Synthesis and characterization of PDA-PEG-DOX nanoparticles
PDA was synthesized from dopamine hydrochloride according to our previous protocol [31]. Transmission electron microscope (TEM) showed that PDA nanoparticles presented uniform spherical morphology (Figure 2A), while the average diameter was around 200 nm as revealed by dynamic light scattering (DLS) (Figure 2B). In order to improve the biocompatibility of PDA nanoparticles, we functionalized PDA with amino polyethylene glycol (PEG) at pH 10.0, and the obtained PDA-PEG nanoparticles showed excellent biocompatibility in different physiological solutions (Figure 2C). Due to PEG coating, the particle size of PDA-PEG nanoparticles became a little larger compared to PDA (Figure 2B). However, the spectrum absorption characteristics from ultraviolet to near infrared in PDA-PEG solution is similar to that in PDA (Figure 2D).
To test the ability of PDA-PEG to load therapeutic agent, chemotherapy drug DOX was loaded on the surface of PDA-PEG via hydrophobic interaction and π-π stacking. As shown in Figure 3A, the hydrodynamic size of PDA-PEG nanoparticles increased after drug loading, indicating that DOX was successfully loaded on the surface of PDA-PEG to form the PDA-PEG-DOX nanocomposites. Moreover, the zeta potential increased from -34.4 mV to -17.5 mV (Figure S1A). The characteristic absorption peaks appeared in PDA-PEG-DOX solution at 480 nm (Figure S1B), which all proved the successful loading of DOX. In order to explore the drug loading efficiency, the PDA-PEG solution was mixed with different dose of DOX at pH 8.0. The loading ratio of DOX on PDA-PEG nanoparticles was calculated by their UV-vis-NIR spectrum. As shown in Figure 3B, a new absorption peak of 480 nm was found on the PDA-PEG adsorption spectrum after DOX loading, further indicating that DOX was successfully loaded. DOX loading ratio (DOX: PDA-PEG, w/w) increased with the increase of DOX amounts (Figure 3C). When the weight ratio of DOX: PDA-PEG was 2:1, the DOX loading ratio was up to 120%. So PDA-PEG-DOX with DOX: PDA weight ratio of 2:1 was selected for our further experiments.
To examine the drug release behavior of PDA-PEG-DOX, the ultraviolet absorption value of the drug at different pH (5.7 or 7.4) was measured. As shown in Figure 3D, the drug release of PDA-PEG-DOX was accelerated at lower pH. When pH was 5.7, DOX release efficiency could reach 55% at 72 h. When pH was 7.4, DOX release efficiency was less than 15%. This may be due to the protonation of the amino group in the DOX molecule, which makes DOX positively charged and thus enhances hydrophilicity to induce DOX release.
Preparation and characterization of PDA-PEG-DOX-hpDNA nanoparticles
To detect miRNAs in living cells, we synthesized a PDA-PEG-DOX-hpDNA nano-drug delivery system. PDA-PEG nanoparticles were used as the quenching agent to quench the fluorescence of hpDNA/FITC, which specifically recognize miR-21. When endogenous miR-21 is present, FITC-labeled hpDNAs bind to miR-21 and detach from PDA-PEG to generate fluorescence. Firstly, we examined the quenching efficiency of PDA-PEG on DOX. As shown in Figure 4A, DOX exhibited a maximum fluorescence peak at 600 nm. The addition of PDA-PEG reduced the fluorescence intensity of DOX, which was the result of loading DOX onto PDA-PEG nanoparticles. Increasing the concentration of PDA-PEG with DOX at a fixed concentration (50 μg/mL) led to the gradual decrease of fluorescence intensity, indicating that DOX was successfully loaded onto PDA-PEG to form PDA-PEG-DOX nanoparticles. To evaluate quenching efficiency of PDA-PEG on hpDNA, different concentrations (0, 10, 20, 30, 40, 100, 200 μg/mL) of PDA-PEG nanoparticles were incubated with fixed hpDNAs. The FITC fluorescence intensity of hpDNAs decreased with the increased dose of PDA-PEG (Figure 4B, Figure S2A). When the concentration of quenching agent was 24 μg/mL, more than 83% of fluorescence signal was quenched. Similarly, PDA-PEG-DOX nanoparticles also exhibited the effective fluorescence quenching efficiency when incubated with hpDNAs (Figure S2B). To validate the universality of PDA-PEG as quenching agent, Cy5-labeled hpDNA that specifically target miR-21 was loaded on PDA-PEG. We observed that PDA-PEG can efficiently quench more than 70% of Cy5 fluorescence, indicating that the quenching ability of PDA-PEG is independent of the fluorescent dye labeled (Figure 4C).
The PDA-PEG-hpDNA/FITC NPs was then tested for its ability to detect exogenous miR-21 in vitro. Although PDA-PEG-hpDNA was quenched in the absence of target miRNAs, the fluorescence recovered in the presence of perfectly complementary targeted miRNAs. With the increased concentration of miR-21 from 1 nM to 50 nM, the fluorescence intensity of PDA-PEG-hpDNA increased in a dose-dependent manner (Figure 4D). In contrast, no significant fluorescence recovery of PDA-PEG-hpDNA was detected in the presence of negative control miR-124a, confirming the binding specificity of miR-21 to PDA-PEG-hpDNA. We then evaluated the stability of the PDA-PEG-hpDNA by incubation in Opti-MEM medium and PBS buffer for 4 days. During incubation, fluorescence recovery of PDA-PEG-hpDNA occurred only in the presence of exogenous miR-21 (Figure S3).
The specificity of miR-21 recognition by PDA-PEG-hpDNA in cells
To evaluate the theranostic potential of PDA-PEG-hpDNA in cancer cells, we selected 4T1, A549, P19, 7901 cell lines with high miR-21 expression and MCF-10a cell lines with low miR-21 expression. PDA-PEG-hpDNA nanoparticles were introduced into MCF-10a cells. In the absence of exogenous miR-21, PDA-PEG-hpDNA was still in the quenching state. The fluorescence intensity was gradually enhanced as the concentration of exogenous miR-21 increased in MCF-10a cells (Figure 5A). MiR-124a, which is expressed only during neurogenesis, did not lead to the fluorescence recovery. To detect endogenous miR-21 expression in 4T1 cells, different concentrations of PDA-PEG-hpDNA nanoparticles were incubated with 4T1 cells where miR-21 was highly expressed. With the concentration of PDA-PEG-hpDNA increased, FITC fluorescence intensity increased in a dose-dependent manner (Figure 5B). In contrast, PDA-PEG-hpDNA-Ctrl nanoparticles, which immobilized FITC-labeled control hpDNA with random sequences, did not result in significant increase of fluorescence intensity. In addition, we observed the similar increase trend of fluorescence in P19, A549 and 7901 cells after introduction with PDA-PEG-hpDNA nanoparticles (Figure 5C). Confocal microscopy was performed to further evaluate the fluorescence recovery of PDA-PEG-hpDNA nanoprobe in the present of endogenous miR-21 in 4T1 cells (Figure 5D). It was found that the cytoplasm exhibited green FITC fluorescence and the nucleus presented blue DAPI fluorescence, suggesting the effective uptake of PDA-PEG-hpDNA/FITC nanoparticles by the cells and the effective restore of fluorescence activity. However, the control nanoprobe PDA-PEG-hpDNA-ctrl did not exhibit any FITC signal. Taken together, these results confirmed the high specificity of miR-21 recognition by PDA-PEG-hpDNA.
In vitro synergistic therapy in cells with PDA-PEG-DOX-hpDNA NPs
In view of the excellent performance of PDA-PEG-hpDNA in intracellular uptake, we next want to study the potential toxicity of PDA-PEG-hpDNA in various cancer cells. As shown in Figure 6A, PDA-PEG nanoparticles exhibited no significant toxicity in 4T1 cells even at high concentrations (500 mg mL-1), indicating good biocompatibility of PDA-PEG. The similar results were also observed in P19, A549 and 7901 cells after treatment with PDA-PEG nanoparticles (Figure S4). PDA-PEG-hpDNA induced a slight cytotoxicity in 4T1 cells, which may be owing to the inhibition of endogenous miR-21 function via hybridization of miR-21 to hpDNA (Figure 6B). 4T1 or 7901 cells were then incubated with different concentrations of free DOX, PDA-PEG-DOX and PDA-PEG-DOX-hpDNA for 48 h (Figure 6C and 6D). The results showed that free DOX was the most toxic, inhibited the cell growth of 4T1 and 7901 cells in a concentration-dependent manner, suggesting no specific toxicity of DOX. PDA-PEG-DOX exhibited weaker toxicity than free DOX did, this may be due to the gradual release of DOX from PDA-PEG-DOX under acidic conditions. We also observed the similar phenomena in P19 and A549 cells (Figure S5). However, the PDA-PEG-DOX-hpDNA nanoprobe presented synergistic inhibition effect compared with single-agent treatment with PDA-PEG-DOX (Figure 6C, 6D). These results suggest that the theranostic nanoprobe based on PDA-PEG-DOX-hpDNA nanocomposite material showed great synergistic effect in killing tumor cells.
In vivo imaging of miR-21 and combined therapy using PDA-PEG-DOX-hpDNA
In order to evaluate the performance of the PDA-PEG-DOX-hpDNA nanoprobe for in vivo miRNA detection, 4T1 xenografted tumor models were established by subcutaneously injecting 5*106 4T1 cells into the right flank of nude mice. PDA-PEG-DOX-hpDNA nanoprobes were injected into mice via tail vein. Fluorescence changes in tumor sites of the nude mice were detected at 0, 1, 3 and 5 d. As shown in Figure 7A, the FITC fluorescence signal in the right buttock of nude mice clearly increased over time, which is due to the high expression of endogenous miR-21 detached the FITC-hpDNAs from PDA-PEG-DOX-hpDNA NPs through the specific hybridization between NPs and miR-21 in 4T1 cells. The in vivo fluorescence activity of PDA-PEG-DOX-hpDNA NPs demonstrated the excellent tumor diagnosis ability of our nanoprobes to monitor the dynamic expression of endogenous miR-21 in real-time manner.
Due to the great combined therapy efficiency of PDA-PEG-DOX-hpDNA in vitro, we next evaluated the in vivo performance of our nanoprobe as a theranostic probe to achieve synergistic chemotherapy and gene therapy. The 4T1 xenografted tumor models were randomly divided into 4 groups and received the following different treatment: (1) PBS, (2) PDA-PEG-hpDNA, (3) PDA-PEG-DOX, (4) PDA-PEG-DOX-hpDNA. As shown in Figure 7B, compared with control mice treated with PBS, the tumor growth rate in mice receiving PDA-PEG-hpDNA treatment was slightly reduced. In contrast, the PDA-PEG-DOX-hpDNA group showed more effective inhibition of tumor growth than PDA-PEG-DOX or PDA-PEG-hpDNA treated mice, indicating the remarkable synergistic effect of PDA-PEG-DOX-hpDNA nanoprobes in combination therapy. The tumor sizes further confirmed the treatment effect of different groups (Figure 7C). However, the body weight of the mice with different treatments was monitored and exhibited no obvious change during the process, indicating the in vivo toxicity were negligible (Figure 7D).
In addition, we further detected the fluorescence signals in the main organs and tumors from different treatment groups using the IVIS imaging system. The results demonstrated that the tumors from PDA-PEG-hpDNA and PDA-PEG-DOX-hpDNA group exhibited obvious fluorescence enhancement, implying the effective delivery of nanoprobes into 4T1 tumors and target recognition of endogenous miR-21 (Figure 8A). Meanwhile, the livers also showed certain fluorescence signals since they were the main metabolic detoxification organs. To further investigate the potential toxicity of PDA-PEG-DOX-hpDNA, hematoxylin-eosin (H&E) staining was performed to analyze the main organs of mice in the different treatment groups. No obvious morphological changes were observed in the main organs (heart, liver, spleen, lung and kidney) collected from the nanoprobe treatment group, indicating that the nanoparticles had good biological safety (Figure 8B). However, the H&E staining results from tumor sections demonstrated that partial tumor damages were found in mice after treatment with PDA-PEG-DOX or PDA-PEG-DOX-hpDNA (Figure S6). Of note, tumors receiving PDA-PEG-DOX-hpDNA showed the severest morphological damage, further confirming the combined therapeutic efficacy of PDA-PEG-DOX-hpDNA as theranostic agent in cancer treatment. Taken together, these results demonstrate that the PDA-PEG-DOX-hpDNA nanoprobes enables effective imaging of miR-21 and synergistic suppression of tumors in vivo.