2.1 Synthesis of FA-PEG-COOH
1 g of FA and 1 g of EDCI were added into 30 mL DMSO and activated at 40 ℃ for 30 min. After that, 2 g of NH2-PEG-COOH was added, and the reaction continued for 2 days. The above reaction solution was dialyzed in deionized water (DI water) with dialysis bag (2500 kDa), and the DI water was changed every hour. The solution was lyophilized two days later to obtain the products.
2.2 Preparation of FA-PEG-PTX
0.15 g of PTX and 0.5 g of CDI were added into 10 mL DMSO, activated at 40 °C for 4 h, with 1 g of FA-PEG-COOH, 0.5 g of EDCI and 0.5 g of DMAP were added into 20 mL DMSO and activated at 40℃ for 30 min. Then, the two reaction solutions were mixed and continued for two days. After that, the above reaction solution was dialyzed in DI water with dialysis bag (2500 kDa), and the DI water was refreshed every hour. The solution was lyophilized two days later to obtain the products.
2.3
FTIR and
1
H NMR detection
Fourier Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance Spectroscopy (1H NMR) were used to test the synthesis of materials. The samples were prepared by taking a small amount of powder, grinding and pressing into pieces with KBr or dissolving in deuterated DMSO. The infrared spectra of samples were obtained by scanning in the range of 500 ~ 4000 cm−1.
2.4 Preparation of P@FPP NMs
5 mg of FA-PEG-PTX nano-conjugates were dissolved in 5 mL DMSO. Then, the dialysis bag (7000 kDa) was used to dialysis the above liquid in PBS, and then changed the water every hour. The FA-PEG-PTX was obtained using microporous filter membrane (450 nm) after one day for further detection. 5 mg of FA-PEG-PTX and 0.5 mg of PRI were dissolved in 5 mL DMSO. Then the dialysis bag (7000 kDa) was used to dialysis the above liquid in PBS, and then changed the water every hour. The P@FPP NMs were obtained using microporous filter membrane (450 nm) in the next day.
2.5 Characterization of P@FPP NMs
Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were performed to characterize the structure of NMs. The prepared P@FPP NMs were placed into DTS0012 or DTS1070 cell (Malvern Instruments, Malvern, UK), then placed into the DLS granulometer (DLS, Zetasizer ZS90, Malvern Instruments, Malvern, UK). Each sample was tested three times, 1 min/time. The test conditions were argon ion laser, wavelength 658 nm, temperature 25 ± 0.1 °C, and DLS angle 90°. The ζ-potential was determined at the same time. The operating conditions were 11.4 v cm−1, 13.0 mA, and 25 °C. The sample solvent was diluted with distilled water. The NMs samples observed by TEM that were dropped on the copper mesh coated with carbon support film, dyed with 2% phosphoric acid, and dried naturally.
2.6 Drug-loading and release evaluation
The PTX mass fraction was calculated by 1H NMR peak area of FA-PEG-PTX and FA-PEG-COOH as follows:
$$\begin{aligned} {\text{Peak}}\,{\text{area ratio (PAR) = }} & \Delta (FA - PEG - {\raise0.7ex\hbox{${{\text{PTX}}}$} \!\mathord{\left/ {\vphantom {{{\text{PTX}}} {{\text{FA}}}}}\right.\kern-\nulldelimiterspace} \!\lower0.7ex\hbox{${{\text{FA}}}$}} \\ & - {\text{PEG - COOH)}}\frac{{{\text{A}}(3.52{\text{pmm)}}}}{{{\text{A(7 - 8}}_{{{\text{ppm}}}} )}} \\ \end{aligned}$$
$${\text{w}}\% \, = { 1}/\left( {{1} + {\text{PAR}}*\left( {{15}*{44}} \right) \, / \, \left( {{4}*{853}.{91}} \right)} \right) \times { 1}00\%$$
The PRI loading and release of NMs were determined by dual wavelength method. FA-PEG-PTX has a peak at the wavelength of 290 nm, as 425 nm of PRI. Both of their absorbances at 290 nm and 450 nm were measured at five of different concentrations to establish standard curve. Diluted the P@FPP NMs and determined the absorbance at two wavelengths, the absorbance of each point is equal to the sum of the absorbance of FA-PEG-PTX and PRI, so the concentration of FA-PEG-PTX and PRI in P@FPP NMs can be calculated. The PTX loading of P@FPP were calculated by the ratio of 10 mg PTX to FA-PEG-PTX at δ = 2.5 ppm. The drug-loading (LC) was calculated as follows:
$${\text{LC}}_{{{\text{PRI}}}} \% \, = \,\frac{{{\text{C}}_{{({\text{PRI}})}} }}{{{\text{C}}_{{({\text{FA}} - {\text{PEG}} - {\text{PTX}})}} + {\text{C}}_{{({\text{PRI)}}}} }}\, \times \,100\%$$
$${\text{LC}}_{{{\text{PTX}}}} \% \, = {\text{c}}_{{{\text{FA}} - {\text{PEG}} - {\text{PTX}}}} \times \, \left( {{1} - {\text{ LC}}_{{{\text{PRI}}}} \% } \right) \, \times {\text{w}}\% \times { 1}00\%$$
To test the drug release of P@FPP, 5 mL of NMs were placed into the dialysis bag (7000 kDa) and dialyzed in 20 mL PBS, shaking on a 37 °C degree shaker. PBS liquid out of the dialysis bag was removed, and 20 mL of fresh PBS was replaced at 0, 0.5, 2, 4, 8, 16, 24 and 48 h. The volume of PBS which has been taken out at each hour was measured accurately and expressed as Vt, and the concentration of PRI was expressed as Ct.
The concentration of free-PTX was determined by measuring the absorption at the wavelength of 230 nm with ultraviolet visible spectrophotometer. Since PTX in FA-PEG-PTX released less in PBS with pH 7.4, we use the absorbance of FA to represent the concentration of PTX. The drug release of each time was calculated as follows:
$${\text{Q}}_{{\text{t}}} \% \,\, = \,\frac{{\sum\nolimits_{{\text{t = 0}}}^{{\text{n}}} {{\text{V}}_{{\text{t}}} {\text{C}}_{{\text{t}}} } }}{{{\text{V}}_{{{\text{db}}}} {\text{C}}_{{\text{db(PRI)}}} }}\, \times 100\%$$
where Qt denotes the drug release rate at t hour, Vdb represents the volume of PBS in the dialysis bag, Cdb(PRI) is the initial concentration of the NMs sample. (t = 0, 0.5, 2, ⋯, n, ⋯, 48 h, both V0 and C0 are equal to zero).
2.7 Endocytosis verification of P@FPP NMs
To evaluate the cell uptake of P@FPP NMs by A549 cells, the fluorochrome of Indocyanine green (ICG) were used to instead PRI for constructing ICG@FPP (I@FPP) NMs. In brief, A549 cells (1 × 105) were plated and incubated with I@FPP for 2, 4, 6 h, and then the fluorescence under the fluorescence microscope were observed and imaged after nucleus staining with Hoechst 33342.
2.8 Cell culture and cell viability evaluation
2.8.1 Cell culture
Human non-small cell lung cancer (NSCLC) of A549 cells were obtained in house, but are available from American Type Culture Collection (ATCC). A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin in a humidified atmosphere of 95% air and 5% CO2 with the temperature of 37 °C.
2.8.2 Cell viability evaluation
To detect the effects of free-drugs or NMs on cell viability, the A549 cells was dispersed into signal-cell suspension and seeded at a density of 4 × 104 cells/well in 96-well plates for 18–24 h, then, the cells were treated with free-drugs or P@FPP NMs for 48 h. After that, 10 µL of Cell Counting Kit-8 kit (CCK8, YIFEIXUE BIO TECH) was added into each well. The optical density at 450 nm was examined by a Multimode Plate Reader (EnVision, PerkinElmer) after incubation of 60 min.
$${\text{Cell viability }}\left( \% \right) \, = \frac{{\left[ {{\text{OD}}_{{{49}0 \, ({\text{treated groups}})}} - {\text{ OD}}_{{{49}0 \, ({\text{background}})}} } \right]\,}}{{\left[ {{\text{OD}}_{{{49}0 \, ({\text{control groups}})}} - {\text{ OD}}_{{{49}0 \, ({\text{background}})}} } \right]}} \, \, \times { 1}00\% .$$
2.9 Cell migration and invasion assays
Cell migration and invasion of A549 cells were determined using Transwell system (Corning) with or without Matrigel (Corning). A549 cells were administrated with free-drugs or P@FPP NMs for 24 h and then performed Transwell assay. Briefly, cells with density of 5 × 104 (for migration) or 1 × 105 (for invasion) in serum-free culture medium were seeded into the upper chamber and medium containing 10%-serum was added to the bottom chamber. After incubation at 37 °C for 24 h, the noninvading cells in the upper chamber were removed scrubbing, and the migrating or invading cells in the bottom chamber were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet solution. The cells were observed and photographed under the light microscope.
2.10 Quantification real-time PCR
Total RNA was extracted using the RNA isolater Total RNA Extraction Reagent (Vazyme), and 1 μg was used to reverse-transcribe cDNA. The resulting cDNA was used as a template for quantitative PCR in 20 μL reactions containing 2 μL of cDNA, 0.4 μL of a forward primer, 0.4 μL of a reverse primer, 7.2 μL of ddH2O, and 10 μL of 2 × ChamQ Universal SYBR qPCR Master Mix (Vazyme). The temperature program (95 °C for 10 s, 58 °C for 30 s, and 72 °C for 30 s) was repeated 40 times. The primers used to quantify epithelial-mesenchymal transition (EMT)-related genes (E-cadherin, N-cadherin, Vimentin, and Twist) were listed in Additional file 1: Table S1.
2.11 Western blot analysis
The whole cell lysates were obtained from the treatment cells by using 1 × cell lysis buffer (Cell Signaling Technology) with 1 mM phenylmethanesulfonyl fluoride (Sigma-Aldrich) and 1 × protease inhibitor cocktail (Roche). Then, the whole cell lysates were collected and quantified using the BCA protein quantification method. the protein samples were mixed with the loading buffer and denatured by boiling and electrophoresis on 8 or 10% denaturing PAGE gels followed by incubation of the corresponding primary antibodies (Additional file 1: Table S2) and the HRP-conjugated secondary antibodies. After that, the protein bands were visualized using ChemiDoc XRS + with Image Lab software (Bio-Rad).
2.12 In-vivo biodistribution and xenograft inhibition of P@FPP NMs
2.12.1 In-vivo biodistribution of P@FPP NMs
To investigate the biodistribution of P@FPP, we first assembled fluorochrome of ICG-loading I@FPP NMs, and then the A549 tumor-bearing nude mice were intravenously injected with I@FPP. The fluorescence distribution in vivo was monitored 6 h after injection by IVIS system (PerkinElmer). Subsequently, the mice were euthanized and the major organs (heart, liver, spleen, lung, and kidney) and tumor tissue were dissected and the fluorescence distribution were monitored.
2.12.2 Xenograft inhibition of P@FPP NMs
The A549 xenograft bearing nude mice (~ 80 mm3) were randomly divided into five groups (n = 4) and then intravenously injection of the following formulations: PBS, PRI, PTX, PRI in combination of PTX, and P@FPP NMs. The mice were administrated every two days for 20 days. The mouse weight and tumor volume were monitored in the treatment process, and the tumor volume was calculated as the following formula: volume = 0.5 × (length × width2). The mice were euthanized and then, the tumor tissues and the major organs were harvested at the end of experiment for further analysis. All animal procedures were performed under the guidance of the Animal Care and Use Committee of Nanjing University of Chinese Medicine (ethical approval number: 202204A031).
2.13 Histology analysis
The obtained tissues were fixed and embedded with 4% paraformaldehyde and paraffin, respectively, and then cut into 4-µm-thick sections for H&E staining. For immunohistochemical analysis, the tissue sections were preformed according to the manufacturer’s instructions. Briefly, the tumor sections were first stained with the monoclonal anti-E-cadherin, anti-N-cadherin, anti-Ki-67, and TUNEL, and then stained with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin. After that, the sections were observed under bright-field microscope (DMi 8, Leica). The cells with brown granules were considered as the positively stained cells.
2.14 Statistical analysis
All the experiments were repeated three times. The data represented as mean ± standard deviation (SD). To ascertain the significance of the differences between the mean values of the different experimental groups, one-way ANOVA was employed followed. P < 0.05 and P < 0.01 were considered to be significant while P < 0.001 was considered to be highly significant.
