Antitumor effect of poly lactic acid nanoparticles loaded with cisplatin and chloroquine on the oral squamous cell carcinoma

Synthesis and characterization of CDDP/CQ-PLA NPs

To examine the functional nanoparticles, we performed a series of characterization assays. From the TEM image, we found that the diameter of CDDP/CQ-PLA NP was about 20 nm (Figure 1A). Zeta potential and dynamic light scattering measurements were further conducted and confirmed successful synthesis of CDDP/CQ-PLA NP (Figure 1B, 1C). The drug release kinetics showed that CDDP/CQ-PLA NPs were released faster in the acid condition (pH = 5) than neutral condition (pH = 7) (Figure 1D).

Figure 1. Synthesis and characterization of CDDP/CQ-PLA NPs. (A) TEM image; (B) Dynamic light scattering measurements; (C) Zeta potential; (D) drug release kinetics at 0.5 h, 1 h, 2.5 h, 4 h, 4.5 h, 6 h, 8.5 h, 11.5 h and 24.5 h.

CDDP/CQ-PLA NPs reduces the viability of OSCC cells

To evaluated the cytotoxicity on OSCC cell. We treated CAL-27 cells with different concentrations of CDDP/CQ-PLA NPs (0, 5, 10, 20, 40, 80, 160 ug/ml). The result of MTT and colony formation showed that CDDP/CQ-PLA NPs had more cytotoxicity than CDDP -PLA NPs and CDDP. (Figure 2A, 2B and Supplementary Figure 1).

Figure 2. CDDP/CQ-PLA NPs reduces the viability. (A) Cell viabilities in CAL-27 using MTT assay. (B) Colony forming assays with a concentration of 40μg/mL. *P < 0.05. NC: PBS.

CDDP/CQ-PLA NPs induces caspase-dependent apoptosis in OSCC cells

In order to explore the action of CDDP/CQ-PLA NPs’ cytotoxicity. We examined apoptosis and ROS. As shown in Figure 3, CDDP/CQ-PLA NPs leaded to more apoptosis than CDDP -PLA NPs and CDDP through active caspase-3 (Figure 3A), which was confirmed by western blot (Figure 3B and Supplementary Figure 2) and indicated that CDDP/CQ-PLA NPs induces caspase-dependent apoptosis in OSCC cells.

Figure 3. CDDP/CQ-PLA NPs induces caspase-dependent apoptosis. (A) Cell apoptosis in CAL-27 using Annexin-V/PI assay; (B) Western blot of Bax, Bid, Bim, Bcl-2, Caspase-3, and Caspase-9, β-actin as reference. *P < 0.05, NC: PBS.

CDDP/CQ-PLA NPs induces oxidative damage in OSCC cells

Furthermore, we found that CDDP/CQ-PLA NPs induced more ROS production (Figure 4A and Supplementary Figure 3) and MDA (Figure 4B), followed by decreased CAT (Figure 4C), SOD (Figure 4D), and GSH-P activity (Figure 4E), which suggested that CDDP/CQ-PLA NPs induces caspase-dependent apoptosis through oxidative damage in OSCC cells.

Figure 4. CDDP/CQ-PLA NPs induces oxidative damage. (A) ROS production; (B) MDA activity; (C) CAT activity; (D) SOD activity; (E) GSH-P activity; Data are mean ± SD; *P < 0.05, **P< 0.01, NC: PBS.

CDDP/CQ-PLA NPs reduces autophagy in OSCC cells

As CQ is an autophagy inhibitor, we examined the autophagy change caused by CDDP/CQ-PLA NPs. Confocal image show that CDDP/CQ-PLA NPs reduced the GFP-LC3 puncta caused by CDDP or CDDP -PLA NPs (Figure 5A and Supplementary Figure 4A). Western blot showed the consistent results that CDDP/CQ-PLA NPs reduced LC3-II and induced p62 protein expression (Figure 5B and Supplementary Figure 4B), which indicated that CDDP/CQ-PLA NPs inhibits autophagy in OSCC cells.

Figure 5. CDDP/CQ-PLA NPs reduces autophagy. (A) Immunofluorescence confocal image of LC3; (B) Western blot of p62, LC3-I, and LC3-II, actin as reference. *P < 0.05, NC: PBS.

Materials

Antibody against Bax, Bid, Bim, Bcl-2, caspase-3, caspase-9, p62, LC3, β-actin were purchase from Abcam company.

Cell culture

OSCC cell line, CAL-27 purchased from ATCC were cultured in DMEM with 10%FBS and 1% penicillin-streptomycin at 37° C, 5% CO2 condition.

Preparation of CDDP/CQ-PLA NPs

Cisplatin (Sigma, USA) is dissolved in 1 mL of anhydrous methanol, and then the above cisplatin solution and 3mL of PEG-PLA (Sigma, USA) solution dissolved in chloroform are uniformly mixed (the ratio of lipid material to cisplatin is 5: 1). Then the mixed solution of cisplatin and PEG-PLA were transferred into an eggplant bottle, and the organic solvent was removed by a vacuum rotary evaporator to form a dry drug lipid film at the bottom of the bottle. Subsequently, 2mL PBS buffer solution was added to dissolve the drug lipid membrane, and the drug lipid membrane was hydrated in a water bath at 60° C for 30 minutes. The hydrated solution was filtered through polycarbonate membrane (FormuMax Scientific, USA) with a pore diameter of 0.2 micron to obtain cisplatin polylactic acid nanoparticles (CDDP-PLA NPs) solution [27]. CDDP/CQ-PLA NPs were prepared using the same method above using cisplatin and chloroquine instead of cisplatin. PLA NPs were prepared using the same method above using no drug instead of cisplatin.

Surface morphology, ζ-potential and particle size

Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to detect the morphological characteristics and particle size parameters of drug-loaded nanoparticles. The nanoparticle solution was diluted with deionized water. Then we dropped 10 μL of sample onto a carbon-coated copper mesh, dried overnight at room temperature, and volatilized excess water. The sample then was re-dyed with 5 μL of 1% uranyl acetate solution for 30 seconds, and filter paper was used to suck the dyeing solution, the sample was dried at 42° C with a constant temperature dryer. The morphology of the sample was observed with a transmission electron microscope (Tecnai G2 20 STWIN). Different nano micelle solutions were diluted to 0.25 mg/mL with deionized water, and 1mL sample was taken to measure the hydration diameter and Zeta potential of nano particles with a laser particle sizer (Zetasizer 5000) [28]. Determination conditions: laser wavelength 633nm, angle 90, temperature 25° C.

Drug loading and encapsulation efficiency

DMF was used to dissolve the nanoparticles. The absorbance at 703nm was measured by ultraviolet spectrophotometer for cisplatin content and 330nm for chloroquine content. The following equation was applied:

$\text{EE}\%=\text{Wt}/\text{Ws}\times 100\%$

$\text{LC}\%=\text{Wt}/\text{Wo}\times 100\%$

Note: EE: encapsulation efficiency; LC: loading content; Wt: Amount of drug encapsulated in nanoparticles; Wo: Initial Dosage of Drugs; Ws: Number of Freeze-dried Nanoparticles.

Drug release kinetics

4 mg of cisplatin-chloroquine polylactic acid nanoparticles (CDDP/CQ-PLA NPs) were dissolved in the solution, and then put into PBS (pH=5.0) that added HCl and PBS (pH=7.4) to simulate tumor metaacid intracellular body (pH=5.0) and neutral blood environment (pH=7.4), respectively. Then the suspension was transferred into a dialysis bag with a rejection of 3 kDa, and immersed in 20 ml PBS, and incubated at 37° C for 100 rpm under shaking [29]. 5 ml of the release solution was taken out every 1 hour. The absorbance at 703nm was measured with an ultraviolet spectrophotometer for the content of cisplatin, and the absorbance at 330nm was measured with a visible spectrophotometer for the content of chloroquine. At the same time, the same volume of new PBS solution was added to the release medium [30]. The drug concentration and cumulative release were calculated.

Colony-forming assays/ cytotoxicity study

OSCC cell line CAL-27 was cultured overnight in 96-well plates and treated with the following groups: (1) NC group (PBS); (2) PLA NPs; (3) cisplatin; (4) CDDP-PLA NPs; (5) CDDP/CQ-PLA NPs. Set the concentration range according to cisplatin concentration: 5,10,20,40,80,160 μg/mL. Action time: 24h. At a specific concentration of cisplatin, MTT and colony forming assays were used to verify the different cell viability and proliferation among the groups. At 72 h after treatment, we measured the OD value of MTT (DUOXI, China) and took pictures of colony forming assay.

Apoptosis analysis

CAL-27 cells were seeded in 6-well plates and attached overnight, then treated with the following groups: (1) NC group (PBS); (2) PLA NPs; (3) cisplatin; (4) CDDP-PLA NPs; (5) CDDP/CQ-PLA NPs. After treatment with 40ug/ml cisplatin and continuous culture for 24h, Annexin/PI staining (Abcam, UK) was used to detect cell apoptosis.

Assessment of oxidative damage

Biochemical kits were used to detect malondialdehyde (MDA) content, catalase (CAT) activity, antioxidant enzyme superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) activity in CAL-27 cells. Kits can be purchased from Nanjingjiancheng company.

DCFH-DA purchased from Biyuntian company. is used as fluorescent probe and laser confocal microscope is used to detect ROS generation.

Western blot analysis

Total protein was collected from cells with RIPA lysis Mix (Beyotime, China). The protein extract was separated on SDS-PAGE gel and transferred to nitrocellulose membrane (Pierce, USA). The membrane was sealed with skim milk (Abbexa, UK) and incubated overnight with primary antibody. The membrane was then incubated with horseradish peroxidase-bound IgG (Proteintech, USA). Enhanced chemiluminescence (Pierce, USA) is used to detect signals.

Immunofluorescence confocal laser microscopy

CAL-27 cells were cultured on coverslips at a density of 5×10⁴ cells/well in 500 μL of complete medium (Hyclone, USA). After treatments, immunofluorescence analyses were carried out as previously described using LC3 antibodies. Cells were examined under an Olympus FV1000 confocal laser microscope (Tokyo, Japan).

Statistical analysis

Results are expressed as mean values ± standard deviation. Statistical analyses were assessed with Student’s test and one-way ANOVA using GraphPad Prism 7.0 and SPSS19.0. P values of less than 0.05 were considered significant.