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EGFR/EGFRvIII Dual-Targeting Peptide Mediated Drug Delivery for Enhanced Glioma Therapy Jiani Mao, Danni Ran, Cao Xie, Qing Shen, Songli Wang, and Weiyue Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b05617 • Publication Date (Web): 07 Jul 2017 Downloaded from http://pubs.acs.org on July 8, 2017

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ACS Applied Materials & Interfaces

EGFR/EGFRvIII

Dual-Targeting

Peptide

Mediated

Drug

Delivery for Enhanced Glioma Therapy Jiani Maoa†, Danni Rana†, Cao Xiea, Qing Shene, Songli Wanga, Weiyue Lua,b,c,d,* a

Department of Pharmaceutics, School of Pharmacy, Fudan University & Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education, Shanghai 201203, China b Minhang Hospital, Fudan University, Shanghai 201199, China c State Key Laboratory of Medical Neurobiology, The Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China d The Institutes of Integrative Medicine of Fudan University, Shanghai 200040, China e State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China † These authors contributed equally to this work.

Abstract: Tumor-homing peptides have been widely used to mediate active targeted drug delivery. L-AE is a reported targeting peptide, demonstrating high binding affinity to epidermal growth factor receptor (EGFR) and mutation variant III (EGFRvIII) overexpressed on neovasculature, vasculogenic mimicry (VM), tumor cells and tumor stem cells (CSCs). To improve its proteolytic stability, a D-peptide ligand (termed D-AE, the enantiomer of L-AE) was developed. D-AE was confirmed to bind receptors EGFR and EGFRvIII with comparable targeting capability as L-AE. In vivo biodistribution demonstrated the superiority of D-AE in prolonged circulation and enhanced intratumoral accumulation. Furthermore, stabilized peptide modification endowed micelles higher transcytosis efficiency and penetrating capability on BBTB/U87 tumor spheroids co-culture model. When PTX (paclitaxel) was loaded, D-AE-Micelle/PTX demonstrated excellent anti-tumor effect in comparison to Taxol, Micelle/PTX and L-AE-Micelle/PTX. These findings indicated that multi-targeted drug delivery system enabled by D-AE ligand provided a promising way for glioma therapy.

Keywords:

EGFR,

EGFRvIII,

AE,

D-peptide,

multi-targeted

Glioblastoma

ACS Paragon Plus Environment

drug

delivery,


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1. INTRODUCTION Glioblastoma multiforme (GBM) is a highly malignant primary brain tumor. Despite multimodality therapies including surgery, radiotherapy and chemotherapy have been employed, the median survival remains less than 15 months with recurrence rate of 7 months.1 The unsatisfactory therapeutic outcome is largely attributed to the complex microenvironment of glioma. First of all, the biological barriers, the blood-brain barrier (BBB) strictly limits the transport of chemotherapeutic agents into brain.2-4 Similar to the blood-tumor barrier (BTB) of peripheral tumors, the blood-brain tumor barrier (BBTB) located among glioma cells and microvessels, further hinders the accumulation and penetration of chemo drugs into tumor regions. Tumor angiogenesis and vasculogenic mimicry (VM) also play crucial roles in tumor progression and metastases.5-7 In addition, a small population of tumor stem cells (CSCs) is regarded highly responsible for post-treatment relapse, with indefinite self-replication, chemotherapeutic resistance and tumor-initiative ability.8-10 Active targeted drug delivery system has emerged as a promising strategy for glioma therapy by functionalizing with tumor targeting ligands to improve delivery efficiency and anti-tumor efficacy.11-16 Many studies and clinical trials have attempted to identify the specific biomarkers on glioma, of which the epidermal growth factor receptor (EGFR) is the most well-characterized.17,

18

According to The Cancer

Genome Atlas (TCGA) GBM database, ~57% of GBMs patients have EGFR overexpression and/or mutation.19 EGFR activation promotes tumor cell proliferation, migration and angiogenesis via MAPK and PI3K/Akt pathways.20-23 The commonest mutation EGFR variant III (EGFRvIII) was identified in ~25% of EGFR-amplified GBMs, but not in any normal tissues.24, 25 Compared with EGFR, the extracellular domain truncation of amino acids residues 6-273 results in the ligand-independent self-activated status. The consecutive activation of downstream signaling pathway enables tumor cells DNA-repair acceleration and chemo-resistance.26-28 Recent studies reported that high expression of EGFRvIII on CSCs may give rise to tumor initiation and recurrence.29-32 These evidence highlighted that targeting both EGFR and EGFRvIII would be more advantageous in GBM treatment than mono-targeting therapy. A novel hexapeptide ligand L-AE (LFLALLLGLELA), which was identified through mixture-based synthetic combinatorial library, has demonstrated selective binding affinity to EGFR and EGFRvIII.33-35 However, proteolytic instability of L-amino peptides


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in vivo significantly impaired their targeting efficacy.36 All D-peptide strategy is an effective approach to render L peptides noticeable serum stability.37-39 In this study, we designed two D-peptides: D-AE (DFDADLDGDEDA, The enantiomer of L-AE) was developed by substituting D-amino for L-amino without changing the sequence; RI-AE (DADEDGDLDADF, the retro-inverso isomer of L-AE) was synthesized by converting L-amino acids into D-configuration in reverse sequence. Cellular internalization and biodistribution assay were conducted to evaluate bioactivities of both D-peptides. The results indicated that D-AE possess similar multifunctional tumor targeting efficacy as the parent peptide L-AE. Therefore, we hypothesized that D-AE could serve as a potential targeting ligand to achieve glioma-targeted drug delivery through precisely binding to EGFR and EGFRvIII (Figure1). We constructed AE peptide modified PEG-PLA micelles and assessed the tumor targeting efficiency in vitro and in vivo. After encapsulating paclitaxel (PTX), the anti-tumor efficacy of micelles were evaluated in subcutaneous U87 tumor-bearing mice model.

Figure 1. Schematic illustration of EGFR/EGFRvIII dual-targeting AE peptide mediated micelle-based drug delivery. Micelles are designed to specifically penetrate the BBTB and target neovasculature, vasculogenic mimicry (VM), tumor cells and tumor stem cells. D-AE-Micelle with excellent proteolyic stability demonstrated superior targeting ability in vivo compared with L-AE-Micelle.

2. EXPERIMENTAL SECTION



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2.1 Materials Protected Boc-amino acid derivatives were purchased from GL Biochem Ltd. (Shanghai,

China).

MBHA

resin

was

supplied

by

Fluka

(USA).

O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluorophosphate (HBTU) purchased

from

American

Bioanalytical

Co.

(Natick,MA).

was

Mal-FITC

(Fluorescein-5-maleimide) was obtained from Fanbo Biochemicals (Beijing, China). FAM

(5-carboxyfluorescein)

6-diamidino-2-phenylindole)

was

from

Sigma

was obtained

from

(St.

Louis,

Roche

MO).

DAPI

(4,

(Basel, Switzerland).

Coumarin-6 was supplied by Sigma-Aldrich (St Louis, MO, USA). Near infrared dye DiR was purchased from Invitrogen (Grand Island, NY). mPEG2000-PLA2000 as well as mal-PEG3000-PLA2000 were obtained from Advanced Polymer Materials Inc (Hudson County, State of New Jersey). Polyester (PET) membrane Transwell (12mm membrane diameter, 0.4 µm pore size) was obtained from Conrning Incorporated Life Sciences (USA). Growth factor-reduced Matrigel matrix was obtained from BD Biosciences (San Diego, CA, USA). Rat serum was supplied by Guangzhou Jianlun Biotechnology Co. (Guangzhou, China). Rabbit anti-EGFR antibody, rabbit anti-EGFRvIII antibody and rabbit anti-CD31 antibody were purchased from Abcam (USA). PTX was obtained from Dalian Meilun Biotech Co., Ltd (Dalian, China) and Taxol was from Bristol-Myers Squibb Company. Human glioblastoma cells (U87), human umbilical vein endothelial cells (HUVECs) and Human normal liver cells (HL7702 cells) were provided by Shanghai Institute of Cell Biology. Cells were cultured at 37°C in Dulbecco’s modified Eagle medium (Gibco), containing 10% FBS (fetal bovine serum), penicillin (100 U/mL) and streptomycin (100 µg/mL). Male BALB/c nude mice of 20-25 g body weight and Sprague-Dawley (SD) rats were supplied by BK Lab Animal Ltd. (Shanghai, China) and maintained under standard SPF conditions. 2.2 Synthesis and characterization of Peptides and Derivatives L-AE (LFLALLLGLELA), D-AE (DFDADLDGDEDA) and RI-AE (DADEDGDLDADF) peptides were synthesized by solid phase peptide synthesis strategy. Boc-protected amino acids were dissolved in DMF and HBTU. The protected amino groups were deprotected by 100% trifluoroactic acid and washed by N, N-dimethylformamide (DMF) through the synthesis cycle. Crude products were cleaved from resin by HF and purified to homogeneity by preparative C18 reverse-phase HPLC and


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ascertained by HPLC and electrospray ionization mass spectrometry (ESI-MS). Fluorescein labeled peptides and Cyanine7 labeled peptides were synthesized via

sulfhydryl-maleimide

covalent

conjugation.

Peptides were

reacted

with

fluorescein-5-maleimide or Cyanine7-Maleimide (1:1.2 molar ratio) in phosphate buffer (0.1 M, pH 7.2) for 2 h. After purification, products were confirmed by HPLC and ESI-MS. The functional materials, L-AE-PEG3000-PLA2000, D-AE-PEG3000-PLA2000 and RI-AE-PEG3000-PLA2000 were synthesized by the following procedure, in brief, peptides dissolved in phosphate buffer (0.1 M, pH = 7.2) were added to mal-PEG3000-PLA2000 dissolved in DMF. After stirring for 2 h at room temperature, excessive peptides were removed by dialysis (MWCO 3.5 kDa) against distilled water. The pure products were characterized by 1H-NMR. 2.3 In vitro cellular uptake assay of AE peptides For qualitative study, HUVECs and U87 cells were seeded into confocal disc chambered cover glasses. After growing overnight, the culture medium were replaced with 5 µM fluorescein labeled AE peptides or FAM in DMEM (10% FBS) for 4 h. Then cells were rinsed with phosphate buffered saline (PBS), fixed with 4% (wt/vol) paraformaldehyde and stained with DAPI, cellular uptake was imaged using a laser scanning confocal microscope (TCS SP5, Leica, Germany). For quantitative analysis, cells were washed by PBS for three times and the fluorescence intensity was detected by a flow cytometer (FACS Aria, BD, USA). To confirm the tumor-selectivity characteristic of AE peptide, HL7702 cells were set as control and tested as above. 2.4 In vivo tumor targeting ability assay of AE peptides The U87 xenograft model was established by subcutaneously inoculating the tumor cells (1 x 109 cells in 100 µL PBS) into the right flank of mouse. When the tumor size was about 100 mm3, 100 µL Cy7 labeled peptides were injected into mice via tail vein, the fluorescence images at defined time points were captured with an animal imaging system (IVIS Spectrum, Caliper, USA). Then mice were anesthetized with organs harvested for further fluorescence distribution analysis. 2.5 Competitive inhibition and stability assay of AE peptides In order to confirm the specificity pathway of D-AE and RI-AE, competitive inhibition assay was conducted on U87 cells. U87 cells suspension at a density of 1 × 105 cells / 400 µL was distributed in tubes, pre-incubated with 100 µM peptide solution



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(L-AE, RI-AE, D-AE) for 2 h at 4°C and further incubated with fluorescein labeled peptides at the final concentration of 5 µM. After cultivation overnight at 4°C, the fluorescence intensity was determined by flow cytometer. The peptide stability assay was performed in 50% rat serum. Peptides (1 mg) in 0.1 mL phosphate buffered saline were mixed with 0.9 mL 50% rat plasma (diluted in phosphate buffered saline). At various incubation periods of 0, 0.25, 0.5, 1, 2, 4, 6 and 8 h, 100 µL of mixture was sampled and mixed with 20 µL 15% trichloroacetic acid solution. After storing at 4°C for 30 min, samples were centrifuged at 10000 rpm for 10 min. 20 µL of supernatant were analyzed by HPLC to quantify the remaining peptides. 2.6 Molecular Docking Study The crystal structure model of EGFR protein was downloaded from www.pdb.org (pdb code: 4uip). The missing atoms in the protein structure was added by protein prepare package in the software Schrodinger. The three dimensional structure model of L-AE, RI-AE and D-AE peptides were built with the software Schrodinger and optimized in OPLS2005 force field. The designed peptides were docked to EGFR protein by glide package in Schrodinger with default parameters.40-42 The binding energy was predicted by glide. After analyzing the resulting binding modes, the figures presented in paper were made by software pymol. 2.7 Preparation and characterization of AE peptide modified micelles Micelle/PTX, L-AE-Micelle/PTX, D-AE-Micelle/PTX and RI-AE-Micelle/PTX were prepared via thin-film hydration and extrusion method. 20 mg micelle materials (20 mg mPEG2000-PLA2000 or 2 mg AE-PEG3000-PLA2000 plus 18 mg mPEG2000-PLA2000) and 8 mg PTX were co-dissolved in 2 mL acetonitrile, rotary evaporated the organic solvent to form thin-film. After hydrating with physiological saline at 37°C for 0.5 h, micelles were filtered three times against 0.22 µm filter membrane to remove unloaded PTX. Coumarin-6 loaded micelles and DiR loaded micelles were obtained using the same method except that PTX was replaced by 0.2% (w/w) coumarin-6 or 0.2% (w/w) DiR. Micelle sizes were measured by dynamic light scattering (DLS) using Zetasizer Nano (Malvern Zetasizer Nano Series, Malvern, UK). The concentrations of PTX were determined by HPLC. The encapsulation efficiency (EE) and loading capability (LC) were calculated as following equations: Loading efficiency (%) = (Amount of loaded PTX in weight) / (Amount of micelle in weight) x 100% Encapsulation efficiency (%) = (Amount of loaded PTX in weight) / (Amount of


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PTX added in weight) ×100% The concentrations of Coumarin-6 and DiR were analyzed by fluorescence spectrophotometer (Cary Eclipse, Agilent, Australia) at Ex/Em 465/502 nm and 748/780 nm, respectively. PTX release from various micelles were investigated in phosphate buffered saline (0.1 M, pH 7.4) containing 1% Tween-80. 2 mL of release medium was taken at different time points. The concentration of PTX was analyzed by HPLC. 2.8 In vitro penetration assay of AE micelles on BBTB/U87 tumor spheroids co-culture model U87 cells were seeded into 48-well plates (2000 cells per well) pre-coated by agarose and cultured for 10 days to form tumor spheroids. To assess the BBTB penetration efficiency of AE modified micelles, BBTB/U87 Tumor Spheroids co-culture model was established.43 HUVECs were seeded on the upper champers of transwell plates (2 × 104 cells per insert) and U87 cells were plated into the apical compartments (5 × 104 cells per well). Three days later, the U87 tumor spheroids were transferred to the basal compartments and culture medium was replaced by Coumarin-6 loaded micelles in DMEM. After 0.5 h incubation, the tumor spheroids were imaged by a confocal laser microscope to explore the transcytosis efficiency and penetrating ability of AE micelles. 2.9 In vivo biodistribution assay of DiR loaded micelles To investigate the in vivo tumor targeting efficiency of AE modified micelles, U87 tumor-bearing mice were injected intravenously with 100 µL DiR loaded micelles. The biodistribution of micelles were imaged by an in-vivo image system (IVIS Spectrum, Caliper, USA) at defined time points. Mice were put to death at 24 h post injection, organs and tumors were dissected for ex vivo fluorescence intensity analysis. 2.10 Immunofluorescence analysis The subcutaneous U87 tumor-bearing mice model was established as abovementioned. Mice were injected with coumarin-6 loaded micelles and sacrificed 4 h post-injection. To prepare tumor slices, tumors were fixed overnight in 4% paraformaldehyde, dehydrated and frozen-sectioned into 10 µm slices. For labeling microvessels and tumor stem cells, slices were incubated with anti-CD31 antibody and anti-CD133 antibody, respectively. EGFR was stained with anti-EGFR antibody and EGFRvIII was indicated by anti-EGFRvIII antibody. All slices were further stained with DAPI for 10 min and subjected to the confocal microscope.



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In order to validate the excellent tumor-selectivity of AE modified micelles, organs of healthy mice were collected 4 h post-injection with coumarin-6 loaded micelles. 2.11 Cytotoxicity assay HUVECs and U87 cells were seeded into 96-well culture plates (3 × 103 cells / 200 µL per well) and cultured at 37°C for 24 h. Then cells medium were substituted with different concentrations of PTX loaded micelles. After 72 h incubation, the cell viability percentage was determined using MTT assay (PowerWave XS, Bio-TEK, USA) based on the absorbance at 490 nm, normalized by untreated cells group. 2.12 In vitro inhibition assay of VM and HUVECs Tube Formation HUVECs and U87 cells were seeded onto Matrigel-coated 24-well plates and treated with 100 ng PTX loaded micelles.44 PTX-free wells were served as control group. After overnight incubation, tube formation and structures can be visualized by an inverted phase contrast microscope (DMI4000 B, Leica, Germany) and counted by Image J 1.46 version program. 2.13 Pharmacokinetics Study SD rats of 200 g were randomly divided into 5 groups (n = 5) and received different PTX formulations with single dosage of 10 mg/kg via tail vein injection. At defined time points, 0.5 mL blood samples were collected and centrifuged to get serum in upper layer. Then 50 µL of diazepam solution (internal standard solution) and 3mL ether were added, following by centrifugation at 4000 rpm for 10 min to precipitate the proteins. The supernatants were evaporated by nitrogen gas. After residues were reconstituted in 100 µL acetonitrile and centrifuged at 10000 rpm for 10 min. The concentration of PTX was determined by HPLC and pharmacokinetic parameters were calculated with the software DAS 2.0. 2.14 In vivo anti-tumor study BBTB will be formed between tumor tissue and microvessels with the deterioration of glioma, which closely resemble BTB of peripheral tumors and prevents efficient passage of cancer therapeutics. Thus subcutaneous U87 tumor-bearing mice model were constructed to evaluate the anti-tumor efficacy of AE micelles via the BBTB/glioma targeting pathway. When the tumor volume reached around 50 mm3, mice were randomly divided into 5 groups (n = 8) and treated with Saline, Taxol, Micelle/PTX, L-AE-Micelle/PTX, D-AE-Micelle/PTX every other day at a dose of 5 mg/kg (to the body weight, the total dose is 25 mg/kg). Tumor volumes and body weights were monitored every 2 days


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after first injection. The tumor volume was calculated as follow: Tumor volume = 0.5 x (length × weith2). On the 14th day, mice were sacrificed and harvested tumors were weighed. TUNEL assay and CD31/PAS dual staining were performed according to the manufacture’s protocol. TUNEL-positive cells were counted to evaluate the cell apoptosis, CD31-positive cells were calculated to assess anti-angiogenesis efficiency in vivo of different PTX formulations. 2.15 Statistical Analysis All data are represented as means ± SD, difference between two groups was analyzed with student’s t-test. Comparisons among multi-groups were assessed by one-way ANOVA analysis (*p < 0.05, **p < 0.01, ***p < 0.001).

3. RESULTS All D-peptide strategy by replacing the parent L-sequence with D-amino acids, has been approved as a useful method to overcome the enzymatic barrier.36-39, 43, 45 In this work, we synthesized RI-AE and D-AE, aiming at improving the metabolic stability of L-AE peptide as well as preserving its targeting efficacy. RI-AE represents the retro-inverso isomer of L-AE, and D-AE represents the enantiomer of L-AE. 3.1. Characteristics of Peptides and Derivatives We successfully synthesized AE peptides (L-AE, D-AE, RI-AE), fluorescein labeled peptides (L-AE-Fluorescein, D-AE-Fluorescein, RI-AE-Fluorescein) and Cy7 labeled peptides (L-AE-Cy7, D-AE-Cy7, RI-AE-Cy7). All products were characterized by HPLC and electrospray ionization mass spectrometry (ESI-MS) (Figure S1). In peptide stability assay, L-AE with natural L-amino acids, displayed quick degradation and almost disappeared after 4 h incubation with 50% rat serum. As expected, both D-AE and RI-AE demonstrated no observable degradation with 98% presence even after 8 h. The result verified the robust proteolytic stability of D-peptides in vivo (Figure 2).



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Figure 2. Stability of AE peptides in 50% rat serum. L-AE, RI-AE and D-AE were

incubated with 50% rat serum diluted with phosphate buffered saline at 37°C for different time periods. The amount of non-metabolized peptide at each time point was determined by HPLC. Data represent Mean ± SD (n = 3).

3.2. In vitro and in vivo tumor targeting ability of AE peptides To explore whether D-peptides would possess similar biological activities as the parent peptide, cellular uptake assay in vitro was conducted (Figure 3). Both L-AE and D-AE could be effectively internalized into U87 cells and HUVECs. No perceptible fluorescence was observed from cells treated with RI-AE or free FAM. This result was further quantified by flow cytometry analysis. After 4 h incubation, the proportion of positive U87 cells was 90.4% for L-AE-Fluorescein and 89.0% for D-AE-Fluorescein, remarkably in contrast to 0.3% for FAM and 3.4% for RI-AE-Fluorescein. Accordingly, the percentage of fluorescein positive HUVEC cells after treated with L-AE and D-AE was 98.31% and 94.23%, indicating similar targeting efficacy in vitro. In addition, negligible uptake in HL7702 cells underlined the superior specificity and targeting ability of AE peptides to tumor cells and neovasculature (Figure S2). The results of in vivo biodistribution further validated the superior targeting ability of D-AE. Fluorescent living images of 2 h post-injection showed the highest intratumoral fluorescence intensity in D-AE group (Figure 4A-B). In contrast, RI-AE rarely distributed in tumor regions, with no significant difference from that of free Cy7 group. Semi-quantitative analysis of ex vivo organ images showed that D-AE led to noticeably higher fluorescence accumulation in tumor compared with L-AE, which could be attributed to the prolonged circulation time of full protease-resistant


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D-peptides (Figure 4C-D).

Figure 3. Cellular targeting ability of AE peptides in vitro. U87 cells (A and C) and HUVEC

cells (B and D) were incubated with fluorescence labelled peptides for 4 h at 37 °C. Quantitive cellular uptake were measured by flow cytometer. Scale bar represents 10 µm.



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Figure 4. Targeting ability of AE peptides in subcutaneous U87 tumors. (A) In vivo

fluorescence imaging of subcutaneous U87 xenograft bearing mice 2 h after intravenously injected with 100 µL Cy7 labelled AE peptides. (B) Semi-quantitative analysis of the fluorescence intensity. (C) Ex vivo near-infrared imaging of dissected organs 2 h post-injection. (D) Semi-quantitative analysis of the fluorescence intensity in different organs and tumors. (Mean ± SD, n = 3). **p < 0.01, ***p < 0.001.

3.3 Characterization of functional materials and micelles L-AE-PEG3000-PLA2000, D-AE-PEG3000-PLA2000 and RI-AE-PEG3000-PLA2000 were synthesized via sulfhydryl-maleimide coupling method.46 In the 1H-NMR spectrum (Figure S3), the characteristic peak of maleimide group at 6.7 ppm disappeared in the spectra of AE-PEG3000-PLA2000, indicating successful conjugation between mal groups and thiol groups of AE peptides. Micelles were prepared by the thin-film hydration method and characterized by dynamic light scattering (Figure S4 and Table S1). The average vesicle sizes were around 30 nm with narrow distributions (PDI < 0.1), which are favorable for extravasation into tumors via EPR effect.47 Both plain micelles and AE micelles exhibit similar sustained release profile of PTX in vitro as shown in Figure S5, suggesting that modification of targeting ligands did not influence the physical properties of micelles. 3.4 Targeting ability of AE micelles on BBTB and U87 tumor spheroids in vitro


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3D

tumor

spheroids

in

vitro

model

is

recognized

microenvironmental heterogeneity close to that of tumor in vivo.

for 48, 49

recapitulating

To evaluate the

transcytosis efficiency of different micelles, the HUVECs/U87 tumor spheroids co-culture model was established to imitate the blood-brain tumor barrier in vitro.43 As shown in Figure 5B-C, only D-AE and L-AE functionalized micelles favourably traversed BBTB and permeated to the core of spheroids with significantly enhanced distribution within tumor spheroids. Pre-incubation in 50% rat serum considerably impaired the penetration ability of L-AE-Micelle. Comparatively, D-AE-Micelle displayed unchanged transcytosis efficiency owing to its robust proteolytic stability. Neither RI-AE-Micelle nor plain micelles penetrated beyond the outer periphery of tumor spheroids, showing inferior tumor targeting ability. These results demonstrated that D-AE peptide modification could facilitate micelles traverse BBTB and achieve efficient drug-tumor penetration.

Figure 5. Transcytosis of AE modified micelles in BBTB/U87 model in vitro (A).

Penetration of different Coumarin-6 loaded micelles in U87 tumor spheroids with or without pre-incubation with rat serum in the BBTB/U87 tumor spheroid co-culture model (B-C). Tumor spheroid penetration was examined by a confocal microscope with a 5 µm interval between consecutive slides.

3.5 In vivo targeting ability of AE micelles The biodistribution of various DiR loaded micelles was monitored in subcutaneous U87 tumor-bearing nude mice (Figure 6A). D-AE-Micelle/DiR specifically localized in glioma region with highest accumulation at all predetermined time points. The ex vivo organs imaging and quantitative ROI analysis consistently confirmed that D-AE functionalized micelles display stronger fluorescence intensity within tumor in contrast to other groups (Figure 6B-C). The immunofluorescence images of tumor slides could illustrate the targeting mechanism of micelles (Figure 7). D-AE-Micelle and L-AE-Micelle showed targeting capability to EGFR and/or EGFRvIII overexpressed cells, evidenced by largely



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co-localized with EGFR and EGFRvIII. In healthy mice, the accumulation of AE modified micelles in normal organs had no specificity (Figure S6). Co-localization with the tumor blood vessel marker CD31 illustrated the targeting ability to tumor neovasculature. CD133 serves as a marker for tumor stem cells (CSCs) harboring tumorigenesis and chemtherapy resisitance. The result shown in Figure 7C suggested that L-AE and D-AE could effectively drive micelles target tumor stem cells via EGFRvIII-mediated pathway. Undetectable fluorescence in RI-AE-Micelles group suggested its rare accumulation in tumor regions. The statistical analyses of co-localization were shown in Figure S7.

Figure 6. Targeting ability of AE modified micelles in subcutaneous U87 tumors. (A) In vivo fluorescence images of subcutaneous U87 xenograft bearing mices at different time

points. (B) Ex vivo near-infrared imaging of organs and tumors 24 h post-injection. (C) Semi-quantitative ROI analysis of the mean fluorescence intensity from the DiR loaded micelles in tumor and organs 24 h post-injection. (Mean ± SD, n = 3). **p < 0.01, ***p < 0.001.


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Figure 7. Distribution of AE modified micelles in tumor tissue of subcutaneous xenografts

U87 tumor-bearing mice 4 h after i.v. administration. EGFR was stained with anti-EGFR (A); EGFRvIII was stained with anti-EGFRvIII (B); CD133 antigen was labeled with anti-CD133 (C); Blood vessels were labeled with anti-CD31 (D). Blue: nuclei stained with DAPI. Green: the Coumarin-6 loaded micelles.

3.6 Targeting specificity of AE peptides There has been consensus that retro-inverso isomer was supposed to have similar bioactivities as the parent L-peptide.50-52 On the basis of such unexpected binding of D-AE, competitive inhibition assay was conducted on U87 cells to investigate the binding site(s) of RI-AE and D-AE (Figure 8A). After pre-incubation with high concentration of L-AE for 2 h, D-AE group showed remarkable decrease in cellular uptake, and also exhibited strong inhibitory effect on the internalization of L-AE. The result validated that efficient cellular internalization of D-AE and L-AE was mediated by the same binding sites (EGFR and EGFRvIII). Noteworthy, both cellular uptake of L-AE and D-AE were unaltered with the pre-treatment of RI-AE, indicating little affinity with targeting receptors of RI-AE. Molecular docking study was performed with Schrodinger software package to explore the binding mode between peptide and receptor. As shown in Figure 8C-D, three peptides bound to the domain 3 of extracellular motif of EGFR, which is a



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common motif in EGFR and EGFRvIII. The result of computational binding affinity evidenced that L-AE and D-AE possess considerable affinity to the domain 3. The binding affinities of L-AE and D-AE is about 50-folds and 100-folds higher than that of RI-AE, respectively, which explains the inefficiency of RI-AE. In the binding mode of L-AE (Figure 8C), hydrophobic interaction is generated between Phe1 and Leu325. Leu3 of L-AE stretched into the hydrophobic pocket formed by Thr357, Thr360 and Thr330 of the domain 3. Meanwhile, Glu5 interacts with Lys322 via ion-interaction . As for D-AE (Figure 8E), cation-π interaction is formed between DPhe1 and Lys322. The amino group in N-terminal generates ionic interaction with Asp323. Moreover, hydrophobic interactions are formed between DLeu3 and Leu325, and also between D

Ala6 and Leu348. Additionally, the hydrogen bond formed between

D

Glu5 and

His409. The various stable interactions that D-AE and L-AE have to bind with domain 3 ensure the high affinity. Conversely, effective interaction is hard to be developed between RI-AE and receptors. As shown in Figure 8D, the interactions contribute to the affinity are only the hydrophobic interaction between DLeu4 and Leu325, the hydrogen bond between DGlu2 and Asn328 as well as the interaction between DPhe6, Thr406 and the backbone of Lys407. These results proved that D-AE and L-AE with similar binding affinity to EGFR and EGFRvIII could mediate the nanocarriers to improve the anti-tumor efficacy.

Figure 8. Binding mode between AE peptides and receptors. (A) Cellular uptake of

fluorescein labelled peptides with or without treatment with three corresponding peptides. (Mean ± SD, n = 3). (B) Experimental determined and predicted free energies of peptides


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binding with domain 3 of extracellular motif of EGFR (∆G=1.3636logKi). Interaction of L-AE peptide (green, C), RI-AE (blue, D), D-AE (yellow, E) with receptors. Domain 3 of extracellular domain of EGFR were represented by cartoon. The interacting residues in binding pocket and the peptides were shown in stick.

3.7 Cytotoxicity of AE micelles in vitro In vitro cytotoxicity of various PTX formulations on U87 cells and HUVECs were examined by MTT assay. The functional materials exhibited no cytotoxicity at all tested concentrations to both cell lines (Figure 9A-B). D-AE-Micelle/PTX showed the lowest IC50 values of 0.02 µM on U87 cells, in comparison to those of Taxol (0.31 µM), Micelle/PTX (0.21 µM) and L-AE-Micelle/PTX (0.05 µM). The inhibitory effect on HUVEC cells was in line with U87 cells, registering the IC50 values of 0.85 µM (Taxol), 0.7 µM (Micelle/PTX), 0.21 µM (L-AE-Micelle/PTX) and 0.06 µM (D-AE-Micelle/PTX), respectively. The cytotoxicity results confirmed that D-AE and L-AE modification facilitated cellular internalization of micelles and enhanced the anti-proliferation effect of chemotherapeutic drugs subsequently.

Figure 9. Cytotoxicity of PTX loaded micelles in vitro. Cytotoxic effect of micelle functional

materials on U87 cells (A) and HUVEC cells (B), respectively. Cytotoxicity of various PTX loaded micelles on U87 cells (C) and HUVEC cells (D). All cells were conducted with 72 h incubation and examined by MTT assay (mean ± SD, n = 3).

3.8 Inhibition efficacy on U87 VM channel and HUVEC tube in vitro



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Angiogenesis is indispensable during glioma progression and metastases. Notably, highly aggressive tumor cells would mimic endothelial cells and form patterned vascular channels for further tumor perfusion. Hence, the destroying ability of different PTX loaded micelles on the formation of U87 VM channel and HUVEC tube in vitro was also evaluated in vitro. As shown in Figure 10, D-AE-Micelle/PTX displayed exceptional inhibitory effect with the lowest degree of HUVEC tubes formation, presenting an attractive antiangiogenic activity. The similar trend was observed in U87 VM channel formation, D-AE-Micelle/PTX group displayed sporadically distribution on matrigel, in contrast to tube-like structure in other groups. Quantitative

analysis

further

verified

the

highest

inhibitory

activity

of

D-AE-Micelle/PTX.

Figure 10. Inhibitory efficacy of PTX loaded micelles on the tube formation in vitro. (A)

Destroying ability on HUVEC tube and U87 VM channel after overnight incubation with different PTX loaded micelles, respectively. (B-C) Quantification of HUVEC tube inhibition and U87 VM inhibition with PTX loaded micelles. Data are presented as the percentages of the control group, which was set at 100%. **p < 0.01, ***p < 0.001.

3.9 Pharmacokinetic characteristics of AE micelles Pharmacokinetic studies of AE micelles were performed in SD rats. The blood clearance curves of PTX formulations were shown in Figure S8. Taxol was


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immediately eliminated from circulation system and almost undetectable at 4 h post injection. While all micelle formulations showed extended circulation in vivo. Additionally, the blood concentration-time profiles and pharmacokinetic parameters of AE micelles were similar to that of unmodified micelles, indicating that AE ligands barely alter circulation properties of drug delivery systems in vivo. 3.10 In vivo anti-tumor efficacy of AE micelles In vivo anti-tumor efficacy of AE micelles was assessed on nude mice bearing the subcutaneous U87 tumors. The tumor volume change curves (Figure 11A) revealed that AE modified micelles hold distinct advantage over Taxol and unmodified micelle in inhibition of tumor growth. Noteworthy, D-AE-Micelle/PTX achieved outstanding tumor-suppression effect with tumor size remained around 100 mm3. The excised tumor image of each group validated that D-AE targeting ligand remarkably improve the therapeutic efficacy with tumor inhibitory rate up to 89%, which can be ascribed to selective glioma targeting and enhanced accumulation within tumors (Figure 11C-D). Moreover, the body weight of mice in each group showed no obvious difference during experiment period (Figure 11B). TUNEL and CD31/PAS immunohistochemical staining analysis could better reveal the intratumoral effect of PTX formulations in vivo. Negligible cell apoptosis was

detected

in

saline

and

Taxol

groups

(Figure

12A-B).

In

contrast,

D-AE-Micelle/PTX induced the highest degree of TUNEL-positive cells, suggesting its remarkable cell-apoptosis ability. CD31/PAS dual staining was adopted to assess the formation of angiogenesis and VM channels. D-AE-Micelle/PTX exerted the strongest inhibition towards angiogenesis (angiogenesis inhibition rate of was up to 94 %), and less VM structures (CD31-negative and PAS-positive) were observed than that in other groups (Figure 12C-D). These results emphasized the advantage of stabilized peptide ligand (D-AE) in achieving the anti-tumor efficacy of PTX loaded micelles.



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Figure 11. Anti-tumor effects of PTX loaded micelles in vivo. (A) Growth curves of

subcutaneous U87 tumor in nude mice administrated i.v. with various PTX formulations on the day 0, 3, 6, 9, and 12, the total dose of PTX is 25mg/kg. (B) Changes in body weights. Relative body weight was calculated as the ratio of body weight at each time point to the initial body weight. (C) Photographs of tumors at the end of treatments. (D) Tumor inhibitory rate of various PTX formulations at the end of treatment (day 14). Tumor inhibitory rate was calculated as the ratio of tumor weight to the saline group. Data represent mean ± SD, n = 8. (**p < 0.01, ***p < 0.001).


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Figure 12. Immunohistochemical analysis of the subcutaneous U87 tumor after treatment

with various PTX loaded formulations. (A) TUNEL immunohistochemical staining images of tumor slices. (B) Quantitive analysis of the percentage of TUNEL positive cells. (C) CD31/PAS dual staining images of tumor slices. (D) Quantification of angiogenesis inhibition in tumors. Scale bar represents 100 µm. Three random fields were selected under a light microscope. Data are represent mean ± SD, n = 3. *p < 0.05, **p < 0.01.

4. DISCUSSION EGFR amplification is the potent driver of glioma development and defines the classical glioma subtype.53 The activation of corresponding intracellular downstream signaling pathways give rise to cell proliferation, migration and angiogenesis. In the past decade, a variety of EGFR-targeted drug delivery systems have been utilized to improve therapeutic outcomes.54-57 Nevertheless, these approaches only delivered modest results, for the oncogenic mutation EGFRvIII was often neglected.58, 59 Being one of the few tumor-specific antigens, EGFRvIII correlates with enhanced tumorigenicity, cell invasion as well as resistance to apoptosis. Therefore aberrant expression of EGFR and EGFRvIII collectively contribute to the dismal prognosis of GBM patients.60 And we believed that co-targeting these two receptors is of great importance for glioma treatment.



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In this study, L-AE peptide, previously validated as the PET tracer for glioma imaging, is able to effectively bind to EGFR and EGFRvIII overexpressed on VM, neovasculature, glioma cells and CSCs.32,34,28-30 In terms of cancer therapy as a targeting ligand, the property of susceptible to proteolytic degradation in vivo would undoubtedly attenuate its targeting capability. All D-peptide strategy has been recognized as an efficient way to improve stability and targeting efficiency by converting linear L-amino peptide into D configuration.35-38, 45-46 Accordingly, two D peptides with excellent anti-proteolysis ability were synthesized. In comparison to the retro-inverso

isomer

RI-AE,

the

enantiomer

D-AE

exhibited

appreciable

glioma-targeting ability with resemblance to L-AE. Furthermore, D-AE modification endowed micelles prolonged circulation and enhanced intratumoral accumulation than L-AE in subcutaneous U87 tumor-bearing mice model. Neither RI-AE nor its modified micelles displayed targeting capability in all experiments. Thus competitive inhibition assay and molecular docking study were conducted to illustrate the targeting specificity and binding mode of AE peptides, D-AE and L-AE hold considerably high binding affinity to EGFR and EGFRvIII and therefore display selective tumor targeting ability. Due to the weak and limited interaction with these two receptors, RI-AE was unable to perform any tumor-targeting behaviors. In order to achieve satisfactory therapeutic outcome, active targeted drug delivery systems were required to bypass various physiological and pathological barriers and deliver adequate therapeutic agent into deeper tumor sites.11-15,61, 62 The BBTB/U87 co-culture model was constructed to mimic the in vivo status. Plain micelle and L-AE-Micelle displayed limited transcytosis efficacy due to the existence of BBTB or enzyme barrier. As expected, D-AE ligand effectively mediated micelles overcome the BBTB followed by penetrating to deeper tumor region, validating its superiority in BBTB/glioma targeting. Additionally, the results of immunofluorescence analysis demonstrated neovasculature and tumor stem

cells

targeting potential of

D-AE-Micelle in vivo, evidenced by co-localization with tumor stem cells marker CD133 and blood vessel marker CD 31. When loaded with PTX, D-AE-Micelle exerted the strongest inhibition on U87 cells, HUVECs and VM tube model in vitro. By specifically targeting EGFR and EGFRvIII overexpressed tumor cells, tumor angiogenesis and CSCs, D-AE functionalized micelle exhibited superior anti-tumor efficacy in vivo.


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5. CONCLUSION In the present study, we developed a novel and promising drug delivery system for glioma therapy. L-AE peptide with high binding affinity to EGFR and EGFRvIII was adopted as targeting ligand, in order to maintain its targeting efficacy in vivo, stabilized D-AE (DFDADLDGDEDA) peptide was designed via all D-peptide strategy. Cellular internalization and biodistribution assays confirmed its multifunctional targeting capability. The modification of D-AE ligand endowed micelles excellent BBTB/glioma targeting efficacy. By precisely targeting neovasculature, vasculogenic mimicry (VM), glioma cells and glioma stem cells via EGFR- and EGFRvIII-mediated pathways, D-AE-Micelle accomplished the best anti-angiogenesis and anti-tumor effects both in vitro and in vivo. Our results suggested that the multifunctional drug delivery system enabled by D-AE remarkably enhanced tumor specificity and therapuetic effectiveness and therefore may provide a feasible alternative approach for tumor therapy.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. HPLC and ESI-MS spectrum of AE peptides and Derivatives; cellular uptake of fluorescein labeled AE peptides by HL7702 cells; the Mal-PEG3000-PLA2000,

L-AE-PEG3000-PLA2000,

1

H-NMR spectra of

RI-AE-PEG3000-PLA2000

and

D-AE-PEG3000-PLA2000; size distributions of different paclitaxel loaded micelles measured by dynamic light scattering; characterization of different paclitaxel loaded micelles; in vitro PTX release profile of different micelles; distribution of Coumarin-6 loaded micelles in healthy mice; statistical analysis of different micelle formulations co-localized

with

EGFR,

EGFRvIII,

CD31

and

CD133;

plasma

PTX

concentration−time profiles of different micelles.

AUTHOR INFORMATION Corresponding author *E-mail: [email protected] (W. Lu). Tel.: +86 21 5198 0006; fax: +86 21 5198 0090. Notes



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The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program, No.2013CB932500), the National Natural Science Foundation of China (No.81690263 & No.81473149), Shanghai international science and technology cooperation project (No.16430723800), and Development Project of Shanghai Peak Disciplines-Integrative Medicine (No.20150407)

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Graphical Abstract 600x400mm (300 x 300 DPI)


EGFRvIII Dual-Targeting Peptide Mediated ... - ACS Publications

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