A near-infrared light-controlled smart nanocarrier with reversible polypeptide-engineered valve for targeted fluorescence-photoacoustic bimodal imaging-guided chemo-photothermal therapy

Despite burgeoning development of nanoplatform made in the past few years, it remains a challenge to produce drug nanocarrier that enables requested on/off drug release. Thus, this study aimed to develop an ideal near-infrared light-triggered smart nanocarrier for targeted imaging-guided treatment of cancer that tactfully integrated photothermal therapy with chemotherapy to accurately control drug release time and dosage. Methods: This delivery system was composed of Ag2S QD coating with dendritic mesoporous silica (DMSN), which acted as nanocarrier of doxorubicin localized inside pores. To provide the nanocarrier with controlled release capability, a polypeptide-engineered that structure was reversible to photothermal effect of Ag2S QD, was covalently grafted to the external surface of drug-loaded DMSN. Results: This nanocarrier with the size of 40~60 nm had satisfactory biocompatibility and photothermal conversion efficiency up to 28.35%. Due to acidity-triggered charge reversal of polypeptide, which significantly extended circulation time and improved targeting ability, fluorescence and photoacoustic signals were still obvious at tumor site post-24 h by tail vein injection and chemo-photothermal synergistic therapy obviously enhanced antitumor efficacy. Mild PTT with multiple short-term exposures not only reduced the side effect of overdose drug but also avoided skin damage caused by long-term irradiation. Conclusion: By adjusting irradiation time and on/off cycle, multiple small amount local drug release reduced the side effect of overdose drug and skin damage. This novel approach provided an ideal near-infrared light-triggered nanocarrier with accurate control of area, time, and especially dosage.

The fluorescence spectra of DOX were obtained by LS-55 fluorescence spectrophotometer (PerkinElmer, USA) and near infrared fluorescence (NIR) spectra were determined by NIRQUEST512-1.7 fiber spectrometer (Ocean Optics, USA). ZS90 ZetaSizer (Malvern, UK) was employed to characterize the size and surface potential of probe, the specific surface area (BET) and pore size distribution (BJH) of nanoparticle were analyzed by ASAP 2020 fully automatic surface area analyzer (Micromeritics Instrument, USA). MDL-III-808-2.5W laser (Changchun New Industries, China) was used for laser irradiation, and thermal imaging was recorded by EasIR-9 infrared thermal imager (Wuhan Guide, China). NIR imaging system [1] and photoacoustic imaging system [2] were built up by our laboratory.

Synthesis of hydrophobic Ag2S QD
The method was modified according to the previous literature [3], the steps were as follows, 76.8 mg Ag(DDTC), 30 g ODE and 6 g DT were heated to 90 º C under Ar protection and maintained for 10 min to remove the water in the system. The reaction was further heated to 150 º C and quenched by n-hexane after maintaining the temperature for 10 min. After the solution was cooled to room temperature, centrifuged (12000 rpm, 10 min) and washed twice with acetone. The hydrophobic Ag2S QD was re-dispersed in chloroform for later use.

Synthesis and purication of the polypeptide
Cys-P and cys-P-RGD plasmids were constructed by conventional genetic engineering method.
The polypeptide was prepared and purified according to previous methods in our laboratory [4,5].
Bacterial was cultured at 37 º C in LB liquid medium supplemented with ampicillin (50 mg/L) and

Photothermal effect and photothermal conversion efficiency
300 µL Ag2S@M at different concentrations (0~8 mg/mL) was irradiated with 808 nm laser (2 W/cm 2 ) for 5 min at a distance of 5 cm, and same concentration (4 mg/mL) Ag2S@M was irradiated at different laser intensities (0.5~2.5 W/cm 2 ). The temperature changes were recorded by thermal 4 imager. In photothermal stability test, 300 µL Ag2S@M was irradiated with laser (2 W/cm 2 ) for 10 min and then naturally cooled to room temperature (n=6). The photothermal conversion efficiency (η) was determined by following equation [6], η= hS(T max -T surr )-Q dis I(1-10 -A 808 ) , I was irradiation power to be 800 mW, A808 was absorption of probe at 808 nm (A808=0.884), Tmax-Tmin was 27.5 º C, τs and hS were calculated to be 154.9 s and 8.134; same method measured water (300 μL) as a control and QDis was calculated to be 26.48. On the basis of data, η could be obtained.

Protein absorption
BSA was used as a model protein to evaluate protein adsorption on nanoparticle in different environments according to previous study [7]. Ag2S@M and Ag2S@M-P-RGD nanoparticles were

Immunological analysis
In order to investigate the effect of reducing immune response after polypeptide modification of nanoparticles, the inflammatory cytokines (IL-6, IL-1β and TNF-α) were analyzed. Ag2S@M and Ag2S@M-P-RGD (100 µg/mL) were incubated with macrophage RAW 264.7 at 37 º C for 12 h. The mRNA expression levels were quantified by real-time reverse transcription polymerase chain reaction

Hemolysis Assay
Blood compatibility was evaluated by hemolysis assay. Fresh mice blood was extracted from orbital vein and stabilized with heparin. 2 mL whole blood was diluted with PBS to 4 mL and centrifuged at 3500 rpm for 5 min to isolate red blood cell (RBCs). The RBCs were further washed and finally diluted to 20 mL PBS. Different concentrations of Ag2S@M and Ag2S@M-P-RGD were incubated with RBCs at 37 º C for 4 h, water as positive control and PBS as negative control. The absorbance of the supernatants from each group was measured using ELX808IU microplate reader (Biotek, USA) at 570 nm. The hemolysis percentage was calculated as follows, hemolysis percentage = (ODtest-ODnegative control)/(ODpositive control-ODnegative control)× 100 %.

Circular dichroism of cys-P-RGD
Polypeptide cys-P-RGD with concentration of 100 µM was scanned by J-810 circular dichroism spectrometer (Jasco, Japan) with temperature gradient in range of 20~70 º C. The wavelength spectra were measured over a range from 190 to 300 nm with a step size of 1 nm.

Cell culture
HeLa and MCF-7 cell lines were cultured in DMEM culture medium (Gbico, Invitrogen) containing 10 % FBS and incubated in humidified atmosphere containing 5 % CO2 at 37 °C. In the experiment, 0.25 % trypsin was used for digestion.

NIR-triggered drug release
Two groups of 5 mg drug-loaded Ag2S@M/D-P-RGD were dispersed in 2 mL PBS (0.01 M, pH 7.4) and incubated in 37 º C water bath and one group was irradiated with laser (2 W/cm 2 , 10 min) at different time points. Both groups were centrifuged (12000 rpm, 10 min) to obtain supernatant and DOX contents were measured according to the standard curve.

Endocytosis and cytotoxicity
6 HeLa and MCF-7 cells were seeded in 6-well plate (5×10 4 cells per well) and grown for 24 h.
Replaced serum-free medium, 100 µg/mL Ag2S@M-P-RGD and Ag2S@M-P were cultured with cells for another 4 h. After terminating the culture, cells were washed with PBS to remove free probe, then immobilized with 4 % paraformaldehyde and collected for fluorescence and photoacoustic imaging.
For confocal imaging of cellular uptake of probe, HeLa and MCF-7 cells were seeded in glassbottom Petri dishes and incubated for 24 h. The cells were treated with Ag2S@M/D-P-RGD and Ag2S@M/D-P (CDOX = 1 µg/mL) for another 4 h and washed with PBS. After irradiation of laser (2 W/cm 2 ) for 10 min or not, different treatment groups were imaged by FV1000 confocal microscopy (Olympus, Japan). The RGD blocking group was pre-incubated with RGD (10 µg/mL) for 2 h before probe addition. DAPI was nuclear dye and LysoTracker® Green DND was lysosomal dye.
In order to evaluate the in vitro safety of probe, MTT assay was used to analysis cell viability of HeLa and MCF-7 cells. Cells were seeded in 96-well plate (1×10 4 cells per well) and incubated overnight.
Subsequently, cells were treated with different amounts of Ag2S@M-P-RGD andAg2S @M-P (25, 50, 100 and 200 µg/mL) for 24 h. After 20 µL MTT (5 mg/mL) was added into each well to incubate for another 4 h, the medium was discarded and 150 µL DMSO was added to solubilize formazan crystal, ELX808IU microplate reader (Biotek, USA) was used to measure the absorption at 490 nm.
Untreated cells were used as negative control, n=4.

Pharmacokinetics and distribution of Ag2S@M-P-RGD
Ag2S@M and Ag2S@M-P-RGD (100 mg/kg) was injected i.v. into female Kunming mice. After 5 min, 1, 3, 6, 12 and 24 h after injection, blood was collected from the mice and dissolved in nitric acid to obtain the total amount of Ag + by graphite furnace atomic absorption spectrometry. HeLa tumor-bearing nude mice were injected with Ag2S@M-P-RGD (100 mg/kg) intravenously, and the fluorescence intensity of different organs was measured by FL imaging system at different time points.

Statistical analysis
All data were presented as mean±SD unless otherwise stated. All the experiments were performed at least in triplicate. The statistical significance was determined using two-tailed Student's test (*p<0.05, **p<0.01) unless otherwise stated.     In order to compare Ag2S-loaded MSN with the same amount of Ag2S nanoparticles, the oilsoluble Ag2S QD was encapsulated by using the hydrophobic structure of Pluronic F-127 to obtain hydrophilic Ag2S nanomicelle. The photoacoustic imaging and photothermal effect of Ag2S@P (η=29.49%) were evaluated by using the same amount of Ag2S (Ag + concentration was measured by graphite furnace atomic absorption).