, with efficient glioma cells, angiogenesis, and VM targeting abilities was chosen for coupling with MC by a facile one-step reaction. The MC-DA7R peptide inherits BBB traversing and glioma targeting features. MC-DA7R was further immobilized to PEGylated liposomes (MC-DA7R-LS) for whole-process glioma targeted drug delivery. 3.1. Targeting Ability of MC-DA7R 3.1.1. Characterization of MC-DA7R. MC was successfully coupled to the

D

A7R peptide

through a one-step peptide coupling, and the coupling product was well characterized and verified by both ESI-MS and HPLC (Figure S1). 3.1.2. Cellular Uptake and Intracellular Distribution of MC-DA7R. Primary BCEC, the main component of BBB, was separated from rat brains. U87 and HUVEC cells were also selected as the most representative human glioma cell line and tumor neovascular endothelial cell model, respectively.43 Fluorescein-labeled MC, DA7R, and MC-DA7R ligands were incubated with these 7



Page 8 of 25

three types of cells to assess the brain, angiogenesis and glioma targeting of MC-DA7R at cell level. Both flow cytometry and confocal imaging were applied to measure the cellular uptake. As shown in Figure 2, DA7R could efficiently internalize into both U87 cells and HUVEC, but not into primary BCEC, which was consistent with our previous study. MC could be taken up by all three types of cells, possibly due to its high hydrophobicity. The uptake of MC-DA7R in all three types of cells seemed to be more efficient, compared with MC and DA7R. Moreover, primary BCEC, U87 cells and HUVEC were stained by LysoTracker to track the intracellular pathways (Figure 3). Most D

A7R peptide colocalized with lysosomes in U87 cells and HUVEC. MC equally distributed in the

whole cytoplasm without any specific colocalization with lysosomes, implying that it might just simply transport into cells by diffusion. However, MC-DA7R was well colocalized with lysosomes in every situation, revealing that the internalization of MC-DA7R underwent endosome-lysosome pathway. The results confirmed MC-DA7R processes spontaneous BBB penetration and brain targeting capability. 3.1.3. Distribution of MC-DA7R in Intracranial U87 Xenograft Glioma. To assess in vivo targeting capability of MC-DA7R, different Fluorescein-labeled ligands were intravenously administrated into intracranial glioma bearing nude mice. Brains were obtained for immunofluorescence staining 1 h after injection. As shown in Figure 4, MC-DA7R demonstrated highest glioma accumulation. Tumor neovasculature was visualized by anti-CD31 antibody labeling. High colocalization of MC-DA7R with blood vessels demonstrated its targeting to glioma neovasculature. CD133 is the most common marker of GSC which are responsible for tumorigenesis and drug resistance.44 MC-DA7R also showed strong colocalization with CD133-positive cells, illustrating its targeting ability to GSC. 3.2. Characterization of Functional Materials and Liposomes. Sulfhydryl-maleimide coupling was

applied

to

synthesize

functional

materials

including

MC-DA7R-PEG3400-DSPE,

MC-PEG3400-DSPE and DA7R-PEG3400-DSPE. Successful ligand conjunctions were confirmed by 1

H-NMR spectra of functional materials shown in Figure S2. It is noteworthy that different

DOX-loaded liposomal formulations displayed similar size distribution (110 nm), zeta potential (-10.5 mV) and DOX loading efficiency (95%) (Figure S3), which indicated MC-DA7R decoration would not alter the physical characteristics of liposomes. Additionally, all DOX-loaded liposomes showed similar DOX-release profiles in both PBS (pH 7.4) and mouse serum as shown in the Table 8


Page 9 of 25 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


S1. 3.3. Targeting Property of MC-DA7R-LS. 3.3.1. Cellular Uptake of MC-DA7R-LS. To investigate whether MC-DA7R grafting could enhance cellular uptake of modified liposomes, three types of cells (i.e. primary BCEC, HUVEC and U87 cells) were treated with FAM-loaded liposomal formulations (Figure 5). As expected, MC-DA7R functionalized liposomes showed significantly better internalization in all three cell types, which validates the feasibility of MC-DA7R-LS in brain, angiogenesis, and glioma targeting. 3.3.2. In Vitro BBB and BBTB Penetration of MC-DA7R-LS. In vitro BBB model was firstly conceived in accordance with reported protocols to explore the BBB transcytosis efficiencies of different formulations. As shown in Figure 6A, MC-DA7R-LS displayed best BBB penetration at every tested time point. For instance, after 2 h incubation, 1.80 ± 0.06% of MC-DA7R-LS and 1.53 ± 0.06% of MC-LS penetrated through BBB. The values were significantly higher than those of D

A7R-LS (1.12 ± 0.04%) and LS (1.08 ± 0.01%), respectively. In vitro BBTB model was also built

up to further investigate the liposomes’ BBTB penetration (Figure 6B). MC-DA7R-LS demonstrated highest transport percentage at all predetermined time points. After 4 h treatment, 6.93 ± 0.20% of MC-DA7R-LS, 4.90 ± 0.14% of DA7R-LS, and 4.06 ± 0.19% of MC-LS traversed through BBTB monolayer, which were absolutely higher than that of LS (2.82 ± 0.13%). Moreover, BBB and BBTB/U87 tumor spheroids co-culture model were conceived to in vitro intimate glioma microenvironment. With high consistence to previous studies, MC-DA7R-LS effectively traversed BBTB and BBB monolayer and eventually internalized into the U87 tumor spheroids (Figure 6C-D). 3.3.3. In Vivo Targeting Property of MC-DA7R-LS. To evaluate the in vivo brain targeting profile of MC-DA7R-LS, DiR-loaded liposomes were prepared and intravenously administrated into ICR male mice. Mice were sacrificed after 1 h and 4 h. Then ex vivo imaging of vital organs was further performed. MC-DA7R-LS and MC-LS represented remarkably higher accumulation in whole brains in comparison to MC-lacking liposomes (Figure 7A-B). MC modification also increased the accumulation in livers, spleens and lungs (Figure S4). Next, nude mice with intracranial glioblastoma implantation were used to assess the glioma targeting profiles of various liposomes. As shown in Figure 7C-D, liposomes decorated with MC-DA7R showed the highest glioma accumulation among all the formulations at both early (i.e. 6 day) and late stages (i.e. 15 day) 9



of glioma. On the other hand, unmodified liposomes only revealed scarce glioma retention at both stages. DA7R modification only enhanced glioma accumulation at the late stage due to the deferred glioma targeting and BBTB penetration of DA7R. MC-LS also demonstrated intensified glioma accumulation owing to the brain targeting and BBB penetration of MC. Immunofluorescence assay was further performed to confirm the feasibility of MC-DA7R modification (Figure 8). Highest glioma accumulation has been observed in MC-DA7R-LS group, which was consistent with previous results. Furthermore, MC-DA7R-LS colocalized well with CD31-labeled blood vessels and CD133-positive cells, which upheld the practicability of MC-DA7R modification in angiogenesis and GSC targeting. 3.4. Anti-glioma Effect of MC-DA7R-LS. 3.4.1. Cytotoxicity of MC-DA7R-LS. Anti-glioma efficacy in vitro was quantified by anti-proliferation against U87 cells (Figure 9A) and HUVEC (Figure 9B). The half maximal inhibitory concentration (IC50) of each DOX-loaded liposomes and free DOX were calculated after 72 h inhibition. Free DOX showed best anti-proliferative effect in both cell lines due to its expeditious internalization in cells. The IC50 data of unmodified liposomes on U87 cells and HUVEC were relatively higher than any other formulations. DA7R-LS and MC-LS yielded lower IC50 values in both cases, owing to better cellular internalization. MC-DA7R-LS displayed greatest cytotoxicity among all the formulations, with the IC50 values of 0.89 µM on U87 cells and 0.51 µM on HUVEC, supporting MC-DA7R modification could enhance the anti-glioma of liposomes. 3.4.2. Pharmacokinetic Profiles of MC-DA7R-LS. Figure S5 displayed the pharmacokinetic properties of DOX-loaded liposomes. Free DOX was quickly cleared off the circulation system within 4 h after administration. In contrast, liposomal formulations maintained sustained release of DOX which were still detectable at 48 h post injection. The prolonged circulations of PEGylated liposomes were further confirmed by comparisons of pharmacokinetic properties (i.e. half-life (t1/2), area under concentration-time curve (AUC0-48) and mean residence time (MRT)). It is noteworthy that MC-DA7R modification did not alter the pharmacokinetic profiles of liposome systems. 3.4.3. In vivo Anti-glioma Efficacy. We further explored in vivo therapeutic outcome by evaluating survival of intracranial U87 glioma bearing nude mice treated with liposomal formulations. As expected, MC-DA7R-LS/DOX accomplished most remarkable anti-glioma effect, where prolonged median survival time (29 days) was recorded (Figure 9C). It outperformed all the 10


Page 10 of 25

Page 11 of 25 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


other groups including DA7R-LS/DOX (26.5 days), MC-LS/DOX (25.5 days), LS/DOX (23.5 days), free DOX (22 days) and saline (20 days). These data confirmed that the unique targeting advantages of MC-DA7R-LS could translate into enhanced therapeutic efficiency. Immunohistochemical studies (i.e. CD31/PAS, TUNEL, and CD133 staining) were then performed to derive possible anti-glioma mechanisms. Angiogenesis structures were labeled in CD31-positive channel, while PAS-positive but CD31-negative channels were considered as VM. From the results shown in Figure 9 D, E, and H, MC-DA7R-LS/DOX showed the best inhibitory effect on angiogenesis and VM. MC-DA7R-LS/DOX treatment also led to highest TUNEL-positive staining in comparison to other groups, revealing that the excellent anti-glioma outcome was attributed to intensive apoptosis (Figure 9F and I). Furthermore, lowest count of CD133-positive cells was observed in MC-DA7R-LS/DOX group, indicating the improved therapeutic effect was related to the elimination of GSC. These results emphasized the feasibility of MC-DA7R-LS/DOX in anti-glioma treatment, validating that the enhanced outcome was from the synergistic inhibition against angiogenesis, VM, glioma cells, and GSC.

4. CONCLUSION In summary, we designed a myristic acid coupled peptide MC-DA7R with excellent in vitro and in vivo glioma targeting efficacy. Whole-process glioma targeted drug delivery system was then assembled by integration of MC-DA7R to PEGylated liposomes. The formulated MC-DA7R-LS could target angiogenesis/VM, traverse across BBB/BBTB and target glioma cells/GSC with high precision, faultlessly achieving the whole-process glioma targeting strategy. Great improvement in therapeutic outcome was eventually achieved by loading therapeutic paradigms into MC-DA7R-LS, as a result of synergistic effect of active targeting and subsequent drug delivery.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Analysis of MC-DA7R and MC-DA7R-Fluorescein; 1H-NMR spectra of functional materials; size distributions, zeta potentials and loading efficiencies of DOX-loaded liposomes; Leakage of different DOX-loaded liposomal formulations; Ex vivo imaging of harvested vital organs of 11



liposomal formulations in healthy ICR mice; time-DOX concentration characteristics of liposomal formulations.

AUTHOR INFORMATION Corresponding Author *Tel.: +86 21 5198 0094; fax: +86 21 5198 0090 E-mail: [email protected] ORCID Weiyue Lu: 0000-0001-8003-2675 Author Contributions #

These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was funded by National Basic Research Program of China (973 Program, No.2013CB932500), Shanghai International Science and Technology Cooperation Project (No.16430723800), Shanghai Education Commission Major Project (2017-01-07-00-07-E00052) and National Natural Science Foundation of China (No.81473149, No. 81773657 and No. 81690263).

12


Page 12 of 25

Page 13 of 25 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


Figure 1. Schematic diagram for whole-process glioma targeting enabled by myristic acid modified D

A7R peptide. MC- DA7R-LS was designed to target angiogenesis and VM, overcome BBTB and

BBB, then target GSC and glioma cells.

13



Figure 2. Cellular uptake of 5 µM Fluorescein-labeled ligands and FAM by the primary BCEC (A-B), U87 cells (C-D) and HUVEC (E-F) with 4 h incubation. Scale bars are 10 µm.

Figure 3. Colocalization of endocytosed MC-DA7R and lysosomes in primary BCEC (A), U87 cells (B) and HUVEC (C). LysoTracker and DAPI staining was conducted after 1 h incubation of different ligands. Scale bars are 10 µm.

14


Page 14 of 25

Page 15 of 25 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


Figure 4. Distribution of Fluorescein-labeled ligands in murine intracranial U87 xenograft glioma tumor sites. Green, different ligands; red, CD31-stained blood vessels (A) or CD133-positive cells (B); blue, DAPI-stained cell nuclei. Scale bars are 50 µm.

15



Figure 5. Cellular uptake of liposomal formulations by the primary BCEC (A-B), U87 cells (C-D) and HUVEC (E-F) with 4 h incubation. Scale bars are 10 µm.

16


Page 16 of 25

Page 17 of 25 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


Figure 6. In vitro assessment of BBB and BBTB penetration of FAM-loaded liposomal formulations. Transcytosis efficiency of various liposomes in BBB (A) and BBTB model (B). Mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001. The penetration of FAM-loaded liposomal formulations in co-culture model of BBB/U87 tumor spheroids (C) and BBTB/U87 tumor spheroids (D). Original magnification, 200×.

17



Figure 7. In vivo targeting of DiR-loaded liposomal formulations. A-B) ex vivo fluorescence imaging and semi-quantitative measurement of mean fluorescence intensity of healthy brains. C-D) ex vivo imaging and semi-quantitative evaluation of mean fluorescence intensity of brains at day 6 and day 15 post tumor implantation. Mean ± SD, n = 3. *p < 0.05, **p < 0.01.

18


Page 18 of 25

Page 19 of 25 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


Figure 8. Intracranial U87 xenograft glioma tumor distribution of liposomal formulations 4 h post injection. Green, FAM-loaded liposomes; red, CD31-stained blood vessels (A) or CD133-positive cells (B); blue, DAPI-stained cell nuclei. Scale bars are 50 µm.

19



Figure 9. Therapeutic outcome of DOX-loaded liposomal formulations. Cytotoxicity of various DOX-loaded liposomal formulations on U87 cells (A) and HUVEC (B). C) Survival of intracranial glioblastoma bearing nude mice administrated with liposomal formulations of equivalent DOX dose, n = 10. Quantification of angiogenesis inhibition (D), VM inhibition (E), TUNEL positive apoptotic cells (F) and CD133 positive cells (G) in tumors. Immunohistochemical evaluation including CD31/PAS dual staining (H), TUNEL staining (I) and CD133 staining (J). Mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. REFERENCES (1) Stupp, R.; Hegi, M. E.; Mason, W. P.; van den Bent, M. J.; Taphoorn, M. J.; Janzer, R. C.; Ludwin, S. K.; Allgeier, A.; Fisher, B.; Belanger, K.; Hau, P.; Brandes, A. A.; Gijtenbeek, J.; Marosi, C.; Vecht, C. J.; Mokhtari, K.; Wesseling, P.; Villa, S.; Eisenhauer, E.; Gorlia, T.; Weller, M.; Lacombe, D.; Cairncross, J. G.; Mirimanoff, R. O. Effects of Radiotherapy with Concomitant and 20


Page 20 of 25

Page 21 of 25 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


Adjuvant Temozolomide Versus Radiotherapy Alone on Survival in Glioblastoma in a Randomised Phase Iii Study: 5-Year Analysis of the Eortc-Ncic Trial. Lancet Oncol. 2009, 10, 459-466. (2) Ostrom, Q. T.; Gittleman, H.; Fulop, J.; Liu, M.; Blanda, R.; Kromer, C.; Wolinsky, Y.; Kruchko, C.; Barnholtz-Sloan, J. S. Cbtrus Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2008-2012. Neuro Oncol. 2015, 17 Suppl 4, iv1-iv62. (3) Wen, P. Y.; Kesari, S. Malignant Gliomas in Adults. N. Engl. J. Med. 2008, 359, 492-507. (4) Dolecek, T. A.; Propp, J. M.; Stroup, N. E.; Kruchko, C. Cbtrus Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2005-2009. Neuro Oncol. 2012, 14 Suppl 5, v1-49. (5) Stupp, R.; Brada, M.; van den Bent, M. J.; Tonn, J. C.; Pentheroudakis, G. High-Grade Glioma: Esmo Clinical Practice Guidelines for Diagnosis, Treatment and Follow-Up. Ann. Oncol. 2014, 25 Suppl 3, iii93-101. (6) Wei, X.; Chen, X.; Ying, M.; Lu, W. Brain Tumor-Targeted Drug Delivery Strategies. Acta Pharm. Sin. B 2014, 4, 193-201. (7) Bhowmik, A.; Khan, R. Blood Brain Barrier: A Challenge for Effectual Therapy of Brain Tumors. BioMed Res. Int. 2015, 2015, 320941-320961. (8) Liu, Y.; Lu, W. Recent Advances in Brain Tumor-Targeted Nano-Drug Delivery Systems. Expert Opin. Drug Delivery 2012, 9, 671-686. (9) Zhan, C.; Lu, W. The Blood-Brain/Tumor Barriers: Challenges and Chances for Malignant Gliomas Targeted Drug Delivery. Curr. Pharm. Biotechnol. 2012, 13, 2380-2387. (10) Hardee, M. E.; Zagzag, D. Mechanisms of Glioma-Associated Neovascularization. Am. J. Pathol. 2012, 181, 1126-1141. (11) Wong, P. P.; Bodrug, N.; Hodivala-Dilke, K. M. Exploring Novel Methods for Modulating Tumor Blood Vessels in Cancer Treatment. Curr. Biol. 2016, 26, R1161-r1166. (12) Maniotis, A. J.; Folberg, R.; Hess, A.; Seftor, E. A.; Gardner, L. M.; Pe'er, J.; Trent, J. M.; Meltzer, P. S.; Hendrix, M. J. Vascular Channel Formation by Human Melanoma Cells in Vivo and in Vitro: Vasculogenic Mimicry. Am. J. Pathol. 1999, 155, 739-752. (13) Chen, Y.; Jing, Z.; Luo, C.; Zhuang, M.; Xia, J.; Chen, Z.; Wang, Y. Vasculogenic Mimicry-Potential Target for Glioblastoma Therapy: An in Vitro and in Vivo Study. Med Oncol 21



2012, 29, 324-331. (14) Ying, M.; Chen, G.; Lu, W. Recent Advances and Strategies in Tumor Vasculature Targeted Nano-Drug Delivery Systems. Curr. Pharm. Des. 2015, 21, 3066-3075. (15) Kise, K.; Kinugasa-Katayama, Y.; Takakura, N. Tumor Microenvironment for Cancer Stem Cells. Adv. Drug Delivery Rev. 2016, 99, 197-205. (16) Lathia, J. D.; Mack, S. C.; Mulkearns-Hubert, E. E.; Valentim, C. L.; Rich, J. N. Cancer Stem Cells in Glioblastoma. Genes Dev. 2015, 29, 1203-1217. (17)Frosina, G. Nanoparticle-Mediated Drug Delivery to High-Grade Gliomas. Nanomedicine 2016, 12, 1083-1093. (18) Belhadj, Z.; Ying, M.; Cao, X.; Hu, X.; Zhan, C.; Wei, X.; Gao, J.; Wang, X.; Yan, Z.; Lu, W. Design of Y-Shaped Targeting Material for Liposome-Based Multifunctional Glioblastoma-Targeted Drug Delivery. J. Controlled Release 2017, 255, 132-141. (19) Chai, Z.; Hu, X.; Wei, X.; Zhan, C.; Lu, L.; Jiang, K.; Su, B.; Ruan, H.; Ran, D.; Fang, R. H.; Zhang, L.; Lu, W. A Facile Approach to Functionalizing Cell Membrane-Coated Nanoparticles with Neurotoxin-Derived Peptide for Brain-Targeted Drug Delivery. J. Controlled Release 2017, 264, 102-111. (20) Mao, J.; Ran, D.; Xie, C.; Shen, Q.; Wang, S.; Lu, W. Egfr/Egfrviii Dual-Targeting Peptide-Mediated Drug Delivery for Enhanced Glioma Therapy. ACS Appl. Mater. Interfaces 2017, 9, 24462-24475. (21) Ran, D.; Mao, J.; Shen, Q.; Xie, C.; Zhan, C.; Wang, R.; Lu, W. Grp78 Enabled Micelle-Based Glioma Targeted Drug Delivery. J. Controlled Release 2017, 255, 120-131. (22) Ran, D.; Mao, J.; Zhan, C.; Xie, C.; Ruan, H.; Ying, M.; Zhou, J.; Lu, W. L.; Lu, W. D-Retroenantiomer of Quorum-Sensing Peptide-Modified Polymeric Micelles for Brain Tumor-Targeted Drug Delivery. ACS Appl. Mater. Interfaces 2017, 9, 25672-25682. (23) Ren, Y.; Zhan, C.; Gao, J.; Zhang, M.; Wei, X.; Ying, M.; Liu, Z.; Lu, W. A D-Peptide Ligand of Integrins for Simultaneously Targeting Angiogenic Blood Vasculature and Glioma Cells. Mol. Pharmaceutics 2018, 15, 592-601. (24) Ruan, H.; Chen, X.; Xie, C.; Li, B.; Ying, M.; Liu, Y.; Zhang, M.; Zhang, X.; Zhan, C.; Lu, W.; Lu, W. Stapled RGD Peptide Enables Glioma-Targeted Drug Delivery by Overcoming Multiple Barriers. ACS Appl. Mater. Interfaces 2017, 9, 17745-17756. 22


Page 22 of 25

Page 23 of 25 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


(25) Zhang, M.; Chen, X.; Ying, M.; Gao, J.; Zhan, C.; Lu, W. Glioma-Targeted Drug Delivery Enabled by a Multifunctional Peptide. Bioconjugate Chem. 2017, 28, 775-781. (26) Ying, M.; Shen, Q.; Zhan, C.; Wei, X.; Gao, J.; Xie, C.; Yao, B.; Lu, W. A Stabilized Peptide Ligand for Multifunctional Glioma Targeted Drug Delivery. J. Controlled Release 2016, 243, 86-98. (27) Ying, M.; Shen, Q.; Liu, Y.; Yan, Z.; Wei, X.; Zhan, C.; Gao, J.; Xie, C.; Yao, B.; Lu, W. Stabilized Heptapeptide A7R for Enhanced Multifunctional Liposome-Based Tumor-Targeted Drug Delivery. ACS Appl. Mater. Interfaces 2016, 8, 13232-13241. (28) Binetruy-Tournaire, R.; Demangel, C.; Malavaud, B.; Vassy, R.; Rouyre, S.; Kraemer, M.; Plouet, J.; Derbin, C.; Perret, G.; Mazie, J. C. Identification of a Peptide Blocking Vascular Endothelial Growth Factor (VEGF)-Mediated Angiogenesis. EMBO J. 2000, 19, 1525-1533. (29) Ying, M.; Zhan, C.; Wang, S.; Yao, B.; Hu, X.; Song, X.; Zhang, M.; Wei, X.; Xiong, Y.; Lu, W. Liposome-Based Systemic Glioma-Targeted Drug Delivery Enabled by All-D Peptides. ACS Appl. Mater. Interfaces 2016, 8, 29977-29985. (30) Meng, Q.; Yu, M.; Gu, B.; Li, J.; Liu, Y.; Zhan, C.; Xie, C.; Zhou, J.; Lu, W. Myristic Acid-Conjugated Polyethylenimine for Brain-Targeting Delivery: In Vivo and Ex Vivo Imaging Evaluation. J. Drug Targeting 2010, 18, 438-446. (31) Li, J.; Gu, B.; Meng, Q.; Yan, Z.; Gao, H.; Chen, X.; Yang, X.; Lu, W. The Use of Myristic Acid as a Ligand of Polyethylenimine/DNA Nanoparticles for Targeted Gene Therapy of Glioblastoma. Nanotechnology 2011, 22, 435101-435109. (32) Pham, W.; Kircher, M. F.; Weissleder, R.; Tung, C. H. Enhancing Membrane Permeability by Fatty Acylation of Oligoarginine Peptides. ChemBioChem 2004, 5, 1148-1151. (33) Pham, W.; Zhao, B. Q.; Lo, E. H.; Medarova, Z.; Rosen, B.; Moore, A. Crossing the Blood-Brain Barrier: A Potential Application of Myristoylated Polyarginine for in Vivo Neuroimaging. NeuroImage 2005, 28, 287-292. (34) Yan, Z.; Wang, F.; Wen, Z.; Zhan, C.; Feng, L.; Liu, Y.; Wei, X.; Xie, C.; Lu, W. Lyp-1-Conjugated PEGylated Liposomes: A Carrier System for Targeted Therapy of Lymphatic Metastatic Tumor. J. Controlled Release 2012, 157, 118-125. (35) Wei, X.; Gao, J.; Zhan, C.; Xie, C.; Chai, Z.; Ran, D.; Ying, M.; Zheng, P.; Lu, W. Liposome-Based Glioma Targeted Drug Delivery Enabled by Stable Peptide Ligands. J. Controlled Release 2015, 218, 13-21. 23



(36) Zhang,

P.;

Hu,

L.;

Wang,

Y.;

Wang,

Page 24 of 25

J.;

Feng,

L.;

Li,

Y.

Poly(Epsilon-Caprolactone)-Block-Poly(Ethyl Ethylene Phosphate) Micelles for Brain-Targeting Drug Delivery: In Vitro and in Vivo Valuation. Pharm. Res. 2010, 27, 2657-2669. (37) Demeuse, P.; Kerkhofs, A.; Struys-Ponsar, C.; Knoops, B.; Remacle, C.; van den Bosch de Aguilar, P. Compartmentalized Coculture of Rat Brain Endothelial Cells and Astrocytes: A Syngenic Model to Study the Blood-Brain Barrier. J. Neurosci. Methods 2002, 121, 21-31. (38) Zhan, C.; Yan, Z.; Xie, C.; Lu, W. Loop 2 of Ophiophagus Hannah Toxin B Binds with Neuronal Nicotinic Acetylcholine Receptors and Enhances Intracranial Drug Delivery. Mol. Pharmaceutics 2010, 7, 1940-1947. (39) Qu, Y.; Zhang, L.; Rong, Z.; He, T.; Zhang, S. Number of Glioma Polyploid Giant Cancer Cells (PGCCs) Associated with Vasculogenic Mimicry Formation and Tumor Grade in Human Glioma. J. Exp. Clin. Cancer Res. 2013, 32, 75-95. (40) Scott, A. W.; Tyler, B. M.; Masi, B. C.; Upadhyay, U. M.; Patta, Y. R.; Grossman, R.; Basaldella, L.; Langer, R. S.; Brem, H.; Cima, M. J. Intracranial Microcapsule Drug Delivery Device for the Treatment of an Experimental Gliosarcoma Model. Biomaterials 2011, 32, 2532-2539. (41) Kang, T.; Zhu, Q.; Jiang, D.; Feng, X.; Feng, J.; Jiang, T.; Yao, J.; Jing, Y.; Song, Q.; Jiang, X.; Gao, X.; Chen, J. Synergistic Targeting Tenascin C and Neuropilin-1 for Specific Penetration of Nanoparticles for Anti-Glioblastoma Treatment. Biomaterials 2016, 101, 60-75. (42) Feng, X.; Yao, J.; Gao, X.; Jing, Y.; Kang, T.; Jiang, D.; Jiang, T.; Feng, J.; Zhu, Q.; Jiang, X.; Chen, J. Multi-Targeting Peptide-Functionalized Nanoparticles Recognized Vasculogenic Mimicry, Tumor Neovasculature, and Glioma Cells for Enhanced Anti-Glioma Therapy. ACS Appl. Mater. Interfaces 2015, 7, 27885-27899. (43) Zhu, S.; Qian, L.; Hong, M.; Zhang, L.; Pei, Y.; Jiang, Y. RGD-Modified Peg-Pamam-Dox Conjugate: In Vitro and in Vivo Targeting to Both Tumor Neovascular Endothelial Cells and Tumor Cells. Adv. Mater. 2011, 23, H84-89. (44) Li, B.; McCrudden, C. M.; Yuen, H. F.; Xi, X.; Lyu, P.; Chan, K. W.; Zhang, S. D.; Kwok, H. F. Cd133 in Brain Tumor: The Prognostic Factor. Oncotarget 2017, 8, 11144-11159.

24


Page 25 of 25 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


Table of Contents

25


Myristic Acid Modified DA7R Peptide for Whole ... - ACS Publications

Recommend Documents