Fmoc protected amino acids

EM-1 and EM-2 were prepared by manual solid-phase synthesis using standard N-fluorenylmethoxycarbonyl (Fmoc) chemistry as reported in our previous study [32 (link)]. Fmoc-protected amino acids (GL Biochem Ltd., China) were coupled with Rink amide 4-methybenzhydrylamine (MBHA) resin (Tianjin Nankai Hecheng Science & Technology Co., Ltd., China). The crude peptides were purified by preparative reversed-phase HPLC (RP-HPLC) and determined by electrospray ionization mass spectrometer (ESI-Q-TOF maXis-4G, Bruker Daltonics).
Naloxone, β-FNA, nor-binaltorphimine (nor-BNI), and naltrindole (NTI) were obtained from Sigma-Aldrich. The selective p38 MAPK inhibitor SB203580 was purchased from Beyotime Institute of Biotechnology and dissolved in 1% DMSO in saline. All other drugs were dissolved in sterilized saline and stored at − 20 °C.

Fmoc-protected amino acids were purchased from GL Biochem Ltd (Shanghai, China). HSPC (hydrogenated soy phosphatidylcholine) and mPEG₂₀₀₀-DSPE were obtained from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol was supplied by Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Near infrared dye DiR was obtained from Invitrogen, USA. Sephadex G50 and 5-carboxyfluorescein (FAM) were provided by Sigma (St. Louis, MO). Doxorubicin hydrochloride was supplied by Dalian Meilun Biotech Co., Ltd (Dalian, China). Rat tail collagen Type I was purchased from Shengyou Biological Technology Co. (Hangzhou, China). DNase I and collagenase were supplied by Dingguo Biological Technology Co. Ltd (Shanghai, China).
Human glioblastoma cells (U87), human umbilical vascular endothelial cells (HUVECs) and brain capillary endothelial cells (bEnd.3) were obtained from Shanghai Institute of Cell Biology, cultured in special Dulbecco’s modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). ICR mice and BALB/c nude mice aged 4-6 weeks were supplied by Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China) and housed under SPF conditions. All animal experiments were carried out in accordance with the guidelines evaluated and approved by the Ethics Committee of Fudan University.

Fmoc-protected amino acids were purchased from GL Biochem LTD (Shanghai, China). DIC and Oxyma Pure were obtained from Iris Biotech GmbH (Marktredwitz, Germany). The H-Rink Amide-ChemMatrix® resin was acquired from Sigma Aldrich (Taufkirchen, Germany). DMF used for peptide synthesis was supplied by Fisher Scientific (Schwerte, Germany) and was of peptide grade quality. Acetonitrile used for HPLC was supplied by Fisher Scientific (Schwerte, Germany) with HPLC grade quality. Water used for HPLC and reactions was obtained by purifying deionized water with the purification device Simplicity from Millipore. All other reagents were supplied by Sigma Aldrich (Taufkirchen, Germany), Thermo Fisher Scientific (Langenselbold, Germany), VWR International (Darmstadt, Germany) and Carl Roth (Karlsruhe, Germany). All reagents were of synthesis grade quality and were used as supplied. Unless otherwise stated, biophysical measurements were performed in phosphate buffered saline (PBS, 8.2 mM Na₂HPO₄, 1.8 mM K₂HPO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.4). Peptide concentrations were determined by UV-absorbance at 280 nm (ε₂₈₀(Trp) = 5690 mol^–1 cm^–1, ε₂₈₀(Tyr) = 1280 mol^–1 cm^–1), and, alternatively, at 214 nm using a NanoDrop 2000 spectrophotometer from Thermo Scientific. Extinction coefficients at 214 nm were calculated according to Kuipers et al.50 (link)