NIR‐Activated Polydopamine‐Coated Carrier‐Free “Nanobomb” for In Situ On‐Demand Drug Release

Abstract Carrier‐free nanoparticles with high drug loading have attracted increasing attention; however, in situ on‐demand drug release remains a challenge. Here, a novel near‐infrared (NIR) laser‐induced blasting carrier‐free nanodrug delivery system is designed and fabricated by coating doxorubicin (DOX) nanoparticles (DNPs) with a polydopamine film (PDA) that would prolong the blood circulation time of DNPs and avoid the preleakage of the DOX during blood circulation. Meanwhile, the NH4HCO3 is introduced to trigger in situ “bomb‐like” release of DOX for the production of carbon dioxide (CO2) and ammonia (NH3) gases driven by NIR irradiated photothermal effect of PDA. Both in vitro and in vivo studies demonstrate that the carrier‐free nanovectors with high drug loading efficiency (85.8%) prolong tumor accumulation, enhance chemotherapy, achieve the synergistic treatment of chemotherapy and photothermal treatment, and do not induce any foreign‐body reaction over a three‐week implantation. Hence, the delicate design opens a self‐assembly path to develop PDA‐based NIR‐responsive multifunctional carrier‐free nanoparticles for tumor therapy.


Cell culture
The human cervix carcinoma (HeLa) cells were grown in DMEM medium with 10% fetal bovine serum and 1% antibiotics (penicillin-streptomycin, 10000 U mL -1 ) at 37 o C in a humidified atmosphere of 5% CO 2 .

TEM observation
The morphologies of the fabricated DNPs/N@PDA dispersed on a carbon-coated copper grid were investigated using a transmission electron microscope (TEM) (JEOL JEM-2100F, Japan) with an acceleration voltage of 80 kV. Before visualization, a droplet of the sample solution was placed on a copper grid with formvar film.

Particle size and zeta potential measurement
Dynamic light scattering (DLS) was used to determine the hydrodynamic size and zeta potential of the nanoparticles at 25 o C by Nano-ZSZEN3600 (Malvern Instruments). Data were shown as ± standard deviation (SD) based on three independent measurements.

Fluorescence spectra and XRD patterns of the nanoparticles
The fluorescence spectra of the nanoparticles were obtained by Fluorescence spectrometer (Edinburgh F920, UK). XRD patterns were obtained using an X-ray diffractometer (Brooker, D8 Advanced, Germany) to confirm the NH 4 HCO 3 was successfully encapsulated.

Determination of drug loading efficiency (DLE)
The Fluorescence intensity of DNPs dispersion in DMSO solution (FI (DNPs)) was measured after separation by centrifugation. The fluorescence intensity of DOX in DMSO solution (FI (DOX)) was also measured. By comparison, the DLE was calculated based on the formula: DLE% = FI (DNPs) / FI (DOX) × 100%. The fluorescence absorbance intensity of DOX was measured at 600 nm by a F920 fluorescence spectrometer (Edinburgh) with excitation wavelength of 488 nm.

In vitro drug release test
The

In vitro cytotoxicity
The in vitro cytotoxicity of DNPs/N@PDA against HeLa cells was assessed by the standard MTT assay. First, HeLa cells were seeded into a 96-well plate at a density of 6.0×10 3 cells/well and then incubated in 100 μL DMEM containing 10% FBS for 24 h at 37 o C prior to samples addition. After the cells were incubated with different concentration of DNPs, DNPs@PDA and DNPs/N@PDA for 4 h, the medium was removed and the cells were further incubated in the fresh DMEM for 48 h. To investigate the photocytotoxicity of the formulations, the cells co-incubated with the samples at pH 5.0 were treated with NIR irradiation (808 nm, 5 W cm -2 , 1 min) while other operations were the identical unless noted.
After that, the medium was removed and 200 μL of fresh DMEM containing 10% FBS and 20 μL of MTT (5 mg/mL in PBS buffer solution) was added and the cells were further incubated for 4 h at 37 o C. Subsequently, the medium was replaced by 150 μL of DMSO. The absorbance intensity at 570 nm was measured using a microplate reader (Bio-Red, Model 550, USA). The relative cell viability was calculated as: Cell viability (%) = (OD(sample)/OD(control)) × 100% Where OD(control) and OD(sample) were obtained in the absence and presence of the samples respectively. Data were expressed as mean ± standard deviation (SD) based on three independent measurements.
For the photothermal ablation assay, the HeLa cells were treated with the same method as mentioned above. The DOX concentration was fixed at 5 μg/mL. After incubation for 48 h, the medium was replaced by fresh medium and stained with LIVE/DEAD Viability/Cytotoxicity Kit according to the directions. Finally, the cells were detected with a fluorescent microscope (OLYMPUS, Japan).

In vitro cellular internalization assay
Confocal laser scanning microcopy was used to determine the in vitro cellular uptake of the DNPs, DNPs@PDA and DNPs/N@PDA in HeLa cells under three different conditions: (Ⅰ) pH 7.4 (neutral environment), (Ⅱ) pH 5.0 (acid environment), (Ⅲ) pH 5.0 (acid environment) with NIR irradiation. The HeLa cells were seeded in a single dish with a density of 1.0×10 5 cells/well and incubated in 1 mL of DMEM containing 10% FBS for 24 h at 37 o C. Then the medium was replaced by fresh medium containing DNPs, DNPs@PDA and DNPs/N@PDA with the pH value of 7.4 or pH 5.0, respectively (the concentration of DOX was 10 μg/mL).
After incubated for 4 h, the medium was removed and fresh culture medium was added, and the HeLa cells treated with the materials at pH 5.0 were exposed under laser irradiation (808 nm, 5 W cm -2 ) for 5 min. Then the cells were further cultured for another 2 h. After removing the medium, the cells were washed with PBS for several times and 1 mL of DMEM containing 10 μL Hoechst 33342 was added and the cells were incubated for 15 min at 37 o C.
Prior to the imaging under a confocal laser scanning microscopy (Nikon C1-si TE2000, Japan, excitation filter 488 nm and emission cut-off filter 570-630 nm for red light), the cells were washed several times by 1 mL of PBS. It was noted that the exposure to strong light should be avoided in the whole process to protect the fluorescent dyes.

Flow cytometry analysis
Flow cytometry was used to quantitatively evaluate the cellular internalization of DOX.
Briefly, the HeLa cells were seeded in the 24-well plates with a density of 6 × 10 4 cells/well and incubated for 24 h. After that, the cells were treated with 1 mL of fresh DMEM containing the as-prepared samples. The cells were incubated for 4 h with/without NIR irradiation (808 nm, 5 W cm -2 , 5 min). Then the cells were further incubated for another 2 h, and the medium was removed. Immediately, the cells were washed by fresh PBS thrice. Then the cells were trypsinized with 0.25% trypsin for 3 min at 37 o C. Subsequently, the cells were collected, washed and resuspended in 500 μL PBS. The fluorescence intensity was detected by a flow cytometer (BD LSRFortessa, USA). The blank cells served as a negative control.

Animals and tumor models
Male BALB/c nude mice (5 weeks old, ca. 20 g body weight) were purchased from Chinese

In vivo and ex vivo fluorescence imaging
The in vivo and ex vivo fluorescence imaging was performed in male BALB/c nude mice bearing HeLa tumor on right hind limb. The as-prepared DNPs and DNPs/N@PDA formula at a DOX dosage of 3 mg kg -1 were intravenously injected into the mice bearing HeLa tumor via tail vein.
For in vivo fluorescent imaging and biodistribution assays, the mice were anesthetized by trichloroacetaldehyde hydrate (10%) with a dosage of 40 mg/kg body weight at 1 h, 4 h and 24 h post-injection, respectively. The DOX-related fluorescence was detected by a singlefilter set with the excitation wavelength of 480 nm and emission wavelength of 600 nm in the absence of autofluorescence produced by the skin and blood vessels. For ex vivo fluorescence imaging and biodistribution assay, after 24 h post-injection, the mice were sacrificed and the tumors and major organs were dissected, washed with cold saline and then subjected to Xtreme imaging system to obtain the fluorescence images. Tumors and organs were stored at -80 o C for further use.

Statistical analysis
All data were expressed as the mean ± standard deviation (S.D.) (n = 3~5). The statistical significance between different groups was evaluated with Student's t-test. p<0.05 was considered to be statistically significant.