Materials
Peptides 89WP (RQIKIWFWWRRMKWKK), N-terminal cysteine capped 89WP (Cys-89WP, CRQIKIWFWWRRMKWKK) and C-terminal FAM-labeled 89WP (89WP-FAM, RQIKIWFWWRRMKWKKK-FAM) were synthesized by Kangbeibio (Ningbo, China). Hydrophilic cyanine-5 maleimide derivative (sulfo-Cy5-Mal) was purchased from Ruixi Biological Technology (Xi’an, China). Fmoc-protected α-amino acids were from GL Biochem (Shanghai, China). Wang resin (Substitution = 0.54 mmol/g) was from Xi'an Innovision Bioscience (Xi'an, China). Fmoc N-hydroxysuccinimide ester (Fmoc-OSu), N,N’-diisopropylcarbodiimide (DIC), 4-dimethylaminopyridine (DMAP), O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluorophosphate (HBTU), 1-Hydroxybenzotriazole anhydrous (HOBT) and N,N-diisopropylethylamine (DIEA) were purchased from Aladdin Reagent (Shanghai, China). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), DMEM/Nutrient Mixture F-12 (DMEM/F12), Roswell Park Memorial Institute 1640 Medium (RPMI 1640), antibiotics (Penicillin-Streptomycin, 10,000 U/mL) and LysoTrackerTM Red DND-99 were purchased from Thermo Fisher (Massachusetts, United States). Melphalan, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Cell Counting Kit-8 (CCK-8) and 4,6-diamidino-2-phenylindole (DAPI) were from Dalian Meilun Biotechnology (Dalian, China). All the other reagents used in this work were of analytical grade, except chromatographic grade for liquid chromatography.
Cell cultivation
Human corneal epithelial cells (HCEC, BNCC337876) and Human umbilical vein endothelial cells (HUVEC, ATCC® CRL-1730™) were cultivated in DMEM medium supplemented with 10% FBS and 1% antibiotics at 37°C in a 5% CO2 humidified atmosphere. Human retinal pigment epithelial cells (ARPE-19, GNHu45) were cultivated in DMEM/F12 medium supplemented with 10% FBS and 1% antibiotics at 37°C in a 5% CO2 humidified atmosphere. Human retinoblastoma cells (WERI-Rb-1, TCHu213) and Fluc/GFP-Rb-1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotics at 37°C in a 5% CO2 humidified atmosphere. ARPE-19 and WERI-Rb-1 cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences. Fluc/GFP-Rb-1 cells were obtained in our previous work16.
Animals
Animals used in this work were obtained from the Experimental Animal Center of Fudan University and maintained at 22 ± 2°C on a 12 h light−dark cycle with access to food and water ad libitum. The animals for the experiments were treated according to protocols that were evaluated and approved by the Ethical Committee of Fudan University, and were acclimatized to laboratory conditions for 1 week prior to experiments.
Synthesis, purification and characterization
Conjugation of 89WP and hydrophilic Cy5 was through Michael addition reaction of thiol and maleimide. Briefly, Cys-89WP was mixed with sulfo-Cy5-Mal (1.2 eq.) in 10 mM phosphate buffer (PB, pH7.2) under nitrogen protection and stirred overnight at room temperature. Purification was implemented via Pre-HPLC (Waters, United States) on a reversed-phase C18 column (Waters, United States). The conjugate Cy5-89WP was obtained with a further freeze-drying process, and characterized by HPLC (Agilent, United States) and ESI-MS (AB, United States). An ODS column (YMC, 4.6 × 150 mm, 5 μm) was used at 25°C for HPLC analysis. The mobile phase was acetonitrile (0.1% trifluoroacetic acid, TFA):distilled water (0.1% TFA) at 5%–65% gradient in 30 min, at a flow rate of 0.7 mL/min. UV absorbance of the effluent was monitored at 214 nm. In ESI-MS analysis, the mobile phase was methanol (80%):distilled water (19.9%):formic acid (0.1%), at a flow rate of 0.3 mL/min, capillary voltage was 3000 V, and flow rate of drying gas at the temperature of 350°C was 12 L/min. Cysteine was conjugated to sulfo-Cy5-Mal using above method to obtain hydrophilic Cy5-Cys (abbreviating to Cy5 in this work).
Solid phase synthesis was used to accomplish conjugation of 89WP and melphalan (89WP-Mel) using Wang resin according to Fmoc methodology. Fmoc-protected melphalan (Fmoc-Mel) was synthesized via mixing melphalan (1.0 eq.) with Fmoc-OSu (1.2 eq.) in dioxane at 50°C with agitation overnight. The mixture was directly used for next procedure after the solvent dried under vacuum. Fmoc-Mel was added to Wang resin (0.5 eq.) after 5 min of pre-activation in dichloromethane (DCM) using DMAP (1.2 eq.) and DIC (1.0 eq.) as catalysts. After washing with dimethylformamide (DMF), acetic anhydride (5.0 eq.) and pyridine (5.0 eq.) were introduced to block unreacted active sites of the resin. The resin was immersed in piperidine/DMF (2:8) solution for 15 min twice to remove Fmoc-group and then rinsed with DMF. Further peptide condensation was facilitated by using excess Fmoc-protected amino acid (8.8 eq. to Wang resin, similarly hereinafter) activated with HBTU (8.8 eq.), HOBT (8.8 eq.) and DIEA (17.6 eq.) for 1 h in DMF. The Fmoc deprotection was implemented as above. The resin bound 89WP-Mel was rinsed 3 times with DMF and 3 times with DCM/methanol (1:1), and dried under vacuum. The conjugate 89WP-Mel were deprotected and cleaved from resin with a cocktail mixture (9.5 mL of TFA, 0.25 mL of triisopropylsilane and 0.25 mL of H2O, per 1.0 g of resin) for 3 h at room temperature. The resulting solution was concentrated to an oil in vacuo and cold diethyl ether was poured over the solutions to precipitate the samples. Purification and characterization of 89WP-Mel were similar with Cy5-89WP.
Cellular uptake, endocytosis mechanism and cytotoxicity
For quantitative cellular uptake evaluation of conjugates, HCEC or ARPE-19 cells cultivated on 24 well plates at a primary density of 20,000 cells/well were incubated with FAM, mixture of 89WP and FAM, or 89WP-FAM for 4 h, and all of them contained 3 μM FAM. After washed with 10 mM PBS containing 0.02 mg/mL heparin sodium three times and trypsinized, the cells were resuspended in 10 mM PBS, and the amount of FAM-positive cells and mean fluorescence intensity were tested by flow cytometer (BD, United States). The cells were also incubated with Cy5, mixture of 89WP and Cy5, or Cy5-89WP and tested as above. The concentration of Cy5 was 3 μM.
To discern conjugates 89WP-FAM and Cy5-89WP distribution in HCEC or ARPE-19 cells, each cells cultivated on 35mm 4-chamber glass bottom dishes at a primary density of 20,000 cells/well were incubated with related molecules for 4 h, respectively. Then the cells were washed with 10 mM PBS containing 0.02 mg/mL heparin sodium and immersed in glycerine/10 mM PBS (1:1, volume ratio) for further observation under confocal laser scanning microscope (Carl Zeiss, Germany).
To identify probable internalization mechanisms of 89WP-engaged conjugates, ARPE-19 cells were pre-incubated at 4°C or treated with inhibitors at 37°C for 0.5 h, then incubated with the 89WP-FAM at 4°C or in the presence of inhibitors at 37°C for another 1.5 h. The cellular uptake of 89WP-FAM at 37°C was used as the positive control (100% uptake efficiency). The results are shown as relative uptake rate of different groups normalized according to the positive control.
Endosomal escape ability of 89WP-engaged conjugates was evaluated via an erythrocyte lysis assay32. Briefly, freshly prepared human erythrocytes were resuspended in sodium citrate buffer (300 mM NaCl, 30 mM sodium citrate) at a concentration of 7 × 107 cells/mL, following incubation with equal volume of 89WP solution at a series of concentrations for 1 h at 37°C. After centrifugation (1000 g × 5 min), 100 μL of the supernatant were transferred to a 96-wells plate and OD450 nm was determined via microplate reader (Bio-Tek, United States). The distilled water was used as the negative control (no hemolysis) while 10% Triton X-100 solution was used as the positive control (100% hemolysis).
HCEC, ARPE-19 or WERI-Rb-1 cells at the logarithmic growth phase were cultivated on 96 well plates at a primary cell density of 2,000 cells/well for 24 h. Cells were then incubated with 89WP, melphalan or 89WP-Mel at concentration gradients for 4 h, following a further cultivation in 200 μL complete medium for 20 h except WERI-Rb-1 cells. After that, cells were incubated with 0.5 mg/mL MTT reagent in each well for 4 h at 37°C, and produced formazan was dissolved in 150 μL DMSO, except that WERI-Rb-1 cells were treated with CCK-8 reagent for 2 h after incubation with 89WP-Mel. The OD490 was measured via microplate reader.
Transport efficiency across in vitro ARPE-19 cell monolayer
The in vitro ARPE-19 cell monolayer model was established as previously reported with slight changes21. Briefly, ARPE-19 cells were seeded on the front of rat tail collagen type Ⅰ coated 6.5 mm polyester membrane filters (pore size 0.4 μM, Corning) at a density of 5,000 cells/well, respectively. Transepithelial electrical resistance (TEER) was measured by an epithelial volt-Ωm (Millipore, USA) to confirm the barrier integrity. Monolayers with TEER over 100 Ω·cm2 were chosen for the further experiments. The apical side of monolayers was incubated with FAM, mixture of 89WP and FAM, or 89WP-FAM in 100 μL D-Hanks balanced salt solution (D-HBSS, pH7.2) while the basolateral side was 600 μL D-HBSS. All of three groups contained FAM at a concentration of 3 μM. An aliquot of 200 μL of sample was extracted from the basolateral side every 0.5 h, and followed with addition of 200 μL fresh D-HBSS. Fluorescence intensity of the extracted sample was measured via microplate reader. Post-transported monolayers were also observed under confocal laser scanning microscope. To evaluate permeability of the conjugate, the apparent permeability coefficient (Papp, cm·s-1) was calculated as previously reported11. The samples in acceptor side were also characterized by HPLC and ESI-MS after a dialysis (MWCO 1000 Da) and freeze-drying process. The HPLC and ESI-MS conditions were same as mentioned above.
In another monolayer model, the acceptor side was seeded with WERI-Rb-1 cells at a density of 20,000 cells/well in RMPI 1640 medium. The FAM and 89WP-FAM in DMEM/F12 medium were added into the apical side and incubated for 4 h at 37°C. Then the WERI-Rb-1 cells were collected for flow cytometry analysis.
Ex vivo corneal and scleral permeability
Permeability of the Cy5-89WP through excised rabbit cornea and sclera was evaluated using horizontal diffusion cells as previously reported11. Animals were euthanized by an overdose of sodium pentobarbital (150 mg/kg) administered through marginal ear vein. Then the cornea and sclera were excised and immediately transferred into normal saline. Each cornea or sclera was placed vertically between the diffusion cells with epithelium orienting to the donor cells. Both donor and acceptor cells contained 3.5 mL normal saline as the diffusion medium, which was maintained at 34 ± 0.5°C by circulating water bath, and 5 μM Cy5 or Cy5-89WP was added in the donor side, respectively. An aliquot of 500 μL of sample was extracted from the acceptor cell every 0.5 h for 4 h, and followed with supplement of 500 μL fresh normal saline. Fluorescence intensity of the samples was measured via microplate reader. To compare ex vivo tissue permeability of Cy5 and Cy5-89WP, the apparent permeability coefficients (Papp, cm·s-1) were calculated as mentioned above.
After the diffusion, the cornea and sclera were fixed in 4% paraformaldehyde, dehydrated in 30% sucrose solution and dyed with hematoxylin-eosin or DAPI for further observation by microscope.
Intraocular distribution and pharmacokinetics
Male mice (18 ~ 20 g) derived from Institute of Cancer Research, United States were used in this experiment. Both 30 μM 89WP-FAM and Cy5-89WP dissolved in 10 μL artificial tear fluid33 were instilled into the conjunctival sac of mice, respectively. After instillation, the mice were sacrificed (injection with the fatal dose of pentobarbital sodium, 150 mg/kg) 0.5 h, 2 h, 4 h, 8 h, 12 h, 18 h, 24 h later and the eyeballs were fixed in Davidson’s solution for 0.5 h, following an overnight dehydration in 30% sucrose. DAPI-stained frozen sections were observed under an inverted fluorescence microscope (Leica, Germany).
Inhibition of intraocular retinoblastoma via topical instillation
Intraocular tumor-bearing mice model were established as previously reported16. Briefly, the male Balb/c nude mice were anesthetized by intraperitoneal injection with pentobarbital sodium (30 mg/kg) in combination with topical application of 0.4% oxybuprocaine hydrochloride. Topical instillation of 0.5% tropicamide was used to dilate the pupil. Fluc/GFP-Rb-1 cells (2 × 104 cells suspended in 2 μL 10 mM PBS) were injected slowly into the vitreous body near retina of the right eye using a microsyringe (33 G, Hamilton), following instillation of 0.25% chloramphenicol. Ocular luminescence of the mice was detected in the following days and the tumor-bearing mice were randomly divided into five groups (n = 5) in the first evaluation and four groups in the second evaluation (n = 8).
In the first evaluation (safety and preliminary pharmacodynamics evaluation), mice were treated with 10 μL of normal saline (N.S.), melphalan (3.0 mg/mL, dissolved in a cocktail of 1, 2-propanediol, ethyl alcohol, sodium citrate and distilled water), 89WP-Mel (containing melphalan 0.3 or 3.0 mg/mL, dissolved in normal saline) via topical instillation once daily from Day 0 to Day 30. Intravitreal injection of melphalan (8.0 mg/mL in 2 μL cocktail mentioned above, once a month) was used as the positive control. After 1-month treatment, the mice were sacrificed (injection with the fatal dose of pentobarbital sodium, 150 mg/kg) and the treated eyes and visceral organs were fixed in 4% paraformaldehyde, dehydrated in 30% sucrose solution and dyed with hematoxylin-eosin (HE) for further observation under microscope. Brain from a survival mouse in topically instilled melphalan group was also treated as above and implemented immunohistochemical experiments using HRP-labeled anti-GFP primary antibody (ab183734, Abcam) for metastasizing tumor cells detection.
In the second evaluation (optimized pharmacodynamics evaluation), mice were treated with 10 μL of normal saline, melphalan (3.0 mg/mL), 89WP-Mel (containing melphalan 3.0 mg/mL) via topical instillation once daily from Day 0 to Day 60. Intravitreal injection of melphalan (0.5 mg/mL in 2 μL medium composed of 10% cocktail and 90% N.S.) once on alternate weeks was used as the positive control. The mice were intraperitoneal injected with 150 mg/kg D-luciferin and anesthesia by isoflurane 15 min before luminescence signal was detected via the IVIS Spectrum system (PerkinElmer, USA). In vivo imaging was carried out on Day 0, 2, 4, 6, 9, 12, 15, 17, 20, 25, and 30. When the mice was moribund (body weights fast decreased to lower than 21 g) or at the end of experiment (Day 60), the whole brain of sacrificed mice was fixed in 4% paraformaldehyde, dehydrated in 30% sucrose solution and was implemented immunohistochemical experiments using HRP-labeled anti-GFP primary antibody (ab183734, Abcam) for metastasizing tumor cells detection.
Statistical analysis
Statistical significances of the quantitative data were analyzed by the One-way ANOVA multiple comparison or t-test. Considered that p > 0.05 was no significant, p < 0.05 was significant, and p < 0.01 even p < 0.001 was highly significant.