Materials

CPT was procured from Dalian Meilun Biology Technology Co., Ltd (Liaoning, China) and used to synthesize DCPT as per our previous report without any modifications [18, 22]. Phenylalanine (Phe), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-ε-Cbz-L-lysine, and 1-hydroxy-2,5-pyrrolidinedione (NHS) were bought from GL Biochem Ltd (Shanghai, China). Ce6 was provided by Frontier Scientific (Utah, USA). Dimethyl sulfoxide (DMSO) was purchased from Aladdin (Shanghai, China). N, N-dimethylformamide (DMF) was purchased from Damao (Tianjin, China). Hydrogen bromide in acetic acid (33 wt%) and bis(trichloromethyl) carbonate were supplied by J & K Scientific Ltd (Beijing, China). Thiazolyl blue tetrazolium bromide (MTT) was purchased from Amresco (Solon, Ohio, USA). PEG6000 were obtained from JenKem Technology Company (Beijing, China). DAPI were purchased from Beyotime Biotechnology (Jiangsu, China).

Synthesis and characterization of PKF-Ce6 conjugate

First, N-carboxyanhydride of phenylalanine (Phe-NCA) and N-ε-Cbz-L-lysine (Lys(Z)-NCA) were synthesized as previously reported [23]. Then, polylysine-polyphenylalanine (PKF) was synthesized as following steps [18, 24]. Briefly, a solution of 3.27 mmol of Lys(Z)-NCA in 15 mL of anhydrous DMF was subjected to ring-opening polymerization (ROP) by adding 0.11 mmol of hexylamine. After 72 h of reaction at 30 °C under nitrogen protection, followed by precipitation, PLys (Z) was obtained and further used to trigger the ROP of Phe-NCA at room temperature to synthesize PLys(Z)-PPhe. Then, purified PKF was attained by deprotecting the carbobenzoxy groups of PLys(Z)-PPhe using trifluoroacetic acid (TFA) and HBr solution, followed by precipitation in pre-cooled ether, dialysis against water, and freeze-dry under vacuum. The synthesis of PKF was confirmed by 1 H NMR spectroscopy (Bruker, USA).

In addition, Ce6 was further grafted onto the PKF polypeptide through the following process: First, Ce6 (18 mg), EDC and NHS (20 mg for each) were added in 3 mL of DMSO and allowed to react for 30 min, thus initiating the carboxyl group of Ce6. Later, the mixture was added into PKF (in DMSO) and reacted for 1 d. Then the crude product was transferred into a dialysis tube containing DMSO for another 24 h. An UV-Vis spectrophotometer (HACH, USA) was employed to characterize the synthesized PKF-Ce6 polymer.

Formulation and characterization of PCD and PPCD micelles

PCD and PPCD micelles were fabricated using dialysis method. In brief, PKF-Ce6 (at an equivalent Ce6 mass of 100 µg) and 500 µg of DCPT dissolved in DMSO were dripped into 2 mL of water and allowed to stir for 2 h. PCD was yielded after dialysis (with a MWCO 3500 Da) and centrifugation (4700 r/min, 5 min). Furthermore, appropriate amounts of α,ω-diformyl poly(ethylene glycol) (OHC-PEG-CHO, about 4–6 times of PKF), which was synthesized according to reported procedures [25], were added to PCD for 30 min of reaction, yielding the PPCD micelles. The particle sizes and zeta potential of PCD and PPCD micelles were assessed using a Nano-ZS Zetasizer instrument (Malvern, UK). The encapsulation efficiency of DCPT and Ce6 were measured by UV-Vis spectrophotometer at 380 or 660 nm, respectively. Then, the morphology of PCD and PPCD was examined using a transmission electron microscope (FEI Tecnai). To assess the pH-sensitivity of the PEG detachment, 0.5 mL of both PCD and PPCD were combined with 0.5 mL of PBS (pH 7.4 or 6.5) and incubated for 15 min. Changes in the zeta potential of the micelles were examined. For in vitro release of DCPT from PPCD, dialysis (MWCO: 3500 Da) of 1 ml of PPCD against 20 ml of PBS with different pH and GSH conditions was performed. The release of DCPT was monitored by drawing 1 ml of sample at various time points. The fluorescence intensity of DCPT in the sample was read by using a SpectraMax microplate reader (λex: 370 nm and λem: 435 nm).

Cell culture

The human lung adenocarcinoma cell line A549 and PC-9 cells were offered by American Type Culture Collection (MD, USA). Cells were incubated in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin under 5% CO2 at 37 °C.

Cellular uptake

A549 and PC-9 cells were planted in 12-well plates with a number of 1.0 × 105 cells per well. Then, DMEM at pH 7.4 or 6.5, containing Ce6, PCD and PPCD micelles (all at an equivalent Ce6 concentration of 1 µM) were added. After 1, 4, or 8 h, the cells were rinsed and harvested to mearsure the fluorescence intensity of Ce6 by a BD FACS Canto™ flow cytometer (NJ, USA).

Additionally, the cellular uptake of micelles was observed using the Leica SP8 CLSM system (Wetzlar, Germany). Firstly, A549 and PC-9 cells were planted with a number of 1.5 × 105 cells per dish in individual confocal petri dish. Subsequently, the cells were exposed to DMEM (pH 7.4 or 6.5) mixed with Ce6, PCD and PPCD micelles (equivalent Ce6 concentration of 2 mM). After 8 h-incubation, cells were fixed and stained with DAPI (5 µg/mL) and observed under a CLSM.

ROS Measurement

The ROS generating ability of PCD and PPCD after laser irradiation were detected by 1,3-DPBF reactive oxygen probes (Merck, USA). Breifly, 20 µg of DPBF and 2 µg of Ce6 (in PCD or PPCD) were added to 996 l of DMF solution and exposed to a laser (660 nm, 100 mW/cm2). The decolarization of DPBF after light irradiation was assessed using a UV spectrophotometer at a wavelength of 415 nm. In addition, intracellular ROS production was evaluated through the application of DCFH-DA ROS assay kit (Beyotime Biotechnology, Jiangsu, China). Firstly, A549 and PC-9 cells were seeded in 12-well plates with a number of 1.5 × 105 cells per well. Subsequently, free CPT, free DCPT, free (CPT + Ce6), free (DCPT + Ce6), PCD and PPCD micelles (equivalent Ce6 1 µM) were introduced and incubated for 8 h. The intracellular ROS was then stained with DCFH-DA (20 µM) for a duration of 20 min. After that, each well was subjected to laser irradiation (660 nm, 100 mW/cm2, 10 min) or left untreated. The fluorescence signals were determined by a flow cytometer.

Cell viability assay

A549 and PC-9 cells were placed in 96-well plates with a number of 8 × 103 cells per well. Once the cells had adhered, DMEM containing various CPT concentrations of free CPT, free DCPT, free (CPT + Ce6), free (DCPT + Ce6), PCD and PPCD micelles were added for cell culture at both pH 7.4 and pH 6.5 with an incubation period of 24 h. Then, medium was substituted with MTT-containing medium (1 mg/mL) and incubated for 4 h. Following this, the medium in each well was discarded and substituted with 100 µL of DMSO to dissolve the formazan crystals. Finally, the absorbance of each well was read by microplate reader at 570 nm. The cell viability was calculated based on at least three independent experiments.

Furthermore, the chemo-photodynamic combination therapeutic effects against A549 and PC-9 cells were carried out. Similarly, cells were subjected to varying concentrations of Ce6, including free Ce6, free (CPT + Ce6), free (DCPT + Ce6), PCD and PPCD micelles, under both pH 7.4 and pH 6.5 conditions. After an incubation period of 8 h, the treated cells were subjected to a laser (660 nm, 100 mW/cm2) for 10 min, followed by an additional 16 h-incubation. At last, the relative cell viabilities were determined using MTT assay and compared to the control group.

Visually observation of combination effects

With the aim to directly visualize the combination therapy efficacy, A549 and PC-9 cells were planted into 6-well plates with a number of 2 × 105 cells per well. Medium containing CPT, DCPT, as well as medium supplemented with free Ce6, free (CPT + Ce6), free (DCPT + Ce6), PCD or PPCD micelles (equivalent Ce6 1 µM) were introduced to replace culture medium. Following an incubation period of 8 h, the subsequent five groups underwent a laser irradiation (660 nm, 100 mW/cm2) for 10 min. After an additional 0.5 h-incubation, the cells were rinsed with buffer solution and then stained with Calcein-AM/PI double stain kit (Yeason, Shanghai, China) for viable and non-viable cells labelling. The corresponding fluorescence images were taken by a GE Healthcare Life Science IN Cell Analyzer 2000 (MA, USA).

Cell apoptosis

The micelle-induced cell apoptosis was further invesigated by a Cell Signaling Technology Annexin V-FITC kit (MA, USA). Briefly, A549 and PC-9 cells were seeded in 12-well plates and exposed to various formulations including free CPT, free DCPT, free Ce6, free (CPT + Ce6), free (DCPT + Ce6), PCD and PPCD micelles. After an 8-h incubation, the latter five groups were subjected to laser irradiation (660 nm, 100 mW/cm2) for 10 min, followed by an additional 12 h-incubation. Sequently, cells were collected and gently suspended in 96 µL of binding buffer, followed by staining with 1 µL of Annexin-FITC together with 12.5 µL of PI. Finally, the apoptosis of cells was investigated using flow cytometry.

Animals

BALB/C mice (female, 6–7 weeks) and BALB/c nude mice (male, 5 weeks) were provided by Gunagdong Medical Laboratory Animal Center (Guangzhou, China). For tumor model establishment, a suspension of 5 × 106 A549 cells in 100 µL of PBS was inoculated into the right flank of each nude mouse.

Pharmacokinetics study

Female BALB/C mice were allocated into 3 groups (n = 3) randomly. Prior to the study, mice were fasted with water for overnight. An equivalent dose of 5 mg per kg body weight of DCPT was administered by different routes (PCD and PPCD: intravenously injection; DCPT: intraperitoneally injection). 10 µl of blood samples were obtained from the tail vein at various time intervals, which was diluted to 50 µL with PBS for measurement. Pharmacokinetic study of Ce6 was performed using another 3 groups of female BALB/C mice. Equivalent 1 mg per kg body weight Ce6 dose was administered by different routes (PCD and PPCD: intravenously injection; Ce6: intraperitoneally injection). Fluorescence intensity of DCPT and Ce6 in blood sample was recorded by using a SpectraMax microplate reader (DCPT: λex = 370 nm and λem = 435 nm; Ce6: λex = 402 nm and λem = 652 nm).

Assessment of therapeutic efficacy in vivo

Once the tumor grew to a size of approximately 100 mm3, the mice were categorized into 5 treatment groups (n = 5): PBS, PCD, PPCD (intravenous administration), DCPT, Ce6 (intraperitoneal administration). The administration does was every 3 days, with total 5 times injection (DCPT dose: 5 mg per kg body weight). Following a 12-h interval post-administration, the tumor site of each mouse was performed laser irradiation (660 nm, 100 mW/cm2) for a duration of 15 min. The tumor size was assessed using a vernier caliper, and body weight was monitored every alternate day. The tumors were calculated according to the formular: V = 0.5 length x width2. After completion of the entire therapeutic regimen, the mice were euthanized to dissect the main organs and tumor tissues for histopathological evaluation. After fixing with 10% (W/V) formaldehyde, embedding in paraffin, slicing, and stainging using hematoxylin and eosin, the 3-µm slices were examined using an Olympus BX43 microscope (Tokyo, Japan).

Statistical analysis

All data are expressed as the mean ± SD of independent measurements unless explicitly stated otherwise. Statistical significance was indicated when p < 0.05 (denoted by *), < 0.01 (denoted by **) and < 0.001 (denoted by ***).