iNGR-Modified Liposomes for Tumor Vascular Targeting and Tumor

Subscriber access provided by University of Newcastle, Australia

Article

iNGR-modified liposomes for tumor vascular targeting and tumor tissue penetrating delivery in the treatment of glioblastoma Jing-e Zhou, Jing Yu, Lipeng Gao, Lei Sun, Ting Peng, Jing Wang, Jianzhong Zhu, Weiyue Lu, Lin Zhang, Zhiqiang Yan, and Lei Yu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00101 • Publication Date (Web): 07 Apr 2017 Downloaded from http://pubs.acs.org on April 8, 2017

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

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

Page 1 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

iNGR-modified liposomes for tumor vascular targeting and tumor tissue penetrating delivery in the treatment of glioblastoma Jing-e Zhou,1, # Jing Yu,1, # Lipeng Gao,1 Lei Sun,1 Ting Peng,1 Jing Wang,1 Jianzhong Zhu,1 Weiyue Lu,2 Lin Zhang,3 Zhiqiang Yan,*,1 and Lei Yu*,1

1

Institute of Biomedical Engineering and Technology, Shanghai Engineering Research Center of

Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China 2

Department of Pharmaceutics, School of Pharmacy, Fudan University & Key Laboratory of Smart

Drug Delivery, Fudan University, Ministry of Education, Shanghai 201203, China 3

Department of Pharmacy, Shaoxing People’s Hospital, Shaoxing Hospital of ZheJiang University,

Shaoxing 312000, People’s Republic of China

#

The two authors contributed equally to this work.

*Corresponding author: Zhiqiang Yan ([email protected]); Lei Yu ([email protected])

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

Abstract

The tumor vascular barrier and tumor stroma barrier become the two main obstacles in the in vivo delivery of nanomedicines. In this study, to overcome the two barriers, we used iNGR, a tumor-penetrating peptide, to modify the liposomes to increase their accumulation and penetration in tumor tissues. Firstly, iNGR-modified sterically stabilized liposomes (iNGR-SSL) were prepared, which showed vesicle sizes of about 100 nm and narrow size distribution. The uptake of iNGR-SSL by U87MG cells and HUVECs were significantly more than that of unmodified liposome. The in vivo imaging study demonstrated that iNGR modification remarkably increased the accumulation of the liposome in orthotopic tumor tissues of animal model. The immunofluorescence staining analysis proved that iNGR-SSL could penetrate through tumor blood vessels and into the deep tumor tissues. The cytotoxicity of iNGR-modified doxorubicin-loaded liposomes (iNGR-SSL/DOX) on U87MG and HUVECs cells in vitro was significantly enhanced than that of unmodified doxorubicin-loaded liposomes (SSL/DOX). The iNGR-SSL/DOX also showed comparatively (p < 0.05) stronger cytotoxicity on tumor than SSL/DOX, which should be resulted from the increased tumor accumulation and penetration mediated by iNGR. This study proved that iNGR peptide modification might be an effective method to enhance the tumor penetrating ability of liposomes in tumor tissue and enhancing their anti-tumor effect. Keywords: tumor-penetrating peptide; nano drug delivery system; targeting; liposomes; doxorubicin


Page 2 of 34

Page 3 of 34

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


1.

Introduction

The proportion of glioblastoma in all central nervous systems tumors and brain tumors was reported to be 29%.1 In the past 30 years, the incidence of primary malignant brain tumors has increased with annual growth rate of about 1.2%. Glioblastoma is a most devastating cancer with rapid progression, high recurrence and poor prognosis. According to the WHO statistics, the median survival time of 4 grade glioblastoma patients was only 12 months.2 The current methods to treat glioblastoma include surgery, radiotherapy and chemotherapy. However, due to the invasive growth of glioblastoma, it is difficult to completely remove the tumor by surgery causing frequent recurring after treatment.3 Radiation therapy tends to cause dementia, and conventional chemotherapeutics often lead to serious systemic side effects due to the lack of selectivity to tumor tissue. Accordingly, we need a new drug delivery system to improve the therapeutic effect to glioblastoma. In recent years, active targeting nano drug delivery systems have shown great advantages in cancer treatment, and have been widely reported to be used for glioma treatment. Among these reports, tumor vessel targeted delivery system has have achieved much advance and targeting new vessels of tumor has become a research hotspot.2, 4-6 It is reported that almost all tumors have rich new vessels, which can provide enough nutrients for malignant growth and proliferation of tumor cells.7, 8 On the surface of tumor blood vessels, many over-expressed tumor-specific receptors have been found, such as VEGF receptor,9 integrin αvβ3

10

and endothelium-selectin. These receptors often play an

important role in promoting the formation, development and metastasis of tumors11. The specific ligands of these receptors, such as RGD or VEGF, have been conjugated to the surface of nano drug delivery systems, to enhance the tumor-targeting efficiency and anti-tumor effect. However, the in vivo transport of nano drug delivery systems are still seriously hampered by the tumor vascular barrier12 and tumor stroma barrier 13. It was reported that the two transport barriers make the majority of tumor cells be not exposed to the drugs, which is considered as the main cause of incomplete therapy and recurrence of tumors14. Therefore, it is important for us to try to enable the drug delivery system to penetrate across the tumor blood vessels and to the deep tumor tissue. In order to achieve effective drug delivery and accumulation into tumor tissues, tumor-penetrating drug delivery system should be a suitable choice. iNGR (CRNGRGPDC sequence), a peptide screened out by phage display technology, has been reported to be a tumor-penetrating peptide. The structure of iNGR was composed of a vascular homing motif, a tissue



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

penetration motif(R/KXXR/K), and a protease recognition site.15 For one thing, it can specifically recognize tumor blood vessels mediated by the receptor CD13, which is over-expressed on the neovascular endothelial cells of glioma, especially in the pathological state. For another, it can be cut by specific enzyme near the tumor into iNGRt peptide (CRNGR sequence), which can specifically penetrate through tumor vessels and into the deep tumor tissues and be internalized by glioblastoma cells. This function is based on the specific interaction between iNGRt and its receptor NRP-1 overexpressed on the tumor vessels and glioblastoma cells16. Therefore, the tumor vessel targeting and tumor penetrating ability may allow iNGR to be able to overcome the tumor vascular barrier and tumor stroma barrier. However, few reports are available on its use as targeting molecule in tumor-targeted drug delivery system. In this work, in order to overcome the two transport barriers of glioblastoma, we conjugated the iNGR peptide to liposomes to enable them to specifically recognize and penetrate across the tumor blood vessels and into the deep tumor tissue, thereby increasing the drug accumulation on tumor. Firstly, we prepared iNGR-modified doxorubicin liposomes, and the vesicle size and morphology were determined. The tumor-targeting ability, anti-tumor effect and tumor-penetrating ability was also investigated on the glioblastoma cells in vitro and on the animal model in vivo. This study may present an effective method to increase the drug delivery to brain tumor.

Figure 1. A tumor vessel recognizing and tumor penetrating system is developed by modifying the iNGR peptide to the surface of liposomes (iNGR-SSL/DOX). The iNGR-SSL/DOX firstly binds to tumor blood vessel by the interaction of iNGR and CD13 receptor. Then iNGR is proteolytically


Page 4 of 34

Page 5 of 34

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


cleaved to iNGRt, which specifically binds to NRP-1 overexpressed on tumor blood vessel. The iNGRt mediates the liposome penetration through tumor neovascularization and tumor tissue, and finally the cellular uptake by glioblastoma cells.

2. Materials and methods

2.1. Materials Fetal bovine serum (FBS) was purchased from Biological Industries Co. DMEM and RPMI1640 medium were from Biological Industries Co. Mal-PEG-DSPE was supplied by Sigma Co. (USA). Cholesterol was from Aladdin. Chemical Reagent Co. (China). HSPC and PEG2000-DSPE were obtained from Degussa Co., Ltd (Germany). DiR was obtained from Sigma Chemical Reagent Co (USA). Rat anti-mouse CD31 was was provided by R&D Systems (UK). Sephadex G50 was supplied by GE Healthcare (UK). Goat anti-rat IgG-R was provided by Santa Cruz (USA). Hochest33342 was supplied by Life technologies (USA). All chemical regents are analytic reagent grade. U87MG cell line was purchased from Shanghai Institute of Cell Biology,

cultured in

DMEM supplemented with 10%FBS, 100U/ml penicillin, and 100ug/ml streptomycin at 37°C under a humidified atmosphere containing 5% CO2. HUVECs were obtained from Shanghai Institute of Cell Biological Technology Co.(Shanghai, China), which was cultured in RPMI1640 supplemented with 10%FBS, 100U/ml penicillin, and 100ug/ml streptomycin. Male Balb/c Nu/Nu mice (20-25 g body weight) of four weeks age were purchased from Shanghai SLAC laboratory animal company (Shanghai, China) and kept in SPF conditions. All mouse work was approved by the ethics committee of East China Normal University. .

2.2 Synthesis of iNGR-PEG-DSPE, NGR-PEG-DSPE and iNGRt-PEG-DSPE Firstly, the three thiolates targeting peptides, including iNGR (CC(Acm)RNGRGPDC(Acm)), NGR (CC(Acm)NGRC(Acm)) and iNGRt (CRNGR), the iNGR peptide was prepared by solid phase peptide synthesis, and then conjugated with Mal-PEG-DSPE as described in the literature

18, 19

. The

solution was dialyzed against water (MWCO 3.5 kDa cut, Spectrum laboratory) and then freeze-dried. At last, iodine oxidation method was used to deprotect the Acm-protected thiol and form the disulﬁde in iNGR and NGR groups. The final products, including iNGR-PEG-DSPE, NGR-PEG-DSPE and iNGRt-PEG-DSPE were characterized by HPLC, 1H-NMR and FTIR.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 34

2.3. Preparation of liposomes The thin-film hydration and extrusion method was used to prepare the liposomes encapsulated with DOX, FAM or DiR.

20

For the peptide-modified liposomes (including iNGR-SSL, NGR-SSL and

iNGRt-SSL), the membrane materials were composed of HSPC, cholesterol, PEG(2000)-DSPE and ligand-PEG-DSPE and the molar ratio was 55:45:2:1; for unmodified liposomes, the ratio was 55:45:2:0. During the preparation, FAM was dissolved in the water phase, and DiR was dissolved in the organic phase. The liposomes loaded with DOX were prepared by using the ammonium sulfate gradient method 21.

2.4. Characterization of liposomes Liposomes were verified by transmission electron microscopy (TEM) (JEM-2100FEF, JEOL, Japan) and the dynamic light scattering (DLS) by evaluating the morphology and the vesicle sizes.. liposomes (Zetasizer Nano-ZS, Malvern Instruments, Westborough, MA). The entrapment efficiency (EE) of liposomes was calculated by the following equation: EE=loaded DOX/total DOX. The in vitro leakage of FAM or DIR from liposomes was tested using dialysis method as described previously.20

The

concentrations

of

FAM

or

DiR

were

measured

by

an

FL

Spectrophotometer(F-1000,Hitachi, Japan) at Ex/Em 494/522 nm and 741/776 nm, respectively.

2.5. Cellular uptake and flow cytometry tests The cellular uptake of liposomes by the U87MG cells and HUVECs was investigated previously.22,

23

as described

The U87MG cells were incubated with iNGR-SSL/FAM, NGR-SSL/FAM,

iNGRt-SSL/FAM or SSL/FAM in DMEM medium with 10% FBS for 2 h. The HUVECs were incubated with iNGR-SSL/FAM, NGR-SSL/FAM, iNGRt-SSL/FAM or SSL/FAM for 2 h in RPMI1640. For the imaging by the laser scanning confocal microscope (DMI4000 B, Leica, Germany), following the incubation, the

cells were washed by PBS, and fixed with 0.5%

paraformaldehyde and stained with the Hochest33342 for about 10 min. For quantitative analysis, at the end of incubation, the U87MG cells and HUVECs were trypsinzed, centrifuged, resuspended and detected with Flow cytometry instrument (Guava EasyCyte, Merck Millipore, USA).

2.6. In vivo fluorescence imaging study Near-infrared in vivo fluorescence imaging method was used to evaluate the targeting ability of iNGR-SSL to intracranial glioblastoma. According to previous reports, the nude mice model of glioblastoma was constructed by the Paxinos brain locator. The mice were injected of 5 × 105


Page 7 of 34

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


U87MG cells via the right striatum. The injecting coordinates based on the anterior fontanel were as follows: 3 mm deep, 1.8 mm lateral and 0.6 mm anterior 24, 25. On the 30th days after the inoculation, 100 µl of iNGR -SSL/DiR were i.v. injected to mice in order to examine the targeting ability of iNGR -SSL to the intracranial glioblastoma, and the NGR -SSL/DiR and SSL/DiR were used as control. The DiR concentration was 20 µg/ml, at a dose of 0.1 µg/g animal weight. After 2, 4, 8 ,12, 24 and 48h, the mice were anesthetized and imaged on an in vivo imaging system (Bruke MISE, USA) with a near-infrared filter set (excitation: 745 nm, emission: 780 nm).

2.7. Immunofluorescence study The immunofluorescence study was carried out to evaluate the in vivo tumor-penetrating ability of iNGR -SSL/ FAM. The glioblastoma-bearing nude mice were administrated i.v. with SSL/FAM and iNGR-SSL/FAM 24 days after inoculation. The concentration of FAM was 3.76 µg/ml at a dose of 0.0188ug/g animal weight. Mice were sacrificed at 6 hours after administration to collected the glioblastoma-bearing brains and fixed with 4% paraformaldehyde, dehydrated in 25% sucrose, frozenand embedded in Tissue-tek O.C.T., and sliced (10 um ). The sections were treated with Triton X-100 solution and then incubated with rat anti-mouse CD31 (1: 10, R & D Systems). After that, they were incubated with rhodamine-conjugated goat anti-rat IgG (1: 100, Santa Cruz), and then stained with Hochest 33342.

2.8. Cytotoxicity assay The in vitro cytotoxicity of iNGR -SSL/DOX was measured by previously

CCK-8 assay as described

26

. U87MG cells (2000/well) and HUVECs (5000/well) were cultured in 96 well plate

overnight, respectively. The cells were incubated with a series of concentrations of SSL/DOX, NGR-SSL/DOX or iNGRt-SSL/DOX for 48 h, followed by the addition of CCK-8. The absorbance was determined by using a microplate reader (Power Wave XS, Bio-TEK, USA) to calculate the percentages of cell viability. Curve fitting analysis software (Graph Pad Prism 6.02) was used to calculate the IC50 values.

2.9. Glioblastoma inhibition study of iNGR-SSL/DOX The glioblastoma-bearing nude mice model was established as demonstrated in section 2.6. 30 animals were randomly divided to five groups (n = 6) and i.v. administered of normal saline (N.S.), DOX, SSL/DOX, iNGR-SSL/DOX and NGR -SSL/DOX at the 16th, 18th, 22nd, 26th day after inoculation, respectively. The total dose of DOX in all groups was 22.5 mg/kg. Then the survival



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

time of each animal was recorded.

2.10. Statistical analysis All data were presented as mean ± SD. Two groups of data was analyzed by using student’s t-test (two-tailed), and p < 0.05 was as significant. Over three groups of data were analyzed with analysis of variance (ANOVA), followed by the multiple comparison of Tukey. The survival analysis was performed with the log-rank method. The p-values of multiple comparison among all groups were corrected by the Bonferroni method. All the analysis was performed with the software Graphpad Prism v6.0. 3. Results and conclusion

3.1. Characterization of CRNGRGPDC-PEG-DSPE The HPLC, H1-NMR and FTIR spectra of Mal-PEG-DSPE, NGR-PEG-DSPE, iNGRt-PEG-DSPE and iNGR-PEG-DSPE were shown in Figure 2. The HPLC spectra (Figure 2A) showed the differences of the retention times of these products. It can be seen that the retention times of Mal -PEG-DSPE, NGR-PEG-DSPE, iNGR-PEG-DSPE and iNGRt-PEG-DSPE are 25.1, 16.8, 19.2 and 22.4min, respectively. Compared with Mal -PEG-DSPE, the latter three ones all showed decreased retention time, which should be attributed to the introduction of hydrophilic peptide into the molecular structure. The NMR spectra of Mal -PEG-DSPE, NGR -PEG-DSPE, iNGR -PEG-DSPE and iNGRt -PEG-DSPE are shown in Figure 2B. In the spectrum of Mal-PEG-DSPE, the solvent peak of CDCl3, the methylene protons of DSPE and the repeat units in PEG exhibited peaks at 7.26 ppm, 1.26 ppm and 3.7–3.8 ppm, respectively. The spectra of Mal -PEG-DSPE shows the characteristic peak of Mal group at 6.77 ppm, while that of iNGR -PEG-DSPE does not shows, which indicated that the thiol group of thiolated iNGR had successfully reacted with Mal-PEG-DSPE. As shown in Figure 2C, the FTIR spectrum of Mal-PEG-DSPE showed a weak and broad peak at 1666.8 cm−1 and a weak peak at 3200–3600 cm−1, which could be responsible for the C=O stretching and N-H stretching in the amide groups of Mal-PEG-DSPE, respectively. Whereas in the spectrum of iNGR-PEG-DSPE, the intensity of the two peaks remarkably increased, which should be resulted from the increased number of amide groups in the conjugated iNGR peptide. The result demonstrated iNGR had been successfully conjugated to Mal-PEG-DSPE.


Page 8 of 34

Page 9 of 34

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 2. The HPLC (A), NMR (B) and FTIR spectra (C) of Mal-PEG-DSPE, NGR-PEG-DSPE, iNGR-PEG-DSPE and iNGRt-PEG-DSPE. 3.2. Characterization of liposomes As shown in the TEM images, all types of liposomes have uniform and spherical morphology



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 3). The vesicle sizes of all types of liposomes were examined by the DLS method. Figure 4 showed the average size of particle is around 100 nm and narrow distributions. Table 1 showed the encapsulation efficiency (EE),

partical sizes, and drug leakage of all types of liposomes. The

results indicated that the morphology and vesicle sizes were not significantly influenced by the modification of targeting peptides. Besides, as depicted in table 1, 2% of FAM was leaked out from liposomes within 4 h, and 0% of DiR was leaked out within 24 h. The above data demonstrated good stability of FAM or DiR-labeled liposomes, which showed the feasibility of the following experiments.27

Figure 3. The TEM images displayed that SSL, iNGR-SSL, SSL/DOX, iNGR-SSL/DOX, NGR-SSL/DOX and iNGRt-SSL/DOX all showed uniform spherical morphology.

Figure 4. The vesicle sizes and size distributions of SSL/DOX, iNGR-SSL/DOX, NGR-SSL/DOX and iNGRt-SSL/DOX.


Page 10 of 34

Page 11 of 34

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 1. The Vesicle size (nm), Encapsulation Efficiency (%) and Drug leakage (%) of liposomes Sample

EE (%)

particle size(nm)

Drug leakage(%)

SSL/DOX

95.9±2.7

100.6±1.7

3.25±1.7

iNGR-SSL/DOX

96.59±1.9

98.2±3.2

4.68±1.9

SSL/FAM

1.5±2.3

99.2±3.8

4.1±1.3

iNGR-SSL/FAM

1.3±1.8

100.9±2.5

2.1±1.1

SSL/DiR

100±0.9

98.2±2.8

N.D

iNGR-SSL/DiR

100±1.1

98.7±2.1

N.D

All data were presented as mean ± SD (n = 3); N.D. = not detectable.

3.3. The in vitro and in vivo targeting ability of iNGR-SSL 3.3.1. Cellular uptake of iNGR-SSL in vitro Figure 5 and Figure 6 showed the images of cellular uptake of SSL/FAM, NGR-SSL/FAM, iNGR-SSL/FAM and iNGRt-SSL/FAM by U87MG cells (Figure 5) and HUVEC cells (Figure 6). In Figure 4, the fluorescent micrograph showed the increased cellular uptake of iNGR-SSL/FAM, iNGRt/FAM and NGR-SSL/FAM by U87MG cells than SSL/FAM. These results indicated that the three targeting peptides, including iNGR, iNGRt and NGR, could mediate the specific binding of liposomes with their receptors overexpressed on U87MG cells. The images of flow cytometry showed that the percentages of fluorescence-positive cells for SSL/FAM, NGR-SSL/FAM, iNGR-SSL/FAM and iNGRt-SSL/FAM were 17.36%, 97.89% 98.74% and 98.58%. The mean fluorescence intensities for them were 34.27, 71.56, 169.77 and 168.86, respectively. We could see that the mean fluorescence intensities of iNGR-SSL and iNGRt-SSL are more than that of NGR-SSL, which should be attributed to the different receptors of these peptides. The receptors of NGR, iNGR and iNGRt peptides were CD13, CD13/NRP-1 and NRP-1, respectively. The less uptake of NGR-SSL by U87MG cells may be attributed to the less CD13 expressed on U87MG cells than NRP-1 receptors.



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 5. Cellular uptake of SSL /FAM, iNGR -SSL/FAM, NGR-SSL/FAM and iNGRt -SSL/FAM by U87MG cells was imaged by confocal microscopy and quantified by flow cytometry. After iNGR modification, the cellular uptake was remarkably enhanced.

The fluorescent micrograph (Figure 6) showed the increased cellular uptake of iNGR-SSL/FAM, iNGRt/FAM and NGR-SSL/FAM by HUVECs compared with SSL/FAM. This should be attributed to the mediated targeting effect of these peptides. The flow cytometry results showed that the percentages of fluorescence-positive cells for SSL/FAM, NGR-SSL/FAM, iNGR-SSL/FAM and


Page 12 of 34

Page 13 of 34

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


iNGRt-SSL/FAM were 28.12%, 99.73% 100% and 99.97%, and the mean fluorescence intensities for them were 15.91, 39.29, 71.84 and 61.65, respectively. The results showed that the peptide modification significantly increased the cellular uptake of liposomes by HUVECs, which indicated that there may be both CD13 and NRP-1 receptors overexpressed on HUVECs.

Figure 6. Cellular uptake of SSL /FAM, iNGR -SSL/FAM, NGR-SSL/FAM and iNGRt -SSL/FAM by HUVECs was examined by fluorescent microscopy and flow cytometry. After iNGR modification, the cellular uptake was remarkably enhanced.



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

3.3.2. The targeting ability of iNGR-SSL to glioblastoma in vivo. The SSL/DiR, NGR-SSL/DiR, iNGR-SSL/DiR were i.v. administered to test the targeting ability to glioblastoma in vivo, and the representative imaging results of them were shown as the left, middle, and right nude mice in Figure 7, respectively. The results showed that the accumulation of NGR-SSL/DiR and iNGR-SSL/DiR were significantly increased compared with unmodified liposomes, indicating the good targeting ability of NGR and iNGR peptides. Besides, iNGR-SSL/DiR showed a significantly prolonged and increased distribution in glioblastoma compared with NGR-SSL/DiR, which should be attributed to the penetrating effect of iNGR in tumor tissues.


Page 14 of 34

Page 15 of 34

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 7. The in vivo fluorescence images of nude mice bearing glioblastoma after i.v. injection of SSL/DiR (left), NGR-SSL/DiR(middle), and iNGR-SSL/DiR (right). The figure showed the iNGR modification increased the accumulation of liposomes in tumor tissues.

3.4. The tumor-penetrating property of iNGR-SSL



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 16 of 34

To further study the tumor-penetrating property of iNGR-SSL in glioblastoma, the distribution of FAM-labeled liposomes in the tumor tissues slices was performed. We labeled the tumor vessels with fluorescently-labeled antibody against the endothelial marker CD31. In the immunofluorescence images (Figure 8), SSL/FAM showed little distribution in tumor tissue, whereas iNGR-SSL/FAM showed remarkable distribution in the deep glioblastoma tissue.

These data demonstrated that

iNGR modification could promote the penetrating ability of liposomes across tumor vessels and into the deep tumor tissue.

Figure 8. The immunofluorescence images of frozen brain tissues bearing glioblastoma after i.v. injection of SSL/FAM and iNGR -SSL/FAM, respectively. SSL/FAM mainly localized adjacent to blood vessels which is CD31 positive (red), while iNGR-SSL/FAM can penetrate into the deep glioblastoma, indicating the good tumor-penetrating property of iNGR -SSL.

3.5. Anti-glioblastoma effect of iNGR-SSL/DOX 3.5.1. In vitro cytotoxicity of iNGR-SSL/DOX The in vitro cytotoxicity of DOX loaded liposomes was evaluated on U87MG cells using the CCK-8 test. The IC50 values of SSL/DOX, NGR-SSL/DOX, iNGRt-SSL/DOX and iNGR-SSL/DOX were 1.401, 0.229, 0.275 and 0.669µM, respectively (figure 9A). The latter three ones showed significantly enhanced cytotoxicity than SSL/DOX, indicating the peptide-mediated targeting effect. The above results are in line with the results of cellular uptake (Fig. 5), which showed the increased uptake of liposomes after modified by iNGR, iNGRt and NGR peptides. Interestingly, the iNGRt-SSL/DOX showed higher cytotoxicity on U87MG cells than iNGR-SSL/DOX, which should


Page 17 of 34

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


be caused by that iNGRt can directly bind with NRP-1 receptors while iNGR cannot. The in vitro cytotoxicity of DOX loaded liposomes was evaluated on HUVECs using the CCK-8 test. The IC50 values of SSL/DOX, NGR-SSL/DOX, iNGRt-SSL/DOX and iNGR-SSL/DOX were 2.552, 1.729, 2.345, and 0.962 µM, respectively (figure 9B). The latter three ones showed significantly enhanced cytotoxicity than SSL/DOX due to the targeting effect of conjugated peptides. Besides, the cytotoxicity of iNGR-SSL/DOX was higher than iNGRt-SSL/DOX, which was possibly resulted from that the HUVECs express less NRP-1 receptors than U87MG cells do. The cytotoxicity of iNGR-SSL/DOX was higher than that of NGR-SSL/DOX, which was possibly attributed to the dual targeting effect of iNGR for CD13 and NRP-1 compared with NGR. The above results were in line with the results of cellular uptake (Fig. 6), which showed that the iNGR, iNGRt and NGR modified liposome increased the uptake compared to SSL/DOX. It should be the increased cellular uptake of iNGR-SSL/DOX that lead to the higher cytotoxicity compared with NGR-SSL/DOX.



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 9. The cytotoxicity of SSL/DOX, iNGR-SSL/DOX,NGR-SSL/DOX and iNGRt-SSL/DOX on U87MG（A）and HUVECs(B) as measured by CCK-8 test. The cytotoxicity of DOX-loaded liposomes was significantly

increased

after iNGR modification.

3.5.2. The in vivo anti-glioblastoma effect of iNGR -SSL/DOX The in vivo anti-glioblastoma effect of iNGR-SSL/DOX was studied on glioblastoma-bearing nude mice model and the results were shown using the Kaplan–Meier survival curve(figure 10). The median survival times for N.S., DOX, SSL/DOX, NGR-SSL/DOX and iNGR-SSL/DOX are 5, 27, 39, 43 and 50 days, respectively. The results show that the survival time of SSL/DOX group (p < 0.05), NGR-SSL/DOX and iNGR-SSL/DOX group (p < 0.001) are significantly prolonged compared with that of N.S. group. Besides, the survival time of iNGR-SSL/DOX group and


Page 18 of 34

Page 19 of 34

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


NGR-SSL group are longer than that of SSL/DOX group. Such superiority should be attributed to the active targeting delivery to tumor mediated by peptide ligands. In particular, the median survival time of iNGR-SSL/DOX group is longer than that of NGR-SSL/DOX. The result suggests that iNGR-modified liposomes exhibited enhanced anti-tumor effect than NGR-modified liposome, which is perhaps resulted from the tumor tissue penetrating ability of iNGR. This inference is also in line with the result of tumor tissue distribution test (Fig. 8).

Figure 10. The Kaplan–Meier survival curve of nude mice bearing glioblastoma following injection of different types of liposomes. The iNGR-modified liposome group showed significantly prolonged median survival time than unmodified liposome group (p < 0.05).

4. Discussion In this work, a tumor penetrating peptide iNGR was used to modify the liposome drug delivery system to improve the liposome penetration across the tumor vessel wall and further into the whole glioblastoma tissue. The results demonstrated that iNGR modification effectively increased the liposome uptake by U87MG cells and HUVECs in vitro, and also increased the inhibitory effect of the two cells. Furthermore, the iNGR-modified liposomes exhibited significantly increased accumulation and improved penetration in glioblastoma tissue in vivo. Finally, compared with ordinary liposomes, the iNGR modified DOX liposomes effectively increased the survival time of gliomastoma animal model. Many scientists have carried out studies on the peptide modified targeting drug delivery systems due to their effective therapy for glioblastoma.25, 28 Many peptides, such as RGD peptide, peptide

30, 31

24, 29

RGE

and Angiopep-2,32 Have been extensively studied in glioblastoma-targeted DDS.

However, most of NDDSs following i.v. injection can only be delivered to the region near the tumor



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 20 of 34

blood vessel, but not throughout the whole tumor tissue18. This problem, the poor penetration of NDDSs across tumor vessels, has seriously limited the treatment of glioblastoma33. iNGR is a tumor-penetrating peptide, and the mechanism of action is as follows. First, iNGR binds to CD13 receptors overexpressed on tumor vessels, then it is proteolytically cleaved to produce a truncated peptide, iNGRt. The iNGRt loses its affinity for CD13 and binds to NRP-1 receptors, which are over-expressed on tumor vessels and tumor cells, activating the tumor tissue penetrating pathway. By contrast, NGR peptide only recognizes CD13 receptors and cannot be activated into truncated peptide to bind with NRP-1 receptors and penetrate through tumor tissue.15,

34

Thus,

although NGR and iNGR binds to the same primary receptor, only iNGR can mediate the liposome penetration into deep tumor tissue. Our results also proved that iNGR showed a better tumor-targeting ability than NGR in vitro and in vivo. For example, the cellular uptake of iNGR-SSL by HUVECs and U87 cells was significantly higher than that of NGR-SSL (Fig. 5 and Fig. 6). The in vitro cytotoxicity of iNGR-SSL/DOX are stronger than NGR-SSL/DOX on HUVECs (Fig. 9B). The in vivo fluorescence imaging results showed that iNGR-SSL produced more increased and prolonged tumor distribution than NGR-SSL (Fig. 7). Compared with iNGRt peptide (CRNGR), which has an exposed CendR motif (RNGR), the iNGR peptide (CRNGRGPDC) has a cryptic CendR motif, since the CendR motif has to be exposed after activation by proteolysis. In other words, iNGR can only mediate the tumor penetration of NDDSs when both CD13 and NRP-1 receptors are overexpressed in the tumor tissue. This is advantageous for tumor targeted drug delivery, since the dual recognition can avoid the penetration of NDDSs in non-targeting tissues.15, 33 Thus, iNGR should have a better selectiveness for tumor tissue than iNGRt, though iNGRt-SSL showed a similar or even better in vitro tumor targeting and inhibitory ability compared with iNGR-SSL (Fig. 9). This problem had also been concerned by other researchers. Therefore, iNGRt-SSL had not been used in our in vivo study. The targeting ability of iNGR for tumor blood vessels is also an advantageous feature. It is well-known that tumor angiogenesis plays a key role in tumor development and metastasis35. Currently, there have been many drugs developed for targeting and inhibiting tumor angiogenesis, such as bevacizumab, Apatinib and Avastin, etc. These drugs showed good anti-tumor effects, and also produced synergistic antitumor eﬀects together with the traditional chemotherapeutics. The iNGR peptide has dual targeting ability to tumor blood vessels based on CD 31 receptor and to tumor


Page 21 of 34

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 based on the NRP-1 receptor, which was evidenced by the above in vitro cellular uptake test (Fig. 5). This may be another reason why iNGR-SSL/DOX showed an enhanced anti-tumor effect compared with NGR-SSL/DOX. In summary, iNGR-SSL can penetrate across tumor vessels and into the whole glioblastoma tissue, and effectively prolong the lifetime of glioblastoma animal models. This study demonstrated that iNGR peptide modification is an effective method for improving the delivery of NDDSs in glioblastoma tissue and enhancing their anti-glioblastoma effect.

Acknowledgements This work was supported by National Basic Research Program of China (2013CB932500), National Natural Science Foundation of China (60976004), "985" grants of East China Normal University (ECNU), Zhejiang Provincial Science and Technology Department Public Technology Application Research Program(2015C33285), Zhejiang Provincial Natural Science Foundation (LY14H300002).

Competing Interests The authors have declared that no competing interest exists.

Reference: 1.

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. Journal of controlled release : official journal of the Controlled Release Society 2015, 218, 13-21. 2.

Yan, Z.; Yang, Y.; Wei, X.; Zhong, J.; Wei, D.; Liu, L.; Xie, C.; Wang, F.; Zhang, L.; Lu, W.; He, D.

Tumor-penetrating

peptide mediation: an effective strategy for improving the transport of liposomes in tumor tissue. Molecular pharmaceutics 2014, 11, (1), 218-25. 3.

Bhowmik, A.; Khan, R.; Ghosh, M. K.

Blood brain barrier: a challenge for effectual therapy of brain tumors.

BioMed research international 2015, 2015, 320941. 4.

Feng, Z.; Zhao, G.; Yu, L.; Gough, D.; Howell, S. B.

Preclinical efficacy studies of a novel nanoparticle-based

formulation of paclitaxel that out-performs Abraxane. Cancer chemotherapy and pharmacology 2010, 65, (5), 923-30. 5.

Draffehn, S. r.; Kumke, M. U.

Monitoring the collapse of pH-sensitive liposomal nanocarriers and environmental

pH simultaneously: A fluorescence-based approach. Molecular pharmaceutics 2016, 13, (5), 1608-1617. 6.

Irby, D.; Du, C.; Li, F.

Lipid-drug Conjugate for Enhancing Drug Delivery. Molecular pharmaceutics 2017.

7.

Yang, D.; Van, S.; Shu, Y.; Liu, X.; Ge, Y.; Jiang, X.; Jin, Y.; Yu, L.

Synthesis, characterization, and in vivo efficacy

evaluation of PGG-docetaxel conjugate for potential cancer chemotherapy. International journal of nanomedicine 2012, 7, 581-9. 8.

Lili X. Peng1, A. I., Sang Van4, Gang Zhao4, Akshay S. Chaudhari1, Lei Yu4,; Stephen B. Howell3, J. A. M., David A.



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

Gough1. 9.

Page 22 of 34

Characterization of a clinical polymer-drug conjugate using multiscale modeling. 2010.

Shein, S. A.; Kuznetsov, I. I.; Abakumova, T. O.; Chelushkin, P. S.; Melnikov, P. A.; Korchagina, A. A.; Bychkov, D. A.;

Seregina, I. F.; Bolshov, M. A.; Kabanov, A. V.

VEGF-and VEGFR2-Targeted Liposomes for Cisplatin Delivery to Glioma

Cells. Molecular pharmaceutics 2016, 13, (11), 3712-3723. 10. Kulhari, H.; Pooja, D.; Kota, R.; Reddy, T. S.; Tabor, R. F.; Shukla, R.; Adams, D. J.; Sistla, R.; Bansal, V.

Cyclic RGDfK

Peptide Functionalized Polymeric Nanocarriers for Targeting Gemcitabine to Ovarian Cancer Cells. Molecular pharmaceutics 2016, 13, (5), 1491-1500. 11. Lv, L.; Jiang, Y.; Liu, X.; Wang, B.; Lv, W.; Zhao, Y.; Shi, H.; Hu, Q.; Xin, H.; Xu, Q.

Enhanced Antiglioblastoma Efficacy

of Neovasculature and Glioma Cells Dual Targeted Nanoparticles. Molecular pharmaceutics 2016, 13, (10), 3506-3517. 12. Clark, P. A.; Al-Ahmad, A. J.; Qian, T.; Zhang, R. R.; Wilson, H. K.; Weichert, J. P.; Palecek, S. P.; Kuo, J. S.; Shusta, E. V. Analysis of Cancer-Targeting Alkylphosphocholine Analogue Permeability Characteristics Using a Human Induced Pluripotent Stem Cell Blood–Brain Barrier Model. Molecular pharmaceutics 2016, 13, (9), 3341-3349. 13. Morshed, R. A.; Muroski, M. E.; Dai, Q.; Wegscheid, M. L.; Auffinger, B.; Yu, D.; Han, Y.; Zhang, L.; Wu, M.; Cheng, Y. Cell-Penetrating Peptide-Modified Gold Nanoparticles for the Delivery of Doxorubicin to Brain Metastatic Breast Cancer. Molecular pharmaceutics 2016, 13, (6), 1843-1854. 14. Pi, Y.; Zhou, J.; Wang, J.; Zhong, J.; Zhang, L.; Wang, Y.; Yu, L.; Yan, Z.

Strategies of overcoming the physiological

barriers for tumor-targeted nano-sized drug delivery systems. Curr Pharm Des 2015, 21, (42), 6236-45. 15. Alberici, L.; Roth, L.; Sugahara, K. N.; Agemy, L.; Kotamraju, V. R.; Teesalu, T.; Bordignon, C.; Traversari, C.; Rizzardi, G. P.; Ruoslahti, E.

De novo design of a tumor-penetrating peptide. Cancer Res 2013, 73, (2), 804-12.

16. Ahmad, A.; Mondal, S. K.; Mukhopadhyay, D.; Banerjee, R.; Alkharfy, K. M.

Development of liposomal formulation

for delivering anticancer drug to breast cancer stem-cell-like cells and its pharmacokinetics in an animal model. Molecular pharmaceutics 2016, 13, (3), 1081-1088. 17. Willem J. M. Mulder, † Gustav J. Strijkers,† Arjan W. Griffioen,‡ Louis van Bloois,§ Grietje Molema,|; Gert Storm, G. A. K., §,⊥ and Klaas Nicolay†.

A Liposomal System for Contrast-Enhanced Magnetic Resonance

Imaging of Molecular Targets. Bioconjugate Chem 2004, 15, 799-806. 18. Yang, Y.; Yan, Z.; Wei, D.; Zhong, J.; Liu, L.; Zhang, L.; Wang, F.; Wei, X.; Xie, C.; Lu, W.; He, D.

Tumor-penetrating

peptide functionalization enhances the anti-glioblastoma effect of doxorubicin liposomes. Nanotechnology 2013, 24, (40), 405101. 19. Hu, Q.; Gu, G.; Liu, Z.; Jiang, M.; Kang, T.; Miao, D.; Tu, Y.; Pang, Z.; Song, Q.; Yao, L.; Xia, H.; Chen, H.; Jiang, X.; Gao, X.; Chen, J.

F3 peptide-functionalized PEG-PLA nanoparticles co-administrated with tLyp-1 peptide for anti-glioma drug

delivery. Biomaterials 2013, 34, (4), 1135-1145. 20. 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. Journal of controlled release : official journal of the Controlled Release Society 2012, 157, (1), 118-25. 21. Yang, F.; Zhang, Q.

A study of liposomal doxorubicin modified by tumor metastasis targeting peptide for its

specificity to highly metastatic breast cancer cells. Journal of Chinese Pharmaceutical Sciences 2014, 23, (2). 22. Yu, Z.; Schmaltz, R. M.; Bozeman, T. C.; Paul, R.; Rishel, M. J.; Tsosie, K. S.; Hecht, S. M.

Selective tumor cell

targeting by the disaccharide moiety of bleomycin. Journal of the American Chemical Society 2013, 135, (8), 2883-6. 23. Li, C.; Wang, Y.; Zhang, X.; Deng, L.; Zhang, Y.; Chen, Z.

Tumor-targeted liposomal drug delivery mediated by a

diseleno bond-stabilized cyclic peptide. Int J Nanomedicine 2013, 8, 1051-62. 24. Zhan, C.; Gu, B.; Xie, C.; Li, J.; Liu, Y.; Lu, W.

Cyclic RGD conjugated poly(ethylene glycol)-co-poly(lactic acid)

micelle enhances paclitaxel anti-glioblastoma effect. Journal of controlled release : official journal of the Controlled Release Society 2010, 143, (1), 136-42. 25. H. Maeda , J. W., T. Sawa , Y. Matsumura , K. Hori a , a a b c *.

Tumor vascular permeability and the EPR effect in


Page 23 of 34

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


macromolecular therapeutics: a review. Journal of Controlled Release 1999. 26. Li, X.; Tian, X.; Zhang, J.; Zhao, X.; Chen, X.; Jiang, Y.; Wang, D.; Pan, W.

In vitro and in vivo evaluation of folate

receptor-targeting amphiphilic copolymer-modified liposomes loaded with docetaxel. International journal of nanomedicine 2011, 6, 1167-84. 27. Wen, Z.; Yan, Z.; Hu, K.; Pang, Z.; Cheng, X.; Guo, L.; Zhang, Q.; Jiang, X.; Fang, L.; Lai, R. Odorranalectin-conjugated nanoparticles: preparation, brain delivery and pharmacodynamic study on Parkinson's disease following intranasal administration. J Control Release 2011, 151, (2), 131-8. 28. Treat, L. H.; McDannold, N.; Vykhodtseva, N.; Zhang, Y.; Tam, K.; Hynynen, K.

Targeted delivery of doxorubicin to

the rat brain at therapeutic levels using MRI-guided focused ultrasound. International journal of cancer 2007, 121, (4), 901-7. 29. Gao, J.; Xie, C.; Zhang, M.; Wei, X.; Yan, Z.; Ren, Y.; Ying, M.; Lu, W.

RGD-modified lipid disks as drug carriers for

tumor targeted drug delivery. Nanoscale 2016, 8, (13), 7209-7216. 30. Renata Pasqualini, 4 Erkki Koivunen, Renate Kain, Johanna Lahdenranta,2 Michiie Sakamoto,3 Anette Stryhn,; Richard A. Ashmun, L. H. S., Wadih Arap,2 and Erkki Ruoslahti4.

Aminopeptidase N Is a Receptor for Tumor-homing

Peptides and a Target for Inhibiting Angiogenesis1. Cancer research 2000. 31. Huang, N.; Cheng, S.; Zhang, X.; Tian, Q.; Pi, J.; Tang, J.; Huang, Q.; Wang, F.; Chen, J.; Xie, Z.; Xu, Z.; Chen, W.; Zheng, H.; Cheng, Y.

Efficacy of NGR peptide-modified PEGylated quantum dots for crossing the blood-brain barrier and

targeted fluorescence imaging of glioma and tumor vasculature. Nanomedicine : nanotechnology, biology, and medicine 2017, 13, (1), 83-93. 32. Xin, H.; Jiang, X.; Gu, J.; Sha, X.; Chen, L.; Law, K.; Chen, Y.; Wang, X.; Jiang, Y.; Fang, X.

Angiopep-conjugated

poly(ethylene glycol)-co-poly(epsilon-caprolactone) nanoparticles as dual-targeting drug delivery system for brain glioma. Biomaterials 2011, 32, (18), 4293-305. 33. 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 applied materials & interfaces 2016, 8, (21), 13232-13241. 34. Luo, Z.; Yan, Z.; Jin, K.; Pang, Q.; Jiang, T.; Lu, H.; Liu, X.; Pang, Z.; Yu, L.; Jiang, X.

Precise glioblastoma targeting by

AS1411 aptamer-functionalized poly (l-γ-glutamylglutamine)–paclitaxel nanoconjugates. Journal of Colloid and Interface Science 2017, 490, 783-796. 35. Wang, J.; Lei, Y.; Xie, C.; Lu, W.; Wagner, E.; Xie, Z.; Gao, J.; Zhang, X.; Yan, Z.; Liu, M.

Retro-inverso CendR

peptide-mediated polyethyleneimine for intracranial glioblastoma-targeting gene therapy. Bioconjugate chemistry 2014, 25, (2), 414-423.



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. A tumor vessel recognizing and tumor penetrating system is developed by modifying the iNGR peptide to the surface of liposomes (iNGR-LS/DOX). The iNGR-LS/DOX firstly binds to tumor blood vessel by the interaction of iNGR and CD13 receptor. Then iNGR is proteolytically cleaved to iNGRt, which specifically binds to NRP-1 overexpressed on tumor blood vessel. The iNGRt mediates the liposome penetration through tumor blood vessel and tumor tissue, and finally the cellular uptake by glioblastoma cells. 140x82mm (300 x 300 DPI)


Page 24 of 34

Page 25 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The HPLC (A), NMR (B) and FTIR spectra (C) of Mal-PEG-DSPE, NGR-PEG-DSPE, iNGR-PEG-DSPE and iNGRtPEG-DSPE. 205x362mm (600 x 600 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The TEM images displayed that SSL, iNGR-SSL, SSL/DOX, iNGR-SSL/DOX, NGR-SSL/DOX and iNGRtSSL/DOX all showed uniform spherical morphology. 72x44mm (300 x 300 DPI)


Page 26 of 34

Page 27 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The vesicle sizes and size distributions of SSL/DOX, iNGR-SSL/DOX, NGR-SSL/DOX and iNGRt-SSL/DOX. 94x48mm (600 x 600 DPI)



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

Cellular uptake of SSL /FAM, iNGR -SSL/FAM, NGR-SSL/FAM and iNGRt -SSL/FAM by U87MG cells was imaged by confocal microscopy and quantified by flow cytometry. After iNGR modification, the cellular uptake was remarkably enhanced. 175x169mm (300 x 300 DPI)


Page 28 of 34

Page 29 of 34

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


Cellular uptake of SSL /FAM, iNGR -SSL/FAM, NGR-SSL/FAM and iNGRt -SSL/FAM by HUVECs was examined by fluorescent microscopy and flow cytometry. After iNGR modification, the cellular uptake was remarkably enhanced. 175x171mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The in vivo fluorescence images of nude mice bearing glioblastoma after i.v. injection of SSL/DiR (left), NGR-SSL/DiR(middle), and iNGR-SSL/DiR (right). The figure showed the iNGR modification increased the accumulation of liposomes in tumor tissues. 80x188mm (300 x 300 DPI)


Page 30 of 34

Page 31 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The immunofluorescence images of frozen brain tissues bearing glioblastoma after i.v. injection of SSL/FAM and iNGR -SSL/FAM, respectively. SSL/FAM mainly localized adjacent to blood vessels which is CD31 positive (red), while iNGR-SSL/FAM can penetrate into the deep glioblastoma, indicating the good tumor-penetrating property of iNGR -SSL. 175x70mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The cytotoxicity of SSL/DOX, iNGR-SSL/DOX,NGR-SSL/DOX and iNGRt-SSL/DOX on U87MG（A）and HUVECs(B) as measured by CCK-8 test. The cytotoxicity of DOX-loaded liposomes was significantly increased after iNGR modification. 150x138mm (600 x 600 DPI)


Page 32 of 34

Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The Kaplan–Meier survival curve of nude mice bearing glioblastoma following injection of different types of liposomes. The iNGR-modified liposome group showed significantly prolonged median survival time than unmodified liposome group (p < 0.05). 73x37mm (600 x 600 DPI)



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

88x34mm (300 x 300 DPI)


Page 34 of 34

iNGR-Modified Liposomes for Tumor Vascular Targeting and Tumor

Recommend Documents