Coupling by Amide Bond Inspires the Brain Metastatic Tumor T

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Structure Reconstruction of LyP-1: c(LyP-1) coupling by amide bond Inspires the Brain Metastatic Tumor Targeted Drug Delivery Xiaoyu Zhang, Fei Wang, Qing Shen, Cao Xie, Yu Liu, Jun Pan, and Weiyue Lu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00801 • Publication Date (Web): 07 Dec 2017 Downloaded from http://pubs.acs.org on December 12, 2017

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Molecular Pharmaceutics

Structure Reconstruction of LyP-1: Lc(LyP-1) coupling by amide bond Inspires the Brain Metastatic Tumor Targeted Drug Delivery Xiaoyu Zhang a, Fei Wang a, Qing Shen a, Cao Xie a, Yu Liu a, Jun Pan a, Weiyue Lu a,b, *

a

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

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

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

System, Minghang Hospital, Fudan University, Shanghai 201199 & Institutes of Integrative Medicine of Fudan University, Shanghai 200040, China

ABSTRACT. The stability and binding affinity of targeting ligands are very important in active targeting drug delivery. Herein we used LyP-1 peptide as a model peptide to investigate chemical-biology-based strategies in the design of peptide ligands for active targeting. LyP-1 is a short peptide cyclized with a disulfide bond. It can specifically bind to tumor cells and tumor lymphatics through the interaction with cell-surface protein p32/gC1qR. Lc(LyP-1), with a same sequence of LyP-1, is coupled by amide bond. It showed better cellular uptake and stability in blood in our previous research. Further, usually D-peptide demonstrates higher stability than L-peptide, and it may contribute to better active targeting ability in vivo. Herein, we designed a retro-inverso isomer of Lc(LyP-1), termed Dc(LyP-1), expecting to inspire brain metastatic tumor targeted drug delivery. However, although Lc(LyP-1) showed lower stability than Dc(LyP-1) in fresh rat bold serum, both the 4T1 cellular uptake capacity (89.20%) and p32 protein binding affinity (7.39×10-6) were significantly

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higher than those (33.41%, 1.37×10-5) of Dc(LyP-1). Further, Lc(LyP-1) modified PEG-PLA micelles displayed much higher in vivo distribution in brain metastatic tumor than Dc(LyP-1). All results suggested that Lc(LyP-1) had a better performance than Dc(LyP-1) in brain metastatic tumor-targeted drug delivery.

KEYWORDS: Lc(LyP-1), retro-inverso isomer Dc(LyP-1), PEG-PLA micelles, targeting drug delivery, brain metastatic tumor

1. INTRODUCTION Brain metastasis from breast cancer is a leading cause of death for women. Triple-negative breast cancer (TNBC) represents 10-20% of all mammary tumors.1,2 Patients with TNBC are at increased risk of brain metastases (BMs).3 60% of TNBC will spread to the brain. Unfortunately, TNBC has no effective targeted treatment choices due to its distinct biological property,4 as they are lack of expression of Estrogen Receptor (ER), Progesterone Receptor (PR) and Human Epidermal Growth Factor Receptor-2 (HER-2).5,6 Meanwhile, considering the existence of the blood brain barrier (BBB) at the early stage of brain metastatic tumor7,8,9 and the expression of p32 protein on most tumor cells surface,10-12 both the BBB and p32 protein can be used as the dual targets of brain metastatic tumor. D

CDX is a D-peptide composed of 16 amino acids, which can specifically bind to

the nicotinic acetylcholine receptor (nAChRs) that is highly expressed on the surface of BBB.13 When modified on the surface of nanomedicine, DCDX can greatly increase the brain distribution of the drug delivery system.13 As a result, DCDX can facilitate


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the

brain-targeted

drug

delivery

through

nAChRS-mediated

transcytosis.

LyP-1(CGNKRTRGC) coupled by a disulfide bond is a cyclic peptide composed of 9 amino acids14,15, which can significantly bind to the cell-surface protein p32/gC1qR of the tumor, and then target to the tumor cells.10,14,16 With both DCDX and LyP-1 modified on the surface of drug delivery system, dual targeting might be realized: crossing BBB and targeting tumor cells. Therefore, the dual-targeted drug delivery system is a promising choice for therapy of brain metastatic tumor from breast cancer. However, the oxidoreductase, aminopeptidase and carboxypeptidase in the blood may degrade LyP-1, followed by reduction of targeting efficiency.17,18 In order to overcome this drawback, we designed a different conformation of LyP-1, here termed as Lc(LyP-1) (AGNKRTRGC) with a same sequence to LyP-1 except for one different amino acid (Ala) which is used for the amino coupling. Our previous study found that L

c(LyP-1) shows better tumor cellular uptake and serum stability than LyP-1.

However, if the transcytosis of Lc(LyP-1) involves enzymatic organelles, degradation of peptide may still lead to deactivation. In this study, we designed a stable D-peptide isomer of Lc(LyP-1) by retro-inverso isomerization, termed as Dc(LyP-1).19 The tumor targeting efficiency of Dc(LyP-1) at the molecular level was evaluated. DCDX and Dc(LyP-1) peptide modified micelles were constructed to evaluate the potential of D-peptide for inspiring brain metastatic tumor targeted drug delivery.

2. EXPERIMENTAL SECTION



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2.1 Materials. mPEG2000-PLA2000 block copolymer and mal-PEG3400-PLA2000 were purchased from Advanced Polymer Materials Inc.(Canada). Coumarin-6 was from Sigma (St. Louis, MO). Fluorescein-5-maleimide was acquired from Fanbo Biochemicals

(Beijing,

China).

(1,1’-dioctadecyl-3,3,3’,3’-tetramethyl

Near

infrared

indotricarbocyanine

iodide)

dye was

DiR from

Invitrogen (Grand Island, NY). 4’,6-Diamidino-2-phenylindole (DAPI) was purchased from Roche (Switzerland). PTX was given by Shanghai Institute of Pharmaceutical Industry. 4T1 cells were obtained from ATCC (Manassas, VA) and maintained in 1640 medium (Roswell Park Memorial Institute) together with 10% FBS (Gibco), 100U/mL penicillin, and 100µg/mL streptomycin at 37 ℃ under a humidified atmosphere containing 5% CO2. BALB/c female nude mice of 4-6 weeks age were purchased from Shanghai Laboratory Animal Co. LTD (Shanghai, China) and kept under SPF conditions. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Fudan University. 2.2 Peptide Synthesis. Lc(LyP-1) and Dc(LyP-1) peptides were synthesized by solid phase peptide synthesis using active ester chemistry to couple Boc-protected amino acid to the deprotected resin.13 The Boc protecting group was removed by 100% trifluoroactic acid (TFA). Dimethylformamide (DMF) was used as both the flow wash and coupling solvent throughout the cycling. The progress of the assembly was detected by ninhydrin monitoring. The crude peptides cleaved from resin with HF were purified to homogeneity and ascertained by HPLC and ESI-MS.


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Both

L

c(LyP-1)

D

and

c(LyP-1)

were

labeled

by

fluorescein

via

sulfhydryl-maleimide conjugation. Briefly, 2mg fluorescein-5-maleimide in DMF was gently added into 8mg peptide dissolved in PBS (0.1M, pH=7.0). After gentle vortex and one hour incubation in the dark at room temperature, the pure target product was obtained by preparative C18 reversed-phase HPLC. 2.3 Peptide Stability Assay in Blood. 120µL of sulfhydryl groups blocked LyP-1 or Lc(LyP-1) or Dc(LyP-1) (1mg/ml) was separately mixed with 1.08 mL of 25% (V/V) fresh rat serum, and then the mixture was incubated at 37 ℃ . 20µL of 15% trichloroacetic aid (TCA) was added into 100µL reaction mixture at 0, 10, 30, 60, 120, 240, 360, 480, 720 min respectively, followed by storage at 4℃ for 20min and centrifugation at 12000rpm for 10min. 20µL of supernatant was analyzed by HPLC to monitor and quantify peptide hydrolysis. 2.4 Computer-aided simulation. The structures of

L

c(LyP-1) and

D

c(LyP-1)

created by SYBYL 6.9 program (Tripos Inc., St.Louis, MO) were subjected to energy refinement in SYBYL 6.9 with the same parameters as that in our previous study. 20 The docking program Hex v6.1 was used for the preliminary protein-protein docking,21 and the flexible peptide docking package in RosettaDock program was further used to probe the possible minimum-energy binding mode on p32 protein.22 Binding affinities were predicted by X-Score 1.2 with empirical scoring function.23 The predicted affinities and all of available experimental data were used for the selection of the final complex. 2.5 Cellular Uptake Assay. 4T1 cells were seeded onto confocal dishes at a



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density of 105 cells/well. After 24h incubation, 5-fluorescein labeled Lc(LyP-1) and D

c(LyP-1) were added in 1640 culture medium without 10% FBS separately, and then

the cells were incubated at 37℃ for 4h. After staining of nuclei by DAPI and rinsing with PBS for three times, the fluorescent intensity was captured by laser scanning confocal microscope and flow cytometry. To investigate intracellular distribution, cells were fixed by formaldehyde and the immunofluorescence staining was carried out before imaging capture. 2.6 Preparation and Characterization of Peptide-conjugated Micelles 2.6.1

Synthesis

of

Function

Materials.

The

functional

material,

L

c(LyP-1)-PEG3400-PLA2000 or Dc(LyP-1)-PEG3400-PLA2000 or DCDX-PEG3400-PLA2000,

was synthesized through the covalent conjugation of Lc(LyP-1) or Dc(LyP-1) or DCDX with mal-PEG3400-PLA2000 by sulfhydryl-maleimide coupling.22 Briefly, 12mg Mal-PEG2000-PLA3400 dissolved in 0.5mL DMF was added into 8mg Lc(LyP-1) or D

c(LyP-1) or DCDX dissolved in 4mL PBS (pH=7.0, 10mM), and the reaction was

monitored by HPLC. The excessive peptide was removed by dialysis against distilled water (MWCO 3.5 kDa) and confirmed by HPLC. 2.6.2 Preparation of Micelles. No matter for the non-targeting, single-targeting or dual-targeting

micelles,

mPEG2000-PLA2000

mPEG2000-PLA2000/Lc(LyP-1)-

PEG3400-PLA2000

mPEG2000-PLA2000/DCDX-PEG3400-PLA2000

or

a

(97/3,

(97/3,

by by

mixture

of

mol)

or

mol)

or

mPEG2000-PLA2000/Dc(LyP-1)-PEG3400-PLA2000 (97/3, by mol) or mPEG2000-PLA2000/ L

c(LyP-1)- PEG3400-PLA2000/DCDX-PEG3400-PLA2000 (94/3/3, by mol) separately in


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acetonitrile

(free

of

D

CDX-PEG3400-PLA2000)

or

chloroform

(including

D

CDX-PEG3400-PLA2000) was rotary evaporated to form a thin film, which was

subsequently hydrated with normal saline (pH=7.4). For loading DiR or PTX, each of them was dissolved in acetonitrile before the preparation of thin film and the free dye or crude drug was removed by gel filtration over a Sephadex G-50 colume eluted with normal saline. 2.6.3 Characterization of Micelles. The particle size and size distribution of micelles were determined by dynamic light scattering (Nicomp 380ZLS Particle Sizer, PSS Corp., USA). The morphology of PTX-loaded micelles was observed using transmission electron microscopy (TEM) (H-7000, Hitachi, Japan). The micelle samples were negatively stained by 4% phosphotungstic acid and dried on carbon-coated grids before observation. 2.7 Evaluation of Brain-targeting Ability of DCDX. In order to evaluate the brain-targeting ability of DCDX, fluorescently labeled peptides were injected into blank nude mice through tail vein injection.13 Each group consisted of 3 mice. 12 hours after injection, mice were sacrificed and organs were harvested for imaging by a small animal imaging system (IVIS Spectrum, PerkinElmer, Waltham, MA). 2.8 Establishment of intracranial Metastatic Tumor from Breast Cancer Model. 4T1 cells (5×105cells suspended in 5µL PBS) were implanted the into the nude mice’s right brain (1.8mm lateral, 0.6mm anterior to the bregma with 3mm depth) with the help of a stereotactic apparatus. 2.9 Biodistribution of Micelles in Vivo. 7 days after establishment of the animal



model, DiR-loaded micelles were injected into model nude mice through tail vein injection. Each group consisted of 3 mice. 12 hours after injection, mice were sacrificed and organs were harvested for imaging by IVIS Spectrum. 2.10 Apoptosis Detection of Metastatic Tumor Tissue from Breast Cancer. 1 control group, 1 blank group and 3 groups of PTX loaded micelles were injected into model nude mice through tail vein jnjection in 7, 10, and 13 days after 4T1 cell implanted into the nude mice. After 18 days, 3 mice in each group were chosen to take their brain out. Tunel method was applied to detect the cell apoptosis.

3. RESULTS 3.1 Retro-Inverso Isomerization of Lc(LyP-1). Lc(LyP-1), which is composed of nine amino acids with amide coupling, can significantly bind to the p32 protein on the surface of tumor cells and then enhance the receptor-mediated transcytosis. Dc(LyP-1), the retro-inverso isomerization of Lc(LyP-1) composed of nine D-amino acids with a reverse sequence of Lc(LyP-1). The cysteine in the N-terminal of the sequence could be used to conjugate with the maleimide group on the functional material.16 Both L

c(LyP-1) and Dc(LyP-1) were synthesized by solid phase peptide synthesis with

Boc-protected amino acids. After detection by HPLC and ESI-MS, the purity and molecular weight of the two peptides were ascertained. 3.2 Stability of Peptides in Blood. Peptide-based ligands have attracted extensive attention for designing targeted drug delivery due to their high binding affinity and specificity to targets.19 However, the key barrier in targeting is the plasma which


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might inactivate LyP-1 peptide ligand and undermine the interaction with p32 protein on the 4T1 cells. Therefore, we investigated the stability of LyP-1 and Lc(LyP-1) and D

c(LyP-1) in the rat serum. All of the three peptides were incubated at 37℃ with

fresh rat serum. It was obvious that Dc(LyP-1) displayed nearly no degradation under the experiment condition (Figure 1). Meanwhile,

L

c(LyP-1) also demonstrated

considerable stability, though not as good as Dc(LyP-1). The control group LyP-1 showed relatively worst stability. There was more than 50% Lc(LyP-1) remaining in blood 400 min after injection. From this viewpoint, when conjugated with drug or modified on the surface of the drug delivery system, both Lc(LyP-1) and Dc(LyP-1) may not be degraded by plasma proteases quickly. They are both expected to keep the tumor targeting efficiency and hold the potential for extending tumor targeting efficiency due to their resistance to proteolysis, especially Dc(LyP-1).

Figure 1. Stability of LyP-1, Lc(LyP-1) and Dc(LyP-1) in rat serum. Fresh rat serum was diluted with phosphate buffered saline and incubated with LyP-1,

L

c(LyP-1) and

D

c(LyP-1) at a

concentration of 0.1mg/mL. After 0, 10, 30, 60, 120, 240, 360, 480 and 720 min incubation at



37℃, 20µL of 15% TCA was added into 100µL of the reaction mixture. The mixture was stored at 4℃ for 20 min and then centrifuged at 12000rpm for 10min. 20µL of supernatant was analyzed by HPLC to monitor and quantify peptide hydrolysis.

3.3 Computer-aided simulation. To deeply understand the potent action of L

c(LyP-1) (CGNKRTRGA) and Dc(LyP-1) (DADGDRDTDRDKDNDGDC) peptides, we

conducted molecular modeling and docking study on the interaction between p32 and these two peptides, in which the crystal structure of p32 (PDB code: 1P32) and the 3D structures of Lc(LyP-1) and

D

c(LyP-1) built by Sybyl 6.9 software (Tripos Inc.,

St.Louis, MO) were used for study (Figure 2). When Lc(LyP-1) or Dc(LyP-1) was superimposed, the backbone of the peptide adopted consisted conformation, while the side chains of the residues in the same sites were orientated in different areas. Not surprisingly, this difference would lead to variation in the binding affinity of our synthesized peptides on p32 protein. p32 protein is a negatively-charged protein with many acidic residues on its surface, which attracted the positively-charged cyclic peptides to bind into the binding pocket. The binding of p32 with Lc(LyP-1) and Dc(LyP-1) was dominated by hydrogen bonds and electrostatic interaction. By examing the interaction between p32 protein and L

c(LyP-1), it was found that Arg5 and Arg7 formed hydrogen-bond network with

Glu284 and Glu125 (Figure 3), perhaps greatly contributing to its binding affinity on p32 protein. By contrast, such electrostatic interaction could not be found in the binding mode of Dc(LyP-1) (Figure 3), perhaps contributable to lower binding affinity of Dc(LyP-1) to p32. To further evaluate the interaction between the peptides and p32,


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their binding affinities were predicted by X-Score, also in good agreement with the experimentally determined Ki (Table 1), validating the reliability of the design of these potent inhibitors of p32 protein.

Figure 2. 3D structures of Lc(LyP-1) (green) and Dc(LyP-1) (blue).

Figure 3. The binding modes of Lc(Lyp-1) and Dc(Lyp-1) on p32 protein.



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Table 1. Experimentally determined vs. predicted free energies of binding of all synthetic peptides with p32 (∆G=1.3636logKi).

Peptide

Experimental ∆G (kcal/mol)

Predicted ∆G (kcal/mol)

-6.99

-7.31

-6.93

-6.98

L

c(LyP-1)

D

c(LyP-1)

3.4 Cellular Uptake. For cellular uptake experiments, Lc(LyP-1)-fluorescein and D

c(LyP-1)-fluorescein were prepared by Michael addition between the thiol group in

L

c(LyP-1) or

D

c(LyP-1) and the maleimide group in fluorescein-5-maleimide. To

compare the capability of Lc(LyP-1) and Dc(LyP-1) of interacting with 4T1 cells, uptake of the two peptides by 4T1 cells was evaluated. 4T1 cells were incubated with 5µM of Lc(LyP-1)-fluorescein or Dc(LyP-1)-fluorescein for 4h at 37℃. After the incubation, confocal laser microscopy imaging and flow cytometry were conducted. As shown in Figure 4, Lc(LyP-1) demonstrated higher cellular uptake efficiency than D

c(LyP-1).

Flow

cytometry

(Figure

4)

showed

that 89.20% cells

were

fluorescence-positive after the treatment of Lc(LyP-1)-fluorescein, while that for D

c(LyP-1)-fluorescein was only 33.41%. p32 protein is overexpressed on the surface

of 4T1 cells. The effective uptake of Lc(LyP-1)-fluorescein can be explained by the specific interaction between Lc(LyP-1) and p32 protein on the membrane of 4T1 cells. Less uptake of Dc(LyP-1)-fluorescein may be related to its lower binding affinity with p32 protein.


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Figure 4. Cellular uptake of Lc(LyP-1) and Dc(LyP-1) by 4T1 cells. Cells were incubated with 5µM fluorescein labeled peptide at 37℃ for 4h, followed by DAPI staining and rinse with phosphate buffered saline. Intracellular fluorescein was detected by confocal laser scanning microscope (A) and flow cytometer (B). (Scale bar = 5µm.)

3.5 Brain Metastatic Tumor-targeting Ability of Micelles 3.5.1 Brain-targeting Ability. To study the brain-targeting ability of micelles, six types of micelles were prepared, including L

c(LyP-1)/DCDX or

D

CDX,

L

c(LyP-1),

D

c(LyP-1),

L

c(LyP-1)/DCDX-modified micelles and plain micelles to

compare the brain distribution. All micelles were loaded with DiR and separately intravenously administered to nude mice to harvest organs for ex vivo fluorescent imaging 12h after the injection (Figure 5A). it is obvious that

D

CDX-modified

micelles (both the single and dual targeting ones) showed significant brain distribution (Figure 5B). Performance of micelles without

D

CDX modification

showed no evident difference from the blank one. Therefore, DCDX is the key to realize brain targeting, and can make contribution together with c(LyP-1) to targeting the tumor in brain.



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Figure 5. Brain distribution of DiR loaded micelles in vivo. A) Ex vivo imaging of dissected tissues including brains of mice 1 h after injection. Brains from left to right are from mice administrated with control, plain micelles, Lc(LyP-1)-PEG-PLA micelles, Dc(LyP-1)-PEG-PLA micelles, D

D

CDX-PEG-PLA

micelles,

L

c(LyP-1)/DCDX-PEG-PLA

micelles

and

c(LyP-1)/DCDX-PEG-PLA micelles. B) Normalized fluorescence intensity of brain in each group.

Mean±SD, n=3.

3.5.2 Brain Metastatic Tumor-targeted Ability. Previous study have revealed that D

CDX had high transcytosis efficiency in an in vitro blood-brain barrier (BBB)

monolayer.13 After being modified on nanomedicine,

D

CDX could facilitate

significant brain-targeted delivery. Therefore, we established the dual targeting delivery system Lc(LyP-1)/DCDX-PEG-PLA micelle and Dc(LyP-1)/DCDX-PEG-PLA micelle, using the brain-targeted ability of DCDX and the tumor-targeted ability of


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L

c(LyP-1) or Dc(LyP-1) to realize targeting the brain metastatic tumor. 4T1 cells were

inoculated into the nude mice’s brain to establish the brain metastatic tumor model. In order to compare the brain metastatic tumor targeting efficiency of Lc(LyP-1) and D

c(LyP-1), we investigated the brain distribution of micelles by using the ex vivo

fluorescence imaging technique. Lc(LyP-1)-PEG-PLA micelles, Dc(LyP-1)-PEG-PLA micelles and

D

CDX-PEG-PLA micelles as well as mPEG-PLA micelles were

fluorescently labeled with a near-infrared dye (DiR). Grouped nude mouse were injected into 100µL fluorescein labeled micelles by tail vein injection. In vivo fluorescent images were obtained at 2, 4, 8 and 12h. 12 hours after injection, all brains and organs were harvested and subjected to ex vivo fluorescent imaging (Figure 6). Both

L

c(LyP-1)/DCDX and

D

c(LyP-1)/DCDX modified micelles improved the

distribution of dye in the tumor of the brain. Lc(LyP-1)/DCDX-PEG-PLA micelles demonstrated the highest brain metastatic tumor targeted capability. In other words, L

c(LyP-1) displayed worse blood stability, but it showed significantly higher cellular

uptake efficiency and molecular binding affinity. High brain metastatic tumor targeting capability of the dual-targeted micelles Lc(LyP-1)/DCDX-PEG-PLA may be attributable to Lc(LyP-1)’s excellent molecular binding efficiency and cellular uptake performance.



Figure 6. Biodistribution of micelles in brain metastatic tumor nude. mPEG-PLA, L

c(LyP-1)/DCDX-PEG-PLA, or

D

c(LyP-1)/DCDX-PEG-PLA micelles labeled with DiR were

separately (n=3) injected into the tail vein of nude mice bearing model brain metastatic tumors. After 12h, all mice were sacrificed and the brains was dissected for ex vivo florescence imaging. The brain distribution of Lc(LyP-1)/DCDX-PEG-PLA micelles was significantly higher than that of mPEG-PLA and Dc(LyP-1)/DCDX-PEG-PLA.

3.6 TUNEL detection of metastatic brain tumor tissue. The TUNEL staining images and semi quantitative result of 5 group brain tissues were obtained. (Figure 7). The area of the red arrow showed the normal brain tissue, and that of the black arrow showed the metastatic brain tumor tissue. The dark brown color showed where the apoptotic cells were. The result told that compared with control and taxol groups, all


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the left 3 groups of micelle loaded with PTX held significantly better efficacy. The efficacy to kill tumor cells of improved

40.18% and

L

c(LyP-1)/DCDX-PEG-PLA/PTX was distinctly

169.65% than

D

c(LyP-1)/DCDX-PEG-PLA/PTX

and

mPEG-PLA/PTX.

Figure 7. TUNEL staining imaging of metastatic brain tumor with saline, taxol, mPEG-PLA/PTX, D

c(LyP-1)/DCDX-PEG-PLA/PTX, and Lc(LyP-1)/DCDX-PEG-PLA/PTX. (n=3)

DISCUSSION LyP-1 has been shown to specially bind with tumor cells and tumor lymphatics in metastatic lymph nodes and tumor-associated macrophages. p32 protein is overexpressed on the surface of most tumor cells. LyP-1 is a peptide ligand of p32 protein and can promote receptor-mediated transcytosis. However, the fresh blood plasma, where always contains various enzymes, constitutes a challenge to targeted delivery based on the peptide ligand. We hypothesize that a stable peptide coupling by



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amido bond and consisting of D-amino acids with high binding affinity to p32 protein can enhance the targeting efficiency and designed two peptides in this study: L

c(LyP-1), cycling with amido bond; and Dc(LyP-1) (made up of D-amino acids), the

retro-inverso isomerization of Lc(LyP-1). Previous study has shown that no matter the cell uptake of D-peptide in vitro was higher than L-peptide or not, the higher stability of D-peptide helped to achieve better in vivo outcomes than L-peptide. In order to see if there is exception or not, we compared the tumor targeting capacity both in vivo and in vitro of the two peptides: Lc(LyP-1) and Dc(LyP-1). D

c(LyP-1) was synthesized by using a retro-inverso isomerization technique, with a

reverse sequence of Lc(LyP-1). On terms of the binding affinity with p32 protein and the cellular uptake efficiency, Lc(LyP-1) demonstrated better results than those of D

c(LyP-1). Further, the computer-aided simulation confirmed this result on molecular

level. However, compared with the

D

c(LyP-1), Lc(LyP-1) displayed lower blood

stability in fresh rat serum. We supposed that the proteolysis of Lc(LyP-1) in rat serum might decrease the transcytosis efficiency. In order to further explore what’s the final result with all of these above factors, we modified Lc(LyP-1) or Dc(LyP-1) together with DCDX on the surface of micelles to construct two dual-targeting drug delivery systems:

L

c(LyP-1)/DCDX-PEG-PLA micelles and

micelles.

D

D

c(LyP-1)/DCDX-PEG-PLA

CDX was for crossing BBB.11 and Lc(LyP-1) or

D

c(LyP-1) was for

targeting brain metastatic tumors. Results showed that Lc(LyP-1)/DCDX-PEG-PLA micelles displayed significantly better brain metastatic tumor targeting efficiency than D

c(LyP-1)/DCDX-PEG-PLA micelles. In other words, Lc(LyP-1) had better tumor


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targeting efficiency in vivo than Dc(LyP-1). Therefore, based on the brain targeting efficiency of DCDX and the tumor targeting capacity of Lc(LyP-1), dual-targeting L

c(LyP-1)/DCDX-PEG-PLA micelles could be constructed the to realize effective

therapy of brain metastatic tumor. On the other hand, the good stability of D-peptide will not always help to achieve better targeting efficiency than L-peptide in vivo.

CONCLUSION In this study, two kinds of peptide: Lc(LyP-1) and Dc(LyP-1) were designed. The higher stability of Dc(LyP-1) did not help to achieve better targeting efficiency than L

c(LyP-1) in vivo. By contrast, Lc(LyP-1) displayed high binding affinity with p32

protein, high uptake efficiency in tumor cells and not-too-bad blood stability, respectively. Micelles dual-modified by

L

c(LyP-1) and brain targeting

D

CDX

efficiently inspired the brain metastatic tumor targeted capacity, providing a promising platform for brain metastatic tumor-targeted therapy in the future.

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

ACKNOWLEDGMENTS This work was supported by National Basic Research Program of China (973



Program, No.2013CB932500), National Natural Science Foundation of China (No.81773657, No.81690263 and No.81473149), Shanghai Education Commission Major Project (2017-01-07-00-07-E00052) and Shanghai international science and technology cooperation project (No.16430723800).

SUPPORTING INFORMATION Detailed description of the synthesis of peptide ligand Binding affinity of peptides with P32 protein Synthesis and characterization of micelles

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473x212mm (72 x 72 DPI)


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Coupling by Amide Bond Inspires the Brain Metastatic Tumor T

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