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Substance P mediated DGLs complexing with DACHPt for targeting therapy of glioma Tao Sun, Xutao Jiang, Qingbing Wang, Qinjun Chen, Yifei Lu, Lisha Liu, Yu Zhang, Xi He, Chunhui Ruan, Yujie Zhang, Qin Guo, Yaohua Liu, and Chen Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b05997 • Publication Date (Web): 19 Sep 2017 Downloaded from http://pubs.acs.org on September 20, 2017

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ACS Applied Materials & Interfaces

Substance P mediated DGLs complexing with DACHPt for targeting therapy of glioma Tao Sun, † Xutao Jiang, † Qingbing Wang, ‡ Qinjun Chen, † Yifei Lu, † Lisha Liu, † Yu Zhang, † Xi He, † Chunhui Ruan, † Yujie Zhang, † Qin Guo, † Yaohua Liu,*,

§,‖

†

and Chen Jiang*,†

Key Laboratory of Smart Drug Delivery of Ministry of Education, State Key Laboratory of

Medical Neurobiology, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 200032, PR China ‡

Department of interventional Radiology, Ruijin Hospital, School of Medicine, Shanghai Jiao

Tong University, Shanghai 200025, PR China §

Department of Neurosurgery, First Affiliated Hospital of Harbin Medical University, Harbin

150001, PR China. ‖

Department of Neurosurgery, Shanghai First People’s Hospital, School of Medicine, Shanghai

Jiao Tong University, Shanghai 201620, PR China.

KEYWORDS: glioma, drug delivery, platinum drug, blood-brain barrier, Substance P

ACS Paragon Plus Environment

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ABSTRACT. Currently, glioblastoma (glioma) is described as the deadliest brain tumor for its invasive natural with exceeding difficulty in surgical excision. Blood-brain barrier (BBB) can restrict the penetration of most therapeutic reagents including platinum (Pt)-based drugs-the most widely used reagents in clinical trials for their revolutionized cancer chemotherapy against a broad range of tumors. Nanomedicine represents a promising strategy for the intravenous delivery of Ptbased drugs into the brain. In this research, with the aim of malignant glioma treatment by Ptbased drugs, a novel nano drug carrier was developed: dendrigraft poly-L-lysines (DGLs) was PEGylated, linked with diethylenetriaminpentaacetic acid (DTPA) to complex (1,2diaminocyclohexane)platinum(II) (DACHPt), and modified with Substance P (SP) as a BBB/glioma dual-targeting moiety. The preparation and characterization of the platform were exhibited in detail. The increased targeting capability and anti-tumor effect was found both in vitro and in vivo. The well-defined chemical composition, rigorously nanoscaled size and the first attempt of using SP as a BBB/glioma dual-targeting group were highlighted. The combined results suggest this strategy may serve as novel formulation for Pt-based drugs with the aim of clinical glioma treatment.


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1. Introduction

Clinically, malignant glioma is recorded as the most aggressive, devastating and commonly encountered primary brain tumor with an estimated 5% five-year survival rate. Among the primary central-nervous-system (CNS) tumors, glioma accounts as high as 40%.1 Glioma therapy currently faces several inherent challenges. The conventional treatment routine is surgery, followed by radiotherapy and chemotherapy.2 As to the natural of infiltration and resistance to radiation, chemotherapy is believed as an indispensable means. 3 However, most chemotherapeutic agents cannot reach glioma sites due to the existence of the BBB, which can prevent the penetration of 98% small molecules and 100% macromolecules into the brain. 4 Another issue that should be addressed in chemotherapy is the virtual accumulation of chemotherapeutic reagents in glioma cells after crossing BBB. The lack of targeted accumulation could result in severe toxicity to normal encranial cells.5 Currently, Pt-based drugs are the most frequently used chemotherapeutic reagents for a wide range of tumors (including testicular, ovarian and gastric tumors) with high efficacy and fine synergism with other drugs.6 The National Comprehensive Cancer Network (NCCN) declared that Pt-based drugs can act as an adjuvant chemotherapy candidate for glioma treatment. However, Pt-based drugs can neither cross BBB, nor accumulate efficiently in glioma cells, which remains as great obstacles for clinical applications on glioma treatment.7 Pt-based drugs normally bypass the cell membrane in a free diffusion way, leading to a low intracellular concentration. 8 Meanwhile, lack of targeting capability, Pt-based drugs always generate an unneglectable systematic toxicity. For example, neurotoxicity is found to be the most severe toxicity associated with Pt-based drug regime observed clinically. Several drug delivery systems were attempted to load Pt-based drugs via physical encapsulation or chemical bonding. 9 Physical encapsulation might confront a rapid leakage upon dilution in bloodstream due to Pt-based drugs’ strong coordination to plasma during the blood circulation,10 while covalent chemical bindings (the prodrug strategy) are sometimes too stable and could result an unsuccessful release from the drug conjugates.11 Another promising strategy to load Pt-based drugs to the nanocarriers is via supramolecular dynamic bonds (e.g. metal-ligand coordination),12 and at the aimed site, Pt-based drugs can be unloaded upon the


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detachment of the non-covalent dynamic bonds.13 The innovation of the nanomedicine casts new lights for Pt-based drugs to effectively cross BBB and highly accumulate at the glioma sites. 14 Based on the extensive theoretical and practical research, a common sense has been reached that the nanomedicines’ size plays a crucial role in determining the in vivo biodistribution, penetration, cellular internalization, clearance, excretion, and finally the overall therapeutic efficacy. 15 Although the sizes of most reported nanomedicines range from 20 to 200 nm, recent research supported that nanomedicines with a smaller size (< 50 nm) exhibited better performance in vivo with enhanced tumor inhibiting ability and stronger tissue penetrating property.16–18 The currently known transport routes across BBB include paracellular aqueous/transcellular lipophilic pathways, protein-based transportors, receptor and adsorption-mediated transcytosis. Among all the outlined routes, nanocarriers with smaller sizes demonstrate sufficient advantages. 19 Meanwhile, nanovehicles with large sizes are more readily cleared during the circulation. 20 As well-defined nano-sized dendrimers, dendrigraft poly-L-lysines (DGLs, Mw. 22000, 6 nm) have been of general interest for drug and gene delivery in the past decade.21 DGLs are biocompatible and biodegradable dendrimers constructed by cascade L-lysine.22 In our previous work, DGLs were found capable to effectively accumulate in a solid tumors by enhanced permeability and retention (EPR) effect. 23 In this work, DGLs were attempted as carriers for Pt-based drugs. We propose two strategies in our system for glioma-targeting delivery of Pt-based drugs. It is known that during the early period of glioma, BBB is the predominant obstructer to escort active reagents to the aimed sites within the CNS.24~26 To endow the system with BBB-crossing and glioma-accumulating ability, linking DGL with specific targeting ligands could be a rational option.27 Substance P (SP, sequence as Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) is an endogenous neuropeptide originated from the endings of sensory nerve fibers and preferentially binds to the neurokinin-1 (NK-1) receptor, which is vastly distributed among the CNS system.28 NK-1 receptor is the key role in SP permeation across the BBB and is also found overexpressed in glioma cells.29 In this study, SP was firstly employed as the targeting moiety to realize a dual effect with both BBB and glioma-targeting capability by conjugating SP to DGL via a bifunctional polyethylene glycol (PEG) linker to obtain SP-PEG-DGL. PEGylation can help the nanocarriers exempt from the agglutination with plasma protein in blood, enhance steric stability and decrease the liver’s eliminating ratio.30 While in the advanced stage of glioma, BBB breach occurs and


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nano-sized DGL-based drug vehicles could effectively accumulate in solid tumors by EPR effect (Scheme 1). In possession of five carboxyl groups, diethyltriaminepentaacetic acid (DTPA), as a classic chelating agent, is used together with Technetium-99m as a nuclear imaging radiopharmaceutical and with Gadolinium as a MRI contrast medium (Magnevist, Bayer Schering Pharma AG).31 DTPA is also used as a decontamination agent in individuals who have ingested radioactive materials. Herein, DTPA was firstly employed as a chelator for Pt-based drugs DACHPt, which is a prodrug of oxaliplatin. DTPA was conjugated to SP-PEG-DGL under a mild condition to obtain SP-PEG-DGL-DTPA, with which DACHPt was then complexed to form a dual-targeting system SDDPt with the aim of efficiently delivering Pt-based drug into the glioma cells. The design and synthesis were described in detail, while the tumor-targeting capacity and anti-glioma efficacy were studied in vitro and in vivo.

Scheme 1. The illustration of the active/passive targeting design and the dual targeting (BBB and glioma tumor) strategy.


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2. Materials and methods 2.1. Materials SP linked with a 5-hexynoic acid on N-terminal (amino-group on arginine) (sequence: hexinRPKPQQFFGLM-NH2) was ordered from China-Peptides Co., Ltd. (Shanghai, China). Dendrigraft poly-L-lysine (DGL, generation = 3, bearing with 123 primary amino groups, 6 nm diameter, Mw. 22000) was commercial available from COLCOM (Clapiers, France). Azide-PEGNHS (Mw. 3500) was from Jenkem Technology (Beijing, China). 2-(4-Isothiocyanatobenzyl)diethylenetriaminepentaacetic acid (p-SCN-Bn-DTPA), as a derivative of DTPA, functionalized with an isothiocyanate group, was from Macrocyclics (Dallas, TX, USA). 6-(((4,4-Difluoro-5-(2pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl) aminohexanoic acid, succinimidyl ester (BODIPY) and 4,6-diamidino-2-phenylindole (DAPI) were from Molecular Probes (Eugene, OR,

USA).

Filipin,

colchicines,

phenylarine

oxide

(PhAsO)

and

(1,2-

Diaminocyclohexane)platinum(II) chloride (DACHPt) were from Sigma–Aldrich (St. Louis, MO, USA). Oxaliplatin was from Meilunbio Pharmaceutical Co., Ltd. (Dalian, China). Other chemicals and all solvents were purchased from Sinopharm Group Co. Ltd. (Shanghai, China). All materials were directly used as received unless mentioned otherwise.

2.2. Cells The primary cultured brain capillary endothelial cells (BCECs) isolated from BALB/C mouse were kindly provided by Prof. J.-N. Lou (the Clinical Medicine Research Institute of the ChineseJapanese Friendship Hospital, Beijing, China). Primary BCECs were cultured as previously described.32 Briefly, cultured at 37 °C under a humidified atmosphere containing 5% CO2, BCECs were expanded and cultured in special Dulbecco’s modified Eagle medium (DMEM) (Sigma– Aldrich, St. Louis, MO, USA) supplemented with 20% heat-inactivated fetal calf serum (FCS), 100 μg/mL streptomycin, 100 g/mL epidermal cell growth factor, 100 U/mL penicillin, 40 μU/mL insulin, 20 μg/mL heparin and 2 mmol/L lglutamine. All cells used in this study were between generation 15 to 30. Glioma U87 cells expressing photinuspyralis luciferase (U87-Luci) were a gift from Dr. N. Zhang (CaliperLife Sciences, A PerkinElmer Company, Waltham, Massachusetts, USA). U87 cells were


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purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). U87-Luci and U87 cells were cultured at 37 °C in a humidified 5% CO2 atmosphere with DMEM, supplemented with streptomycin (100 mg/mL), penicillin (100 units/mL) and fetal bovine serum (FBS) (10%). The other cell cultural media and reagents were from Life Technologies Corporation (Thermo Fisher Scientific, Carlsbad, California, USA). All cells used in this study were between generation 10 to 20.

2.3. Animals Male Balb-c mice (Slac, 4~5 weeks) with 20~25 g body weight and nude mice of 20~25 g body weight were obtained from the Department of Experimental Animals, Fudan University, and maintained under standard housing conditions. All the animal experiment guidelines were carried out, approved, and monitored by the Institutional Animal Care and Use Committee (IACUC), School of Pharmacy, Fudan University. Glioma model nude mice were prepared by a classic intracranial injection method on a stereotaxic apparatus (striatum, 1.8 mm right lateral to the bregma, 3 mm of depth) of 1 × 105 U87/Luci cells suspended in 5 μL of isotonic phosphate buffered solution (PBS 7.4) into male nude mice with a body weight of 20~25 g, followed by careful sterilization and suture. We recorded more than 90% mice could well recover from the surgery, while the glioma incidence was 100%.

2.4. Synthesis of the dendritic drug carrier NHS-PEG3.5k-N3 was grafted to DGL (1:10 mol. ratio) in PBS (pH 8.0) by stirring at 25 °C for 2 h. The NHS moiety of the bifunctional PEG can specifically react with the primary amino groups on the DGL surface. The obtained conjugate, DGL-PEG, was further purified by an ultrafiltration through a filtering membrane and the buffer was replaced by PBS (pH 7.0). DACHPt was then suspended into distilled water and reacted with silver nitrate (AgNO3:DACHPt = 1:1, mol. ratio) to obtain DACHPt nitrate chloride. In the dark, the suspension was stirred at 25 °C for 24 h. AgCl precipitation was removed by centrifugation. The supernatant was further treated by passage through a filter (0.22 μm). Then, DACHPt nitrate chloride was reacted with p-SCN-Bn-DTPA at a 5:1 molar ratio and reacted at 25 °C for 24 h to obtain (p-SCN-Bn-DTPA)-DACHPt. Then this


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solution was reacted with N3-PEG-DGL (p-SCN-Bn-DTPA:DGL = 20:1, mol. ratio) in PBS (pH = 8.2) and stirred in HEPES 8.5 for 24 h at 25 °C to form PEG-DGL-DTPA-DACHPt (DDPt). The product was purified by a dialysis membrane (MWCO 5000) against distilled water (MWCO 5000) and then dried by a freeze-drier. SP was coupled to the terminus of N3-DDPt through a CuI-catalyzed azide+alkyne “click” reaction.33 N3-DDPt and hexyne-SP (5 eq.) were dissolved in DMF under a N2 atmosphere. CuI (2.5 eq.), sodium ascorbate (5 eq.), and DIPEA (5 eq.) dissolved in DMF was added to the mixture and the suspension was stirred at 30 °C for 12 h. The product, SP-PEG-DGL-DTPA-Pt (SDDPt) was purified by a dialysis membrane (MWCO 5000) against EDTA-2Na (aqueous, 10 mM, pH 7.0) for 24 h, then against deionized water for 24 h, followed dried by a freeze-drier. To investigate the cellular uptake and internalization mechanism, Red-BODIPY (λex/em = 650/665 nm) labeled dendrimer were prepared following below steps: DDPt or SDDPt dissolved in PBS 7.0 was reacted with BODIPY-NHS (2 eq.) dissolved in DCM at 25 °C in dark for 24 h. BODIPY-DDPt or BODIPY-SDDPt in the upper aqueous phase was collected by a separatory funnel, further purified by a Sephadex gel column chromatography using absolute methanol as the eluent, and diluted into several samples for further use. BODIPY-DDPt and BODIPY-SDDPt samples were stored under -20 °C at dark to avoid possible fluorescence quenching.

2.5. Characterization of the dendritic drug carrier SDDPt was characterized by nuclear magnetic resonance (NMR) spectroscopy in detail. SDDPt was freeze-dried, re-solubilized in D2O and determined by an NMR spectrometer (600 MHz, Bruker, Billerica, MA, USA) at 25 °C. The mean diameter was measured by dynamic light scattering (DLS) (Zetasizer Nano-ZS, Malvern, U.K.). The morphology was assessed by transmission electron microscope (TEM, JEM-2010, JEOL, Akishima, Tokyo, Japan). DACHPt loading efficiency were determined by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES, Varian, Palo Alto, CA, USA).

2.6. In vitro DACHPt release study


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SP-PEG-DGL-DTPA-DACHPt with a certain concentration (in Pt form) in a dialysis bag (MWCO 3500) was clamped and suspended in a centrifuge tube containing phosphate buffered saline (0.15 M NaCl, PBS 7.4, close to the physiological condition), which was shaken at 37 °C with a speed of 100 rpm to mimic the in vivo condition. An aliquot of solution was withdrawn from the tube and free PBS was replenished at various time intervals. The parent drug DACHPt was used as the control to prove the dialysis bag has no influence on the drug leak. The drug concentration was measured by ICP-AES.

2.7. Cellular uptake and internalization mechanism study The BCECs and U87-Luci cells were respectively seeded with a density of 2 × 104 per well in 24well plates (Corning-Coaster, Tokyo, Japan), incubated at 37 °C for 72 h, and monitored under microscope until similar confluency and morphology. BCECs and U87-Luci cells were respectively cultured with different concentration of BODIPY-DDPt or BODIPY-SDDPt in the DMEM medium at 37 °C for 0.5 h. Then, the cells were washed thrice with PBS 7.4 and checked by fluoresce microscope (Leica, Wetzlar, Germany). As to flow cytometry analysis, BCECs and U87-Luci cells were respectively seeded with a density of 1 × 105 cells/well in 6-well plates, cultured for similar confluency and morphology. BCECs or U87-Luci cells were cultured with various concentration of BODIPY-DDPt or BODIPY-SDDPt in the DMEM medium at 37 °C for 0.5 h. The cells were washed thrice by PBS 7.4, trypsinized, then centrifuged at a speed of 1,500 rpm for 5 min to obtain a cell pellet, followed by suspending in PBS 7.4, and subsequently analyzed with a flow cytometer (FACSCalibul, BD, USA). The fluorescence intensity of the labeled BODIPY was collected at 650 nm. 10,000 events were recorded and analyzed for each sample. Cells incubated under normal conditions were used as the control. To investigate the mechanism of cellular internalization, the cells were pre-incubated with four inhibitors, including colchicine (1 μg/mL), phenylarsine oxide (0.4 μg/mL), filipin complex asendocytic inhibitors (0.5 μg/mL) and parent SP (100 folds, as a competitive inhibitor) at 37 °C for 15 min. Then, the cells were washed thrice with isotonic PBS 7.4 and further incubated with BODIPY-DDPt or BODIPY-SDDPt (10-5 mol/L, PBS 7.4) for another 0.5 h. The cells were


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washed thrice with isotonic PBS 7.4, then observed/photographed by fluorescent microscope. Quantitative experiment was conducted by using flow cytometry analysis, with the same experimental procedure described above. 2.8. Transport study across the BCECs monolayer by the transwell assay In a 24-well plate, transwell filters (polycarbonate-based, with 1.0 μm mean pore size and 0.33 cm2 surface area, purchased from FALCON Cell Culture Insert, Becton Dickinson Labware, Becton, New Jersey, USA), BCECs were carefully seeded with a density of 5 × 104 cells/cm2. After a 72-h culturing, the complete confluency of cells were checked under the microscope. The cell monolayer integrity was tested by an epithelial voltohmmeter (MILLICELL®-ERS, Merck Millipore, Billerica, Massachusetts, USA) to detect the transendothelial electrical resistance (TEER). The BCECs monolayers with continuous TEER higher than 200 Ω·cm2 were regarded qualified and used for the transport study. To further monitor the integrity of BCECs monolayers, FBS-free medium (500 μL) was dripped into the donor chamber, which should maintain a higher level than the receptor for 2 h. In the receptor chamber, FBS-free medium (800 μL) was added. In the donor chamber, 200 μL solutions (with a Pt concentration as 0.5 mg/mL) of SDDPt, DDPt or SDDPt/SP (with excess SP (100 times) as inhibitor) in FBS-free medium were also added separately (n = 4). The 24-well plate was incubated at 37 °C on a platform rocker at a speed of 50 rpm. At every time point, 100 μL solution was withdrawn from the receptor chamber, with a replenishment of fresh FBS-free medium (100 μL). TEER was tested at every time point to monitor the monolayers’ integrity. The withdrawn sample (100 μL) from receptor chambers were treated with HNO3 (65%, 1 mL) overnight at room temperature on a platform rocker at a speed of 50 rpm. H2O (1 mL) was added to the sample and submitted for Pt-determination measured by ICP-AES. Notably, every time the replenished fresh FBS-free medium contains no Pt, which should be accounted during the calculation. 2.9. In vitro cytotoxicity study MTT assay is a reliable method and widely employed to evaluate in vitro cytotoxicity. Briefly, U87-Luci cells were seeded in 96-well plates with a 5 ×103/well density and cultured at 37 °C for 24 h. Upon achieving a 60-70% confluence, the cells were cultured with oxaliplatin, DDPt and SDDPt dendrimers at various Pt concentrations in DMEM medium (n = 4). After a 48-h incubation,


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the medium was removed and the cells were washed thrice with PBS 7.4. Then, MTT solution (100 μL, 0.5 mg/mL in PBS) was dripped and mixed into each well and the cells were cultured at 37 °C for 4 h. Then, the solution was removed and DMSO (100 μL) was added into each well. The mixture was shaken on a shaking table at a speed of 50 rpm for 10 min. The UV absorbance of the formed formazan crystals in solution was read at 570 nm using a microplate spectrophotometer (Bio-Tek, Synergy 2, Winooski, VT, USA). The cells without any treatment were employed as the control. In vitro cytotoxicity of the dendrimers was assessed by the same procedure with different concentrations.

2.10. In vivo imaging study On the 16th day after the implantation of glioma, the IVIS luminescence intensity is among 6~10×e4 and the tumor-bearing nude mice were intravenously injected with BODIPY-labled dendrimers at the same dose of 15 mg/kg DGL per mouse. At 2, 12 and 24 h after the injection, the treated mice were anesthetized, visualized and photographed under in vivo IVIS spectrum imaging system (Caliper, Newton, MA, USA) at λex/em = 768/789 nm. Then mice were executed by the decapitation method and the main organs were separated for further visualization.

2.11. Biodistribution study Biodistribution study was carried out on tumor-bearing nude mice (n = 6). Oxaliplatin and dendrimers (5 Pt mg/kg) were intravenously injected into the tumor-bearing mice. The mice were executed at 2 and 24 h by decapitation. Main tissues and organs (brain, heart, liver, spleen, lung, kidney and tumor) were carefully excised and collected. These organs and tissues were rinsed and weighed. The selected samples were carefully dissolved in concentrated HNO3 and evaporated to dryness in air. The Pt concentrations were then determined by ICP-AES. BBB penetration study on healthy mice were carried out following a similar procedure.

2.12. In vivo antitumor efficacy study The tumor-bearing mice were randomized to four groups (n = 8). On the 21st, 28th and 35th day after implantation, the IVIS luminescence intensity is among 8~12×e4 and the model mice were


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administrated of saline, free oxaliplatin, SDDPt and DDPt (Pt, 5mg/kg). On the 23 st, 30th and 37th days, the mice were observed through IVIS Spectrum in vivo imaging system.

2.13. In vivo apoptosis study In vivo apoptosis study was carried out on tumor-bearing nude mice (n = 8). On the 21st day, the model mice were intravenously injected with saline, oxaliplatin and the prepared dendrimers. The mice were anesthetized after 48 h using Et2O and executed by the decapitation method. The brains were directly excised, fixed in a 4% (v/v) paraformaldehyde solution (48 h), placed in 15% sucrose solution (in PBS 7.4, 24 h) until subsidence, then in 30% sucrose solution (in PBS 7.4, 48 h) until subsidence. Then, the brain was frozen in the OCT embedding medium (Sakura, Torrance, CA, USA) at -80 °C. The sliced frozen section with a 20-μm thickness were obtained on a cryotome Cryostat (Leica, CM 1900, Wetzlar, Germany). The apoptotic cell detection was performed using an in situ apoptosis detection kit on basis of the TUNEL method. The sliced samples were cultured with proteinase K for 15 min at 25°C. The endogenous peroxidase was blocked with a PBS solution and 3% H2O2 for 10 min. Afterwards, the slides were cultured with digoxigenin-conjugated nucleotides in solution and terminal deoxynucleotidyl transferase (TdT) at 30 °C for 1 h. Subsequently, the anti-digoxigenin antibody was used and cultured for 0.5 h at 25 °C. Detecting the antigene antibody link was prepared via the immune peroxydase and then diaminobenzidine (DAB) chromogenic. The samples were counterstained with hematoxylin, washed with distilled water, visualized and photographed by the fluorescence microscope.

2.14. Histology and immunohistochemistry Histology and immunohistochemistry studies were carried out on tumor-bearing nude mice (n = 8). Prepared dendrimers and free oxaliplatin (5 Pt mg/kg) was administered intravenously into the tumor-bearing mice on the 21 st day. The mice were killed at 48 h post injection. The main tissues and organs were collected. The excised samples were frozen in liquid nitrogen for immunohistochemistry or fixed in a 4% (v/v) paraformaldehyde solution, then placed in paraffin to obtain the tissue sections for H&E staining. As to the immune histochemical staining, frozen samples were sectioned at a 10-μm thickness in a cryostat Cryostat, fixed in acetone, and treated


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with the protein blocking solution. The sliced sections were further treated with antimurine PECAM1 monoclonal antibody, rabbit polyclonal antibody against PDGFRβ, and monoclonal anti-α-SMA antibody. Subsequently, the samples were stained with the secondary antibody conjugated with Alexa Fluor 488, 594, or 647 anti-rat/rabbit IgG (Invitrogen Molecular Probes, Eugene, Oregon, USA). The samples were visualized and photographed by microscope (Olympus, AX80, Shinjuku, Tokyo, Japan) for H&E staining and a confocal microscope (Zeiss, LSM510 Meta, Oberkochen, Germany) for the immunohistochemistry analysis.

2.15. Statistical analysis Analysis was carried on GraphPad Prism 6 or Origin 7.5 softwares. Statistical comparisons were assessed by single-way ANOVA. Herein, data are represented as means ±SD.

3. Results and discussion 3.1. Design, synthesis and characterization SP, as a neuropeptide generated from the sensory nerve fibers’ ending, is the ligand for the tachykinin NK-1 receptor. The NK-1 receptor is widespread throughout CNS and CNS tumors. From previous study,34 it was demonstrated the NK-1 receptor are overexpressed in numerous tumor cell lines, such as malignant gliomas, breast carcinoma and metastatic melanomas. Mediated by NK-1, the permeation across BBB and tumor-targeting of SP or SP-derivate can be greatly improved. Furthermore, on both in vitro and in vivo models, it was proved that SP serves as an important role in a chain of molecular events in disturbing brain endothelial tight junctions, which could result a temporary open-window of BBB.35 Lunte et. al. provides enough evidence on a molecular level for a carrier-mediated mechanism in transporting SP molecules and analogue across the BBB.36 SP also possesses other clinical advantages, including fine biocompatibility, convenient modification and limited steric hindrance. Pt-based reagents, as the most extensively used anticancer drugs in clinic, are still facing problems including unexpected serious systematic toxicity, considerable drug resistance and lack of tumor targeting. The specially-designed nanosized carriers could help drugs shield from undesired


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premature activation, cross physiological barriers to aimed cells, and finally accumulate at the targeted sites on-demand in vivo. In previous studies, Pt-based drugs were well studied to be loaded on carbon nanotubes, liposomes, nanogels, gold nanoparticles, magnetic iron oxide and polymers, as well as by forming oxidative prodrugs, with the hope in treating human colorectal cancer,37 nonsmall cell lung cancer,38 etc. It is noteworthy if Pt-based drugs were attempted as a chemotherapy candidate for glioma treatment, drug vectors with glioma-targeting ability, less complicacy and well-defined nanosize are in urgent need. The third generation Pt-based drug DACHPt is a promising anticancer reagent with an expanded spectrum of activity.39 DACHPt can be activated into oxaliplatin, which already shows excellent anti-cancer effect against several cancer models.40 However, DACHPt’s poor solubility and low targeting capability greatly confine its clinical application. Herein, we employed commercial available PEG/DGL/DTPA to effectively complex Pt-based drugs with high efficacy and less complicacy. SP as the selected active targeting group was modified to the platform with the aim of dual BBB-crossing and glioma-targeting ability. A modular design and stepwise synthesis were carried out as illustrated in Fig. 1A. DGL (the 3rd generation, 3 nm) was firstly modified with bifunctional PEG to obtain N 3-PEG-DGL. DACHPt was reacted with AgNO3 (1 eq.) to form DACHPt-NO3, which was then complexed with p-SCNBn-DTPA to obtain DTPA-DACHPt. DTPA-DACHPt was conjugated with N3-PEG-DGL to yield N3-PEG-DGL-DTPA-DACHPt (N3-DDPt). The molar composition ratio of DGL:DTPA:PEG in DDPt is optimized to be 1:20:15. From the 1H NMR spectrum, the characteristic peaks of DGL at 1.0-2.0 ppm (CH2), PEG at 3.5 ppm (CH2CH2O), and DTPA at 7.3 ppm (phenyl groups) were found (Fig. 1B), suggesting a successful conjugation. Considering a 100% modification with targeting moiety to PEG could result a fast removal by the immune system upon in vivo, the optimized mol. ratio of DGL:DTPA:PEG:SP in SDDPt was 1:20:15:8. A conjugation of hexin-SP to the end of N3-PEG-DGL-DTPA-DACHPt was performed via “click” reaction, a widely used bioconjugation approach, which is precise, highly efficient and completed under mild conditions. The formed triazole ring, which can be clearly observed in 1H NMR spectrum of SP-PEG-DGLDTPA-DACHPt (SDDPt, Fig. 1C), is regarded stable in physiological conditions. The amount of chelated Pt metal was determined using ICP-AES. The percentages (w/w) for Pt in DDPt and SDDPt were determined to be 16.8% (calculated as 16.7%) and 14.4% (calculated as 15.3%),


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respectively, revealing a fine chemical linking efficacy of the designed strategy. Both the 1H NMR and ICP-AES results confirm the well-defined chemical structures.

Figure 1. A) The synthesis steps for DDPt and SDDPt: i) PBS (aq. 10 mM, pH 8.0), 25 °C, 2 h, dark; ii) AgNO3 (aq. 1 eq.), 25 °C, 24 h, dark; iii) p-SCN-Bn-DTPA (0.2 eq.), PBS (aq. 10 mM, pH 8.2), 25 °C, 24 h, dark; iv) HEPES (aq. 10 mM, pH 8.5), 25 °C, 24 h, dark; v) CuI (2.5 eq.), VcNa (5 eq.), DIPEA (5 eq.), DMF, N2, 30 °C, 12 h, dark; B) 1H NMR spectrum of DDPt in D2O;


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C) 1H NMR spectrum of SDDPt in D2O; D) The obtained dendrimer’ size distribution of SDDPt in PBS analyzed by DLS; E) Characterization of SDDPt dendrimer by TEM (scale bar = 50 nm).

The zeta-potential of DDPt and SDDPt were respectively positively charged with a surface charge of 3 ± 0.5 and 3.5 ± 0.9 mV, resulting from the protonated amino group at the surface of DGLs. The vessels of the BBB are assembled of specialized endothelial cells lacking fenestration, which allows a rapid chemical exchange between vessels and tissue.41 The extensive tight junctions that severely restrict the permeability require that the designed nanocarriers should possess an essentially nanosized distribution for BBB-penetrating and tumor-accumulation.42 Nanomedicines with smaller sizes (< 50 nm) allow a better BBB-crossing ability and deeper tumor-tissue penetration.43 The obtained SDDPt dendrimer size was determined by DLS and TEM. DLS (Fig. 1D) gave a 19.65 ±4.65 nm diameter with a narrow distribution (PDI < 0.2). TEM (Fig. 1E) images further confirmed the obtained dendrimer with a favorable dispersivity. The average size measured by TEM is around 15 ±3 nm, the number of which is smaller than DLS results. This can be ascribed to that DLS analysis indicates the hydrated value, while TEM measures the solid substance of the particles. Though the TEM characterization was carried on a carbon net resulting a lower contrast, the fine dispersivity and nano-ranged size can still be well observed.

3.2. In vitro drug release assay The drug loading rate in the prepared dendrimers is as high as 32.7 ± 1.7 % determined by ICPAES (in the form of DACHPt). The binding of DACHPt to DGL-DTPA is quite stable in distilled water, while the drug release could be triggered in NaCl solution via an ion-exchange reaction occurred between chloride ions and carboxylate radicals on DTPA (Fig. 2A).44 Other low molecular weight counterions (such as H3O+, CO32-, CH3COO-, etc) could also activate the Pt prodrug. In the cell, DACHPt can be hydrolyzed to mono-aquated or bis-aquated Pt complex, which then enters the nucleus, blocks the DNA replica and eventually induces cell apoptosis. A simple drug diffusion for the parent drug DACHPt from the dialysis bag was found, and 89 ±8.5% drug leak was monitored at 12 h.


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On the contrary, an interesting two-stage drug release profile for SDDPt in presence of NaCl was proposed as illustrated in Fig. 2B: during the first 16 h of exposure, a rapid drug-release to 40% was noticed; from then to 90 h, a linear rise to 70% was recorded. This kind of profile is beneficial to oncotherapy, because the “burst-release” in the early stage is helpful in causing a rapid cell apoptosis of tumors, while the subsequent sustained release can maintain the drug concentration to kill the relict tumor cells.45 The competition between anions and DTPA is the decisive trigger for the drug reconversion. 46 As known, the metal-ligand complexing interaction belongs to supramolecular forces, which are not as strong as covalent bonds. In physiological conditions, no matter in blood circulation and tumor cells, anions including Cl- are indispensable and always balanced, allowing a sustained release for SDDPt.47 Admittedly, there is risk that during the blood circulation after injection, unexpected drug-leak (

Substance P mediated DGLs complexing with ... - ACS Publications

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