Self-Assembled Tumor-Penetrating Peptide ... - ACS Publications

Subscriber access provided by UNIV OF ESSEX

Article

Self-Assembled Tumor Penetrating Peptide Modified Poly (L-#-glutamylglutamine)-Paclitaxel Nanoparticles Based on Hydrophobic Interaction for Treatment of Glioblastoma Jing Yu, Lei Sun, Jinge Zhou, Lipeng Gao, Lijuan Nan, Shimin Zhao, Ting Peng, Lin Han, Jing Wang, Weiyue Lu, Lin Zhang, Yiting Wang, Zhiqiang Yan, and Lei Yu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00519 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 3, 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.

Bioconjugate Chemistry 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

Bioconjugate Chemistry

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 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

Page 2 of 34

Self-Assembled Tumor Penetrating Peptide Modified Poly (L-γ-glutamylglutamine)-Paclitaxel Nanoparticles Based on Hydrophobic Interaction for Treatment of Glioblastoma Jing Yu,

†,#

Lei Sun, †,# Jinge Zhou, † Lipeng Gao, † Lijuan Nan, † Shimin Zhao, † Ting

Peng, † Lin Han, † Jing Wang, † Weiyue Lu, ‡ Lin Zhang,

§

Yiting Wang,

†

Zhiqiang

Yan,*, † and Lei Yu, *,†

†

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, P.R. China. ‡

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

Laboratory of Smart Drug Delivery, Fudan University, Ministry of Education, Shanghai 201203, P.R. China. §

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

ZheJiang University, Shaoxing 312000, P.R. China. #

These authors contributed equally to this work.

Corresponding

Authors:

Zhiqiang

Yan

([email protected]);

([email protected])

ACS Paragon Plus Environment

Lei

Yu

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


Abstract To enhance the tumor penetrating ability and targeting therapeutic effect of polymer drug

conjugates

(PDCs),

poly(L-γ-glutamylglutamine)

tumor

penetrating

-paclitaxel

peptide

RGERPPR

(PGG-PTX)

modified

nanoparticles

(RGE-PEG/PGG-PTX NPs) were prepared by using a so-called “modular” design strategy. In brief, a RGERPPR conjugated targeting material DSPE-PEG-RGERPPR was firstly synthesized, and assembled with PGG-PTX into RGE-PEG/PGG-PTX NPs based on the hydrophobic interaction between the group of DSPE and PTX. The NPs exhibited a uniform spherical morphology with particle size of around 90 nm as shown by the dynamic light scattering and transmission electron microscopy results. The NPs showed a good in vitro stability at 4 °C for over three weeks, sustained drug release within 120 h, and good hemocompatibility. The cellular uptake study displayed that the NPs showed increased uptake by U87MG cells and HUVECs compared with the unmodified PGG-PTX. The cytotoxicity test demonstrated that RGE-PEG/PGG-PTX NPs produced a stronger growth inhibitory effect against U87MG cells and HUVECs than PGG-PTX, which was consistent with the cellular uptake results. Finally, the pharmacodynamic study proved that RGE-PEG/PGG-PTX NPs significantly prolonged the median survival time of nude mice bearing intracranial glioblastoma. The results indicated the effectiveness of RGE-PEG/PGG-PTX NPs in the treatment of glioblastoma, as well as the feasibility of the “modular” design strategy in the preparation of active targeting PDCs. Keywords: tumor penetrating peptide; poly (L-γ-glutamylglutamine)-paclitaxel; polymer drug conjugates; hydrophobic interaction; modular design; glioblastoma



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

TOC


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


Introduction Glioblastoma multiforme (GBM) is one of the most common brain tumors with a high aggressiveness and mortality.1 Due to its infiltrative growth and proximity to central nervous system (CNS), it is difficult to remove GBM precisely and completely by surgery.2 Moreover, the traditional chemotherapy often leads to serious side effects because of the poor selectivity to tumor. To address this issue, researchers are trying to develop new drug delivery systems (DDS) that can precisely deliver drugs to GBM as well as minimize the toxicity to normal tissues and the adjacent neuronal cells and glial cells. Recently, nano-drug delivery systems (NDDS) have been widely reported as the most promising DDS.3 The most commonly reported nanocarriers include liposomes, micelles, nanoparticles,4 and polymer-drug conjugates (PDCs) etc,5-7 among which PDCs have attracted attention for their controllable drug release and low toxicities due to their covalent linkage of drug instead of physical encapsulation.8 Correspondingly, we have developed several PDCs for tumor targeting therapy, such as poly(L-γ-glutamylglutamine)-paclitaxel (PGG-PTX), which is formed by introducing hydrophilic glutamic acids to the backbone of poly(L-glutamic acid) followed by conjugation of PTX (The structure of PGG-PTX is shown in Figure 1).9, 10 PGG-PTX can self-assemble11, 12 into a nano-core-shell structure with PTX as the hydrophobic core and PGG as the hydrophilic shell. In addition, it has shown a series of advantages such as high water solubility and drug loading capacity, good biocompatibility, low toxicities, improved tumor-targeting ability and anti-tumor efficacy.13, 14 To increase the tumor targeting ability of PDCs, the most common way is to conjugate a targeting moiety to the polymer backbone by covalently chemical modification8, 15, 16 to prepare a so-called actively targeting PDCs. For example, conjugation of iRGD to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer17, 18 or doxorubicin-polymer conjugates19 resulted in an enhanced tumor accumulation and penetration. However, the synthesis of actively targeting PDCs often needs a series of chemical reactions to conjugate the targeting molecules to the backbone of PDCs. Considering the complex structure and large molecular weight of PDCs, the chemical modification is relatively less controllable, complicated and easy to inactivate drugs and PDCs.18, 20 This problem



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

has, to some extent, limited the basic research and clinical translation of PDCs. Therefore, it is significant to find a more convenient and widely applicable modification strategy to prepare the actively targeting PDCs, which may promote the development of PDCs and other NDDSs. Although many studies have proved that the modification by targeting moiety can increase the tumor cellular uptake and anti-tumor effect of NDDS,21, 22 the in vivo tumor accumulation of NDDS still mainly relies on the enhanced permeability and retention (EPR) effect.23, 24 However, the strength of EPR effect is relevant to tumor sizes, types, tumor locations, and the physiological status of the patient, etc., which is also known as the heterogeneity of EPR effect.25 In addition, the EPR effect may be weakened due to the presence of elevated interstitial fluid pressure (IFP) in tumor tissue,26 which is particularly obvious in the intracranial tumors. This means that the EPR effect is not very stable and reliable for NDDS to achieve tumor accumulation. We need a more effective strategy to improve the accumulation and penetration of the NDDS, especially into the GBM tissue.27 Correspondingly, a series of targeting peptides with the structure of a CendR (C-end Rule) motif (R/KXXR/K) are recently identified by phage display technology, which are called “tumor penetrating peptides” (TPPs). These peptides, such as LyP-1, iRGD,28 iNGR, RGERPPR and RPARPPR, can penetrate tumor vessels and deep into the tumor sites.29, 30 The penetrating ability relies on the mediation of its receptor, Neuropilin-1 (NRP-1), which is specially overexpressed on tumor cells and tumor blood vessels. TPP modified NDDS can penetrate into the deep tumor tissue, enter tumor cells and kill them. For instance, iRGD modification significantly enhanced the anti-tumor efficacy of doxorubicin-polymer conjugates.19 We also previously proved that modification with the TPPs, such as iNGR31 and RGERPPR,32 could effectively increase the accumulation, penetration and anti-tumor efficacy of nanocarriers in GBM. Based on the above considerations, in this study, a TPP targeting material DSPE-PEG3400-RGERPPR was firstly synthesized, and assembled with PGG-PTX, by virtue of the hydrophobic interaction between the group of DSPE and PTX, to prepare RGERPPR

modified PGG-PTX

nanoparticles (RGE-PEG/PGG-PTX


Page 6 of 34

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


NPs)(Figure 1). Then the vesicle size, morphology, stability, in vitro drug release and hemocompatibility of RGE-PEG/PGG-PTX NPs were studied. Finally, the cellular uptake in vitro and the anti-tumor effect in vivo were evaluated. The results indicated the effectiveness of RGE-PEG/PGG-PTX NPs in the treatment of GBM and the feasibility of the method of preparing RGE-PEG/PGG-PTX NPs. Compared with direct chemical modification of PDCs, this method can avoid the possible structure destruction of drugs or polymers, and provide a useful idea for the preparation of active targeting PDCs.

Figure 1. The illustration of preparation of RGE-PEG/PGG-PTX nanoparticles Results Characterization of RGERPPR-PEG-DSPE Figure

2

shows

the

RGERPPR-PEG-DSPE.

NMR In

the

and

FITR

spectra

of

Mal-PEG-DSPE

and

1H-NMR

spectra

of

Mal-PEG-DSPE

and

RGERPPR-PEG-DSPE, the methylene protons of DSPE at 1.26 ppm, the repeat units of PEG at 3.7−3.8 ppm and solvent peak of CDCl3 at 7.26 ppm were contained. The peak standing for PEG around 3.50 ppm became smaller after conjugation, which should because that the addition of RGERPPR decreased the proportion of PEG in the whole substance. The disappearance of the characteristic resonance of maleimide in the 1H-NMR spectra of RGERPPR-PEG-DSPE indicated the successful conjugation. FTIR spectrum of Mal-PEG-DSPE contains a wide but week N-H stretch band at 3200~3600 cm-1 and a C=O stretch band at 1666.8 cm-1. The enhanced intensity of the two bands in RGERPPR-PEG-DSPE should be attributed to the plentiful N-H and C=O



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

structures in C-RGERPPR, also indicating the successful reaction of Mal-PEG-DSPE and C-RGERPPR.

Figure 2. The molecular structure (A), NMR spectra (B) and FTIR spectra (C) of Mal-PEG-DSPE and RGE -PEG-DSPE Characterization of RGE-PEG/PGG-PTX NPs As presented in Figure 3A, the dynamic diameters of PGG-PTX and RGE-PEG/PGG-PTX NPs were 30 nm (PDI=0.221) and 90 nm (PDI=0.197), respectively. Morphological characteristics of the two NPs were determined using transmission electron microscopy (TEM) (Figure 3B), and both showed uniform spherical morphology. Besides, PGG-PTX and RGE-PEG/PGG-PTX NPs showed particle sizes of around 25 nm and 80 nm from the TEM images, respectively. The both sizes were smaller than those determined by dynamic light scattering detector (DLS), which may be due to the different mechanisms and preparation processes between the


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


two methods. The detection by DLS was performed in aqueous state, and the particle size was considered as “hydrated diameters”. Whereas the detection by TEM was performed in dehydrated state, possibly leading to smaller particle sizes as above. This is also in accordance with many previous reports.13, 14 According to the drug loading equation, the PTX loaded in PGG-PTX, mPEG/PGG-PTX and RGE-PEG/PGG-PTX NPs were 35.1%, 32.5% and 31.6% respectively, indicating that the modification of RGE peptide through physical encapsulation did not affect the loading capacity of the PGG-PTX delivery system. In addition, our drug delivery system showed a good stability in vitro over three weeks (Figure 4).

Figure 3. The dynamic diameters (A) and the TME images (B) of PGG-PTX and RGE-PEG /PGG-PTX NPs



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. In vitro stability of PGG-PTX and RGE-PEG/PGG-PTX NPs for over three weeks In Vitro Drug Release As shown in Figure 5, the RGE-PEG/PGG-PTX NPs depicted continuous and a little higher PTX release compared with PGG-PTX conjugates at pH 7.4 within 72 h. This may be attributed to the decreased hydrophobic interaction among PTX groups caused by the insertion of DSPE into the hydrophobic core. The sustained release may be explained by the fact that PTX is attached to the PGG via a stable ester bond, which is consistent with the previous reports.9, 10

Figure 5. In vitro release profiles of PTX from PGG-PTX and RGE-PEG/PGG-PTX NPs in PBS (pH 7.4) at 37 °C Cellular Uptake and Flow Cytometry Tests Figure 6 and 7 showed the cellular uptake of PGG-PTX/DiO, mPEG/PGG-PTX/DiO and RGE-PEG /PGG-PTX/DiO by U87MG and HUVEC cells, respectively. As imaged by laser scanning confocal microscope, there was a significant enhancement of cellular


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


uptake

of

RGE-PEG/PGG-PTX/DiO

than

that

of

PGG-PTX/DiO

and

mPEG/PGG-PTX/DiO for the both cells. In U87MG cells, the percentages of DiO-positive

cells

for

PBS,

PGG-PTX/DiO,

mPEG/PGG-PTX/DiO

and

RGE-PEG/PGG-PTX/DiO were 3.17%, 32.44%, 36.98% and 97.71%, and the mean fluorescence intensities for them were 3.45, 9.98, 10.37 and 151.62, respectively. Similar to the results above, in HUVEC cells, the percentages of DiO-positive cells for PBS, PGG-PTX/DiO, mPEG/PGG-PTX/DiO and RGE-PEG/PGG-PTX/DiO were 0.12%, 16.95%, 17.65% and 98.95%, and the mean fluorescence intensities for them were 2.35, 14.62, 13.12 and 253.05, respectively. These data indicated that the modification of RGERPPR significantly enhanced the cellular uptake of PGG-PTX in both U87 MG cells and HUVECs.

Figure 6. The representative images and flow cytometry of U87MG cells incubated with

PBS

(A),

PGG-PTX/DiO

(B),

mPEG/PGG-PTX/DiO

(C)

and

RGE-PEG/PGG-PTX/DiO (D). The cellular uptake of PGG-PTX conjugates by U87MG cells was remarkably enhanced with the modification of RGERPPR.



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

Page 12 of 34

Figure 7. The representative images and flow cytometry of HUVECs incubated with PBS

(A),

PGG-PTX/DiO

(B),

mPEG/PGG-PTX/DiO

(C)

and

RGE-PEG/PGG-PTX/DiO (D). The cellular uptake of PGG-PTX conjugates by HUVECs was remarkably enhanced with the modification of RGERPPR. Hemocompatibility Study Hemolytic profiles of PGG-PTX and RGE-PEG/PGG-PTX at various lipid concentrations are presented in Figure 8. Little hemotoxicity was observed in PGG-PTX and RGE-PEG/PGG-PTX at the tested concentrations, indicating the good compatibility of PGG-PTX and RGE-PEG/PGG-PTX.

Figure 8. Hemocompatibility studies of PGG-PTX and RGE-PEG/PGG-PTX NPs at


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


various lipid concentrations In Vitro Cytotoxicity Assay The in vitro cytotoxicity of PTX, PGG-PTX, mPEG/PGG-PTX NPs and RGE-PEG/PGG-PTX NPs was tested on U87 MG cells and HUVECs. After incubation for 48 h, the IC50 in U87 MG cells were 0.550 µM for PTX, 8.610 µM for PGG-PTX, 8.750 µM for mPEG/PGG-PTX NPs and 2.143 µM for RGE-PEG/PGG-PTX NPs (Figure 9A), respectively. The IC50 in HUVECs were 2.286 µM for PTX, 21.136 µM for PGG-PTX, 18.393 µM for mPEG-DSPE/PGG-PTX NPs and 8.568 µM for RGE-PEG-DSPE/PGG-PTX NPs (Figure 9B), respectively. The minimal IC50 for PTX in both cells means the highest cytotoxicity, which should be due to the inadequate PTX release from the NPs compared with free PTX. In addition, RGE-PEG/PGG-PTX NPs showed significantly increased cytotoxicity compared with PGG-PTX and mPEG-DSPE/PGG-PTX NPs, suggesting the superior anti-glioblastoma ability after the modification by RGERPPR. This is also in agreement with the results of cellular uptake test (Figure 6 and 7).

Figure 9. In vitro cytotoxicity of PTX, PGG-PTX, mPEG/PGG-PTX NPs and RGE-PEG/PGG-PTX NPs in U87 MG cells (A) and HUVECs (B)



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 14 of 34

Anti-Glioblastoma Study of RGE-PEG/PGG-PTX NPs Animal body weight changes of glioblastoma-bearing mice could be used to evaluate the side effect of different treatments. As is shown in Fig.10A, the body weight of animals was almost increased. This indicated our NDDSs might be safe for the treatment of glioblastoma. The anti-glioblastoma effect was presented in a Kaplane Meier plot and the differences among survival curves were analyzed by the log-rank test. As shown in Figure 10B, the median survival times of mice treated with normal saline, PTX, PGG-PTX and RGE-PEG/PGG-PTX NPs were 22, 29, 36 and 47 days, respectively.

Compared

to

normal

saline

and

PTX,

PGG-PTX

and

RGE-PEG/PGG-PTX NPs significantly prolonged the survival time, possibly benefiting from not only the prolonged blood circulation time, but also the passive targeting delivery to tumor via EPR effect. Importantly, RGE-PEG/PGG-PTX NPs showed stronger anti-glioblastoma efficacy than PGG-PTX, which should be attributed to the active targeting and tumor penetrating ability of RGERPPR peptide.

Figure 10. Anti-glioblastoma efficacy in vivo. (A) Animal body weight changes of U87MG glioblastoma-bearing nude mice. (B) The Kaplan–Meier survival curve of U87MG

glioblastoma-bearing

nude

mice.

The

median

survival

time

of

RGE-PEG/PGG-PTX NPs group was remarkably longer than that of PGG-PTX group (p < 0.05, log-rank analysis) Discussion and Conclusion In

this

study,

we

successfully

synthesized

the

targeting

material

RGERPPR-PEG3400-DSPE, which was assembled with PGG-PTX, by virtue of the hydrophobic interaction between the group of DSPE and PTX, to prepare RGE-PEG/PGG-PTX NPs. The NPs we prepared showed the following characteristics:


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


particle size of about 90 nm with uniform spherical morphology, good stability in PBS, sustained drug release, good compatibility and enhanced tumor cellular uptake and cytotoxicity in vitro. Compared with PGG-PTX, the RGE-PEG/PGG-PTX NPs effectively prolong the lifetime of gliomastoma animal models. Currently, targeting molecule modified PDCs is mostly synthesized by a series of chemical reactions. However, due to the large molecular weight of PDCs, poor controllability of chemical reactions and difficult characterization of the molecular structure, it is not easy to directly link the targeting molecule to PDCs. In this study, the targeting moiety RGERPPR-PEG-DSPE was first synthesized and then assembled with PGG-PTX into RGEPRRP peptide modified PGG-PTX NPs. In this nanostructure, DSPE is inserted into the hydrophobic core formed by PTX groups, and PGG forms a hydrophilic shell, while PEG forms a hydration layer to ensure the physical stability of the NPs and sufficient flexibility of the targeting molecule RGERPPR. We can call this preparation method "modular" design, which means that each functional part of NDDS is separately designed and synthesized as individual module, and then these modules are self-assembled into the final NDDS. Compared with direct chemical modification, this method can avoid the complex chemical synthesis and make the preparation of NDDS more convenient and controllable. Nevertheless, the NDDS prepared by this method may have a decreased stability as the targeting moiety inserted via hydrophobic interaction may drop from the NDDS, resulting in the loss of active targeting ability. The decreased stability may be reflected by the slightly increased drug release from RGE-PEG/PGG-PTX NPs compared with PGG-PTX. To avoid the disassembly, there should be a sufficient interaction between the hydrophobic core of the NPs and the hydrophobic ends of the targeting moiety. In this study, the hydrophobic interaction between PTX and DSPE has been evidenced since there have been many reports on PTX encapsulated DSPE-PEG micelles.9, 10 Besides, as is shown in the experiments (Figure 4), RGE-PEG/PGG-PTX NPs we prepared has sufficient stability. Compared with peripheral tumors, brain tumors have a special development process, which determines the particularity in the design of brain tumor targeted NDDS. The development of brain tumors can be divided into three periods: early stage, middle



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

stage and advanced stage. In the early stage, when the blood vessels in the brain tissue remained intact, the main delivery barrier of the NDDS was the blood-brain barrier (BBB). In the middle stage, the tumor tissue began to break vascular integrity and the main delivery barrier was the blood-brain tumor barrier (BBTB). In the advanced stage, the blood vessels were severely damaged and the NDDS could enter the brain tumor sites via the EPR effect.33, 34 However, possibly due to the limitation of the intracranial space, the EPR effect in the brain tumor is much weaker than that in the peripheral tumor, which requires the particle size of the NDDS not be too large, generally below 100 nm. The RGE-PEG/PGG-PTX NPs we prepared satisfied this requirement. According to our previous experiences, nude mice inoculated with GBM could enter the middle stage of brain tumors at around 13 days post-inoculation. This means that the GBM we treated in this study is mainly at the middle and advanced stages. In this stage, it is not easy for the traditional NDDS to enter the brain tumor tissue due to the existence of BBTB and the relatively weak EPR effect. Many studies have demonstrated that NRP-1, the specific receptors of tumor penetrating peptides, is highly expressed on the surface of tumor vascular endothelial cells and brain tumor cells. Accordingly, in this work, we prepared a tumor-penetrating peptide-modified NDDS, RGE-PEG/PGG-PTX, which can penetrate the tumor vessel and deep into the tumor tissue by the mediation of NRP-1 receptor. This can address the issues imposed by BBTB and weak EPR effect, which has been also validated by the studies of in vitro cellular uptake (Figure 6, 7) and in vivo antitumor effect (Figure 10). Therefore, the TPP-modified NDDS is very advantageous in the tumor-targeted therapy, especially for brain tumor. The anti-tumor effect of NDDSs relies on two of essential factors: high stability and adequate drug release in time. On the one hand, the NDDSs must be adequately stable in the blood circulation without depolymerization or drug release, avoiding side effects on normal tissue. On the other hand, the NDDSs should be able to release the drug adequately and timely within tumor tissue. It is the two sides of a coin, and we need to find a balance between the two. In this study, the results show that the RGE-PEG/PGG-PTX NPs we prepared exhibit good stability but inadequate drug


Page 16 of 34

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


release in vitro. This is because that in the structure of PGG-PTX, PTX is linked to the PGA backbone via glutamate, instead of an environment sensitive linker. An adequate and timely drug release can be designed by adding an acid or enzyme-sensitive linker35 between drug and polymer backbone, based on the specific microenvironment in tumor tissue.23, 36-38 We have also conducted related researches,39 which is also one of our future research interests. In summary, the RGE-PEG/PGG-PTX NPs exhibited an improved targeting therapeutic effect on glioblastoma. Moreover, this study provided a so-called "modular" design strategy, by which targeting material was synthesized and assembled with PDCs into the active targeting PDCs. This is a more feasible and applicable strategy, and may further promote the development of PDCs and even other NDDS. Materials and Methods Materials Poly

(L-c-glutamyl-glutamine)-paclitaxel

(PGG-PTX)

nanoconjugates

were

synthesized as previously described9, 10 and the molecular weight (Mw) of PGG-PTX was 79.19 kDa. RGERPPR was obtained from Shanghai Top-peptide Co., Ltd. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-3400]

(mPEG3400-DSPE)

and

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(poly(ethyleneglycol ))3400] (Mal-PEG3400-DSPE) were purchased from Laysan Bio Inc. (USA). Sephadex G50 was supplied by GE Healthcare (UK). Fetal bovine serum (FBS), RPMI1640 and DMEM medium were from Biological Industries Co. DiO, DiR and streptomycin/penicillin were provided by Sigma Chemical Reagent Co (USA). Hochest33342 was obtained from Life Technologies (USA). All chemical regents are analytic reagent grade. U87MG cell line and HUVECs were purchased from Shanghai Institute of Cell Biology. Male Balb/c Nu/Nu mice (18−22 g body weight) were purchased from Shanghai SLAC laboratory animal company (Shanghai, China) and kept under SPF conditions. All animal experiments were approved by the ethics committee of East China Normal University. Synthesis and Characterization of RGERPPR-PEG-DSPE



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 thiolated targeting peptides (C-RGERPPR) was synthesized by the solid phase peptide synthesis method. After identified using HPLC and LC-MS, C-RGERPPR was reacted with Mal-PEG-DSPE as described previously.32 The reaction mixture was dialyzed (MW cut = 3500 Da, Spectrum Laboratory) against distilled water for 36 h, and then lyophilized. The structure of the final product was characterized by 1H-NMR and FTIR. Preparation of RGE-PEG/PGG-PTX NPs PGG-PTX was prepared in our laboratory according to the previous report.9, 10 The RGE-PEG/PGG-PTX NPs were prepared using the emulsion-solvent evaporation method as follows. PGG-PTX was dissolved in 2 mL of sodium tauroglycocholate (0.5% in water) solution. The mPEG-DSPE and RGERPPR-PEG-DSPE, were dissolved separately in 1 mL of dichloromethane: acetone (3:1), and added into the PGG-PTX solution (PGG-PTX: mPEG-DSPE: RGERPPR-PEG-DSPE=98:2:1, molar ratio). The above solution was mixed by a vortex and the O/W emulsion was obtained after ultrasonication for 2 min. The emulsion was added to 8 mL of 1% sodium tauroglycocholate solution in water and stirred for 10 min. The dichloromethane and acetone were removed by rotary evaporation and the reaction mixture was centrifuged, resuspended and purified to obtain the RGE-PEG/PGG-PTX NPs. In addition, the preparations of unmodified mPEG/PGG-PTX NPs were prepared using the same method except that RGERPPR-PEG-DSPE was not added. The DiO or DiR loaded nanoparticles were prepared using the same method except that DiO or DiR was also dissolved in the dichloromethane/acetone solution before being mixed with the PGG-PTX solution. Characterization of RGE-PEG/PGG-PTX NPs Particle size distribution was tested using dynamic light scattering detector (Zetasizer Nano-ZS, Malvern Instruments, Westborough, MA). The morphology of nanoparticles was imaged by transmission electron microscopy (JEM- 2100FEF, JEOL, Japan). PTX has a maximum absorption peak at 228 nm while the PGG polymer exhibits little absorption. The PTX loading capacity of PGG-PTX conjugates were directly determined via ultraviolet–visible (UV) methods. Considering that because


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


RGE-PEG-DSPE has a UV absorption at 228nm, the RGE-PEG/PGG-PTX NPs were first disassembled in methanol and dialyzed (cutoff Mw = 10 k) against methanol for 48 h to remove RGE-PEG-DSPE and mPEG-DSPE. Then the content of PTX was determined by UV method. In addition, the drug loading of unmodified mPEG/PGG-PTX NPs were measured using the same method. The PTX content was calculated by the linear equation drawn by the standard solutions, and the PTX loading capacity was calculated using the following equation: PTX loading (w/w %) = Weight of loaded PTX/Total weight of conjugates or NPs * 100% The in vitro stability of PGG-PTX and RGE-PEG/PGG-PTX NPs was evaluated by determining the particle sizes in 4 °C for over three weeks. In Vitro Drug Release The release of PTX from PGG-PTX conjugates and RGE-PEG /PGG-PTX NPs was assessed at pH 7.4 according to previous reports. All samples were dissolved in phosphate buffer (0.01 M, pH 7.4) with three replicates in each group. A volume of 1.0 mL of these formulations was placed into dialysis membrane tubing (Sigma, MW cut-off 3500 Da) and dialyzed at 37 °C. Concentration of PTX was determined by HPLC after shaking at 180 rpm for 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 96 and 120 h, respectively. Cellular Uptake and Flow Cytometry Tests U87MG and HUVEC cells were used to evaluate the in vitro targeting ability of RGE-PEG /PGG-PTX NPs. Cells (1000/well) were seeded in 35 mm multi-well plates and RGE-PEG/PGG-PTX/DiO, mPEG/PGG-PTX/DiO and PGG-PTX/DiO were added to different wells after 24 h incubation. For the Cellular uptake, the cells were washed, fixed, stained with Hochest after 2 h treatment and imaged using laser scanning confocal microscope (DMI4000 B, Leica, Germany). For the flow cytometry test, cells were seeded in 6 well plates and after treatment with conjugates, the cells were trypsinized, washed, resuspended in PBS and analyzed by flow cytometry instrument (Guava EasyCyte, Merck Millipore, USA). Hemocompatibility Studies



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

Hemolytic toxicity was performed according to a slightly modified reported procedure.40 Fresh whole blood was collected from male rats, centrifuged at 1800 rpm and washed three times with physiological saline (0.9% w/v). RBCs collected from the bottom were washed with physiological saline (0.9% w/v) until a clear, colorless supernatant was obtained. 2% erythrocyte was prepared to test hemolytic toxicity. Various concentrations of Tween 80, PGG-PTX, RGE-PEG/PGG-PTX, distilled water (positive control), normal saline (negative control) and polyoxyethylated castor oil were mixed with equal volume of 2% RBC suspension and incubated at 37 ºC for 3 h, respectively. After incubation, samples were centrifuged and the supernatants were acquired and measured using UV spectrophotometer at λmax 545 nm. The percent hemolysis was calculated using the following equation: Hemolysis (%) = (Absample – Abnegative)/ (Abpositive –Abnegative) * 100% In Vitro Cytotoxicity Assay Cells (3000/well) were seeded in 96-well plate, incubated for 24 h and treated with different

concentrations

of

PTX,

PGG-PTX,

mPEG/PGG-PTX

or

RGE-PEG/PGG-PTX NPs for 72 h. The cytotoxicity was assayed by CCK-8 method and the IC50 values were calculated by curve analysis software (GraphPad Prism 7.02). Anti-Glioblastoma Study of RGE-PEG/PGG-PTX NPs Glioblastoma animal models were established in 5 week-old male BALB/C nude mice by injecting 5 × 105 U87MG cells suspended in 5 µL PBS into the right striatum using a stereotactic ﬁxation device. The coordinates were 1.8 mm lateral and 0.6 mm anterior to the bregma, and 3 mm deep. Four groups (n = 7) of mice were i.v. administrated with normal saline (NS), PTX, PGG-PTX and RGE-PEG/PGG-PTX at the 13rd, 16th, 18th and 20th day post-inoculation. The PTX dose was 50 mg/kg. The body weight and survival times of these mice were recorded. Statistical Analysis Data was shown as mean ± SD and the statistical comparisons of the different groups were analyzed by student t-test for two groups of data, and by one way ANOVA for over three groups of data. Statistical significance was defined as P < 0.05. Acknowledgments


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


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), and Zhejiang Provincial Natural Science Foundation of China (Y14H300002). We wish to thank Yiwen Wang and Bing Ni for their assistance on TEM and CLSM test. Conflict of Interest Statement The authors declare no competing ﬁnancial interest. ORCID Lipeng Gao: 0000-0002-5680-6789 Weiyue Lu: 0000-0001-8003-2675 Zhiqiang Yan: 0000-0002-3176-5757 References (1)

Bush, N. A., and Butowski, N. (2017) The Effect of Molecular Diagnostics on the Treatment of Glioma. Curr Oncol Rep 19, 26.

(2)

Wang, J., Lei, Y., Xie, C., Lu, W., Yan, Z., Gao, J., Xie, Z., Zhang, X., and Liu, M. (2013) Targeted gene delivery to glioblastoma using a C-end rule RGERPPR peptide-functionalised polyethylenimine complex. Int J Pharm 458, 48-56.

(3)

Rajesh, Y., Pal, I., Banik, P., Chakraborty, S., Borkar, S. A., Dey, G., Mukherjee, A., and Mandal, M. (2017) Insights into molecular therapy of glioma: current challenges and next generation blueprint. Acta Pharmacol Sin 38, 591-613.

(4)

Wang, H., Wu, J., Xie, K., Fang, T., Chen, C., Xie, H., Zhou, L., and Zheng, S. (2017) Precise Engineering of Prodrug Cocktails into Single Polymeric Nanoparticles for Combination Cancer Therapy: Extended and Sequentially Controllable Drug Release. ACS Appl Mater Interfaces 9, 10567-10576.

(5)

Alonso, M. J. (2004) Nanomedicines for overcoming biological barriers. Biomed Pharmacother 58, 168-172.

(6)

Jain, K. K. (2010) Advances in the field of nanooncology. BMC Med 8, 83.

(7)

Fouladi, F., Steffen, K. J., and Mallik, S. (2017) Enzyme-Responsive Liposomes for the Delivery of Anticancer Drugs. Bioconjug Chem 28, 857-868.

(8)

Yang, J., and Kopecek, J. (2014) Macromolecular therapeutics. Journal of controlled release : official journal of the Controlled Release Society 190, 288-303.

(9)

Van, S., Das, S. K., Wang, X., Feng, Z., Jin, Y., Hou, Z., Chen, F., Pham, A., Jiang, N., Howell, S. B., et

al.

(2010)

Synthesis,

characterization,

and

biological

evaluation

of

poly(L-gamma-glutamyl-glutamine)- paclitaxel nanoconjugate. Int J Nanomedicine 5, 825-837. (10)

Yang, D., Liu, X., Jiang, X., Liu, Y., Ying, W., Wang, H., Bai, H., Taylor, W. D., Wang, Y., Clamme, J. P., et al. (2012) Effect of molecular weight of PGG-paclitaxel conjugates on in vitro and in vivo



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 22 of 34

efficacy. Journal of controlled release : official journal of the Controlled Release Society 161, 124-131. (11)

Yu, Z., Li, J., Zhu, J., Zhu, M., Jiang, F., Zhang, J., Li, Z., Zhong, M., Kaye, J. B., Du, J., et al. (2014) A synthetic transmembrane segment derived from TRPV4 channel self-assembles into potassium-like channels to regulate vascular smooth muscle cell membrane potential. J. Mater. Chem. B 2, 3809-3818.

(12)

Xu, Q., Lv, Y., Dong, C., Sreeprased, T. S., Tian, A., Zhang, H., Tang, Y., Yu, Z., and Li, N. (2015) Three-dimensional micro/nanoscale architectures: fabrication and applications. Nanoscale 7, 10883-10895.

(13)

Gao, L., Zhou, J., Yu, J., Li, Q., Liu, X., Sun, L., Peng, T., Wang, J., Zhu, J., Sun, J.,et al. (2017) A Novel Gd-DTPA-conjugated Poly(L-γ-glutamyl-glutamine)-paclitaxel Polymeric Delivery System for Tumor Theranostics. Sci Rep 7, 3799.

(14)

Gao, L., Gao, L., Fan, M., Li, Q., Jin, J., Wang, J., Lu, W., Yu, L., Yan, Z., and Wang, Y. (2017) Hydrotropic polymer-based paclitaxel-loaded self-assembled nanoparticles: preparation and biological evaluation. RSC Adv. 7, 33248-33256.

(15)

Yang, J., and Kopecek, J. (2016) Design of smart HPMA copolymer-based nanomedicines. Journal of controlled release : official journal of the Controlled Release Society 240, 9-23.

(16)

Zhan, C., Gu, B., Xie, C., Li, J., Liu, Y., and Lu, W. (2010) 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 143, 136-142.

(17)

Peng, Z.-H., and Kopeček, J. (2015) Enhancing Accumulation and Penetration of HPMA Copolymer–Doxorubicin Conjugates in 2D and 3D Prostate Cancer Cells via iRGD Conjugation with an MMP-2 Cleavable Spacer. Journal of the American Chemical Society 137, 6726-6729.

(18)

Greish, K., Ray, A., Bauer, H., Larson, N., Malugin, A., Pike, D., Haider, M., and Ghandehari, H. (2011)

Anticancer

and

antiangiogenic

activity

of

HPMA

copolymer-aminohexylgeldanamycin-RGDfK conjugates for prostate cancer therapy. Journal of controlled release : official journal of the Controlled Release Society 151, 263-270. (19)

Wang, K., Zhang, X., Liu, Y., Liu, C., Jiang, B., and Jiang, Y. (2014) Tumor penetrability and anti-angiogenesis using iRGD-mediated delivery of doxorubicin-polymer conjugates. Biomaterials 35, 8735-8747.

(20)

Cuchelkar V, Kopečková P, Kopeček J. (2008) Novel HPMA Copolymer-Bound Constructs for

(21)

Huang, L., Xie, J., Bi, Q., Li, Z., Liu, S., Shen, Q., and Li, C. (2017) Highly Selective Targeting of

Combined Tumor and Mitochondrial Targeting. Mol Pharm, 5(5):776-786.. Hepatic Stellate Cells for Liver Fibrosis Treatment Using a d-Enantiomeric Peptide Ligand of Fn14 Identified by Mirror-Image mRNA Display. Mol Pharm 14, 1742-1753. (22)

Wu, J., Jiang, H., Bi, Q., Luo, Q., Li, J., Zhang, Y., Chen, Z., and Li, C. (2014) Apamin-mediated actively targeted drug delivery for treatment of spinal cord injury: more than just a concept. Mol Pharm 11, 3210-3222.

(23)

Danhier, F., Feron, O., and Preat, V. (2010) To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. Journal of controlled release : official journal of the Controlled Release Society 148, 135-146.

(24)

Li, C., Wang, Y., Zhang, X., Deng, L., Zhang, Y., and Chen, Z. (2013) Tumor-targeted liposomal drug delivery mediated by a diseleno bond-stabilized cyclic peptide. Int J Nanomedicine 8, 1051-1062.


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


(25)

Perry, J. L., Reuter, K. G., Luft, J. C., Pecot, C. V., Zamboni, W., and DeSimone, J. M. (2017) Mediating Passive Tumor Accumulation through Particle Size, Tumor Type, and Location. Nano Lett 17, 2879-2886.

(26)

Jin, B. J., Smith, A. J., and Verkman, A. S. (2016) Spatial model of convective solute transport in brain extracellular space does not support a "glymphatic" mechanism. J Gen Physiol 148, 489-501.

(27)

Nehoff, H., Parayath, N. N., Domanovitch, L., Taurin, S., and Greish, K. (2014) Nanomedicine for drug targeting: strategies beyond the enhanced permeability and retention effect. Int J Nanomedicine 9, 2539-2555.

(28)

Wang, J., Wang, H., Li, J., Liu, Z., Xie, H., Wei, X., Lu, D., Zhuang, R., Xu, X., and Zheng, S. (2016) iRGD-Decorated Polymeric Nanoparticles for the Efficient Delivery of Vandetanib to Hepatocellular Carcinoma: Preparation and in Vitro and in Vivo Evaluation. ACS Appl Mater Interfaces 8, 19228-19237.

(29)

Ruoslahti, E. (2017) Tumor penetrating peptides for improved drug delivery. Adv Drug Deliv Rev 110-111, 3-12.

(30)

Yan, Z., Zhan, C., Wen, Z., Feng, L., Wang, F., Liu, Y., Yang, X., Dong, Q., Liu, M., and Lu, W. (2011) LyP-1-conjugated doxorubicin-loaded liposomes suppress lymphatic metastasis by inhibiting lymph node metastases and destroying tumor lymphatics. Nanotechnology 22, 415103.

(31)

Zhou, J. E., Yu, J., Gao, L., Sun, L., Peng, T., Wang, J., Zhu, J., Lu, W., Zhang, L., Yan, Z., et al. (2017) iNGR-Modified Liposomes for Tumor Vascular Targeting and Tumor Tissue Penetrating Delivery in the Treatment of Glioblastoma. Mol Pharm 14, 1811-1820.

(32)

Yang, Y., Yan, Z., Wei, D., Zhong, J., Liu, L., Zhang, L., Wang, F., Wei, X., Xie, C., Lu, W., et al. (2013) Tumor-penetrating peptide functionalization enhances the anti-glioblastoma effect of doxorubicin liposomes. Nanotechnology 24, 405101.

(33)

Chen, C., Duan, Z., Yuan, Y., Li, R., Pang, L., Liang, J., Xu, X., and Wang, J. (2017) Peptide-22 and Cyclic RGD Functionalized Liposomes for Glioma Targeting Drug Delivery Overcoming BBB and BBTB. ACS Appl Mater Interfaces 9, 5864-5873.

(34)

Obermeier, B., Daneman, R., and Ransohoff, R. M. (2013) Development, maintenance and disruption of the blood-brain barrier. Nat Med 19, 1584-1596.

(35)

Kuang, T., Liu, Y., Gong, T., Peng, X., Hu, X. and Yu, Z. (2016) Enzyme-responsive nanoparticles

(36)

Shivhare, K., Garg, C., Priyam, A., Gupta, A., Sharma, A. K., and Kumar, P. (2017) Enzyme

for anticancer drug delivery. Current Nanoscience 12, 38-46. sensitive smart inulin-dehydropeptide conjugate self-assembles into nanostructures useful for

targeted

delivery

of

ornidazole.

Int

J

Biol

Macromol.

http://dx.doi.org/10.1016/j.ijbiomac.2017.08.071 (37)

Kozlovskaya, V., Liu, F., Xue, B., Ahmad, F., Alford, A., Saeed, M., and Kharlampieva, E. (2017) Polyphenolic

Polymersomes

of

Poly(N-vinylcaprolactam)-block-Poly(N-vinylpyrrolidone)

Temperature-Sensitive for

Anticancer

Therapy.

Biomacromolecules 18, 2552-2563. (38)

Kang, Y., Ha, W., Liu, Y. Q., Ma, Y., Fan, M. M., Ding, L. S., Zhang, S., and Li, B. J. (2014) pH-responsive polymer-drug conjugates as multifunctional micelles for cancer-drug delivery. Nanotechnology 25, 335101.

(39)

Han, L., Xiao, Y., Fan, M., Wang, J., Yan, Z., Wang, Y., Yu, L., Peng, H., and Zhu, J. (2017)



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

Synthesis and biological evaluation of an anticancer drug delivery system: Poly( l -γ-glutamyll -carbocisteine)-paclitaxel nanoconjugate. Materials Science and Engineering: C. 81, 113-119 (40)

Jain, V., Swarnakar, N. K., Mishra, P. R., Verma, A., Kaul, A., Mishra, A. K., and Jain, N. K. (2012) Paclitaxel loaded PEGylated gleceryl monooleate based nanoparticulate carriers in chemotherapy. Biomaterials 33, 7206-7220.


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


TOC 90x50mm (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

Figure 1 180x79mm (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


Figure 2 133x223mm (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

Figure 3 135x230mm (600 x 600 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


Figure 4 56x40mm (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



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


Figure 6 242x161mm (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

Figure 7 242x161mm (300 x 300 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





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 99x125mm (600 x 600 DPI)


Page 34 of 34

Page 35 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 10 49x14mm (600 x 600 DPI)


Self-Assembled Tumor-Penetrating Peptide ... - ACS Publications

Recommend Documents