br side effects and toxicity of free DOX
side effects and toxicity of free DOX. However, no apparent weight loss was observed in other group conforming the safety of nanocomposites. H&E staining of the major organs (Fig. S14, heart, liver, spleen, lung, and kidney) further demonstrated nanocarriers with no tissue damage. In vivo safety of BPNs-PDA-PEG-PEITC was verified through blood Puromycin data. Blood biochemistry data is a conventional method to detect the disease and health condition which could be used to evaluate potential biomedical application of the BPNs-PDA-PEG-PEITC for theranostic nanoplatform. The healthy rats were intravenously injected with BPNs-PDA-PEG-PEITC and then blood collection after 1 day, 7 days, and 14 days. The blood of healthy rats without injection were selected as control. Nearly all blood biochemistry data of the rats in-travenously injected with BPNs-PDA-PEG-PEITC were very close to the control group (Fig. S15). These results indicated the promising in vivo application of this BPNs-based nanoplatform for theranostic delivery.
3. Experimental section
The bulk BP was provided by Nanjing XFNANO Materials Tech Co., Ltd. DMSO, MnCl 2, Dopamine, trimethylaminomethane (Tris), DOX, sodium hydroxide (NaOH), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), phenethyl isothiocyanate, 1,3-diphe-nylisobenzofuran (DPBF), H2DCFHDA and N-methyl-2-pyrrolidone (NMP) were purchased from Aladdin Industrial Co., Ltd. PEG-NH2, 4′,6-diamidino-2-phenylindole (DAPI) were provided from Sigma-Aldrich. PBS was obtained from Beijing Solarbio Science & Technology Co., Ltd. Dulbecco minimum essential medium (DMEM), streptomycin, peni-cillin, FBS, were obtained from Thermo Fisher Scientific. MCF-7 cells and MCF-7/ADR cells were obtained from Jiangsu KeyGEN BioTECH Corp., Ltd.
MCF-7 cells and MCF-7/ADR cells were cultured at 37 °C with 5% CO2 in a humidifed incubation chamber. Dulbecco minimum essential medium (DMEM) containing with 10% FBS, 1% streptomycin, and 1% penicillin was used as culture medium.
The morphology of samples was studied by employing TEM, (Tecnai
G2 F30 S-TWIN) and AFM (Bruker Dimension Icon) measurement. The size distribution of the BPNs-PDA-PEG-PEITC was measured through the DLS technology (Malvern Nano-ZS 90 Nanosizer). The FT-IR spectra measurement was performed on a NEXUS670 FTIR spectrometer. Raman spectrometer (LabRam HR800) with 514 nm laser excitation was used to measure the Raman spectra of sample. XRD pattern was performed on a Rigaku Ultima IV powder diffractometer. Agilent 8453 UV–vis spectrophotometer was used to measure the UV–vis spectra of samples. The surface composition of sample was measured through XPS (ESCALAB 250, Thermo Fisher). ESR spectrum of sample was measured by using Bruker EMXplus Spectrometer System.
3.4. Preparation of BPNs
The synthesis of BPNs was similar to previous report through NMP solvent exfoliation . First, 50 mL of saturated NaOH/NMP was mixed with 20 mg of bulk BP. Under ice/water bath, the mixture was sonicated for 6 h. In order to remove the nonexfoliated bulk BP, the mixture was centrifuged for 10 min at 4000 rpm and then the pre-cipitate was discarded. Afterwards, the further centrifugation of sus-pension (5 min at 10,000 rpm) was performed and the precipitate was collected. Chemical Engineering Journal 370 (2019) 387–399
3.5. Preparation of BPNs/DOX
5 mg of BPNs was dispersed in 50 mL of DOX (1 mg/mL) under stirring for one day. After centrifugation, the BPNs/DOX were separated and washed with PBS, and then dried under vacuum.
3.6. Preparation of BPNs-PDA/DOX
For dopamine modification, the 25 mg BPNs/DOX was dispersed in
10 mL Tris buffer (10 mM, pH 8.5) and then 5 mg dopamine hydro-chloride was added in the solution with rotation. After 5 h, the solution was centrifugated to collect the precipitate and then purified by water.
3.7. Preparation of BPNs-PDA-PEG/DOX
The conjugation of PEG-NH2 via Schiff base reaction (Fig. S16),
10 mL of PEG-NH2 solution (1 mg/mL, in Tris buffer) was mixed with 26 mg BPNs-PDA/DOX and stirred for 1 h. Afterwards, the product was obtained by centrifugation and washed with water several times.
3.8. Preparation of BPNs-PDA-PEG-PEITC/DOX
The conjugation of PEITC via π–π stacking (Fig. S16), 27 mg BPNs-PDA-PEG/DOX was dispersed in 10 mL ethanol. Under stirring, 10 mg PEITC was added in dispersion. After 8 h, the mixture was washed with ethanol to remove redundant PEITC. All the washed and separated supernatant in preparation process was collected through measuring the UV absorbance at 480 nm of supernatant to detect the amount of unloaded DOX. The formula of calculating drug loading capacity was as follow: [(M1 − M2)/(M3 − M1 + M2)] × 100% where M1 re-presented the original weight of DOX added, M2 is the weight of un-loaded DOX, and M3 the weight of BPNs-PDA-PEG-PEITC/DOX. The preparation process of nanocarrier without loading DOX was similar to privious section.