Supramolecular peptide constructed by molecular Lego allowing programmable self-assembly for photodynamic therapy

Peptide self-assemblies with multiple nanostructures have great potentials in functional biomaterials, and yet the tedious and costly covalent peptide modification and the lack of facile controllability on self-assembly morphology retard the peptide-related exploration. Here we report a simple approach to fabricate a supramolecular peptide that shows programmable self-assembly with multiple morphologies and application in photodynamic therapy. Pillar[5]arene-based host−guest recognition is used to construct a supramolecular peptide, which simplify the peptide modification and promote the controllability of the self-assembly behavior. Due to the ERGDS sequences on the exterior surfaces and hydrophobic cores of self-assemblies, the nanoparticles formed from the supramolecular peptide are suitable vehicles to encapsulate a photosensitizer for photodynamic therapy. In vitro and in vivo studies demonstrate that the inherent targeting capability and supramolecular strategy greatly boost its photodynamic therapeutic efficiency. This supramolecular peptide holds promising potentials in precise cancer therapy and perspectives for the peptide modification.


Materials and methods
All reagents were commercially available and used as supplied without further purification. Peptide Br-C10H21CO-GGGGGGGCCERGDS (PA) was prepared by solid phase peptide synthesis, purified by HPLC and confirmed by LCMS by Sangon Biotech Co., Ltd (Shanghai, China). 1 H NMR and 13 C HMR spectra were recorded with a Bruker Avance DMX 400 spectrometer using the deuterated solvent as the lock and the residual solvent or TMS as the internal reference. Transmission electron microscopic (TEM) investigations were carried out on a HITACHI HT-7700 instrument. Scanning electron microscopy (SEM) investigations were carried out on a JEOL 6390LV instrument. UV-vis spectra were taken on a PerkinElmer Lambda 35 UV-vis spectrophotometer. The fluorescence experiments were conducted on a RF-5301 spectrofluorophotometer (Shimadzu Corporation, Japan). The ITC experiment was performed on a VP-ITC micro-calorimeter (Microcal, USA). Photographs were taken with a Cannon 550D. Dynamic light scattering was carried out on a Malvern Nanosizer S instrument at room temperature. Fluorescence images were taken with Olympus IX71 inverted fluorescence microscope. 2D and 3D CLSM images were taken on a Zeiss CLSM system. Flow cytometry was carried out on Cytoflex S (Berkman, Germany).
Cells were purchased from ATCC and authenticated by STR. No mycoplasma contamination. The pH of PBS in all experiment was 7.4. The 660 nm laser density in all irradiation procedures was 300 mW· cm -2 .
Cell culture, flow cytometry and confocal laser scanning microscopy. A549 cells were cultured by Dulbecco's modified Eagle's medium/HamF12 (DMEM/F12) containing 10% fetal bovine serum (FBS), 100 μg mL -1 of penicilin and 100 U of streptomycin at 37 o C under a humidified atmosphere of 95% air : 5% CO2. To estimate the endocytosis of TPP@NPs, A549 cells were seeded in a 24-well plate at a density of 6 × 10 4 cells per well and incubated. Then the cells were treated with TPP or TPP@NPs and incubated for another 1 to 6 h. After that, the cells were analyzed by flow cytometry.
To study the endocytosis pathways of TPP@NPs, different endocytosis inhibitors S4 (concentration of inhibitors: amantadine HCl, 10 -3 mM; genistein, 100 mM; amiloride HCl, 2 mM, CytD, 10 μg mL -1 ; c(RGDfK), 50 nM) were used to pretreat A549 cells for 45 min before incubation with TPP@NPs. The distribution of TPP@NPs inside the cells was visualized by 2D and 3D CLSM. For 2D CLSM, A549 cells were seeded in a 6-well plate at a density of 10 4 cells per well and incubated with TPP@NPs for 6 h.
After washing with PBS, the cells were stained with Lyso-tracker green, fixed with 4% formaldehyde and then stained with DAPI. For 3D CLSM, the A549 tumor spheroid was cultured on sphera 96-well U-bottom 3D cell culture plate for 4 days and then stained by TPP@NPs for 12 h, Lyso-tracker Green for 2 h and Hoechst 33342 for 1 h.

Target ability analysis of TPP@NPs. We used endothelial cells (ECs) as normal cells
to study the concentration-dependent light cytotoxicity. The cell culture and PDT process were as the same as them for A549 cells. The cell viability of ECs was estimated by MTT assay.
In vivo PDT. 4T1 tumor-bearing mice (24 mice, initial average tumor volume: 70 mm 3 ; average body weight: 20.0 g) were divided into six groups randomly: (i) control, (ii) irradiation only, (iii) 5 μg of TPP@NPs, (iv) 5 μg of TPP@NPs + irradiation, (v) 25 μg of TPP@NPs + irradiation and (vi) 50 μg of TPP@NPs + irradiation. Group (i) and (ii) were injected with 50 μL of PBS. Group (iii) to (vi) were injected with 50 μL of TPP@NPs with concentrations of 0.1, 0.5 and 1.0 mg mL -1 , respectively. The injection was intratumoral injection. After 30 min, the mice were irradiated under 660 nm laser light at 300 mW cm -2 for 3 min, and another 3 min of irradiation after an interval of 1 min (for avoiding temperature rising under laser). The mice were normally fed after PDT and the body weight and tumor volume were recorded at determined time. After 10 days, the mice were sacrificed and tumors were collected to image and measure the size and weight.
We would like to use in vivo PDT to estimate the anti-tumor efficiency of the supramolecular peptide and to illustrate this therapeutic system definitely having more S5 potentials in cancer treatment. Considering intratumoral injection is more effective, we chose intratumoral injection to dose TPP@NPs.