Polydopamine Nanoparticles for Combined Chemo- and Photothermal Cancer Therapy

Cancer therapy with two different modalities can enhance treatment efficacy and reduce side effects. This paper describes a new method for combined chemo- and photothermal therapy of cancer using poly dopamine nanoparticles (PDA-NPs), where PDA-NPs serve not only as a photothermal agent with strong near infrared absorbance and high energy conversion efficiency, but also as a carrier to deliver cisplatin via interaction between cisplatin and catechol groups on PDA-NPs. Polyethylene glycol (PEG) was introduced through Michael addition reaction to improve the stability of PDA-NPs in physiological condition. A remarkable synergistic therapeutic effect has been achieved compared with respective single treatments. This work suggests that the PDA-based nanoplatform can be a universal scaffold for combined chemo- and photothermal therapy of cancer.


Introduction
Cisplatin that contains platinum is used widely as a means of chemotherapy of cancer [1,2], but its clinical use is limited by its severe toxic effect due to indiscriminate accumulation in normal and cancerous tissues, nonspecific interactions with extra and intracellular proteins, and drug resistance [3]. In parallel to efforts of mitigating toxicity of cisplatin by modifying its chemical structure, an alternate approach is to use delivery vehicles that could overcome these limitations and specifically target cancerous cells [4]. Many carrier systems such as gold [5,6], carbon [7,8], silica [9,10], and polymer [11,12] have been developed, but the long-term toxicity of these carriers remains an issue [13]. A nature-inspired biopolymer, melanin-liked polydopamine (PDA) that has excellent biocompatibility [14][15][16], and free-radical-scavenging activity [17][18][19], has been explored as coating materials for gold nanorods [20,21], iron oxide nanoparticles [15,[22][23][24], and as a substrate for photothermal agent [13], but the photothermal property of PDA nanoparticles has been overlooked. As a kind of semiconducting polymer [25], with a photothermal energy conversion efficiency of 40%, PDA-NPs have shown promising applications in photothermal-based cancer therapy, which is emerging as a powerful technique in cancer therapy due to localized treatment and minimal invasiveness [26][27][28][29].
This paper reports a PDA-NPs based therapeutic platform, where PDA-NPs loaded with anticancer drug cisplatin have been prepared through a mild method. PDA-NPs serve not only as a photothermal agent due to strong near infrared absorption and high photothermal energy conversion efficiency, but also as a carrier to load cisplatin via the interaction between cisplatin and catechol groups on PDA-NPs. Conjugation of cisplatin onto PDA-NPs has been achieved by mixing PDA-NPs and cisplatin in minutes. Polyethylene glycol (PEG), as a Food and Drug Administration (FDA) approved polymer has been introduced via a Michael addition reaction to improve the biocompatibility of the assay was used to examine cell viability as follows. An amount of 10 µL of MTT (5 mg/mL in PBS) was added and cells were incubated for 4 h in the dark. The medium was then replaced with 100 mL of DMSO and the absorbance was monitored using a microplate reader at 550 nm. The cells treated with the same procedure without adding MTT were used as the background control. The cell viability experiment was repeated three times and four parallel wells were used for each group. Figure 1 shows the procedure of making PDA-PEG-CP nanoconstructs. PDA-NPs were made by oxidation and polymerization of dopamine in an alkaline solution, where the color of the solution rapidly turned to yellow (oxidation) and gradually changed to dark (polymerization). Figure 2a,b show the TEM image of PDA-NPs, where the nanoparticles with uniform size (148 nm) are obtained. The nanoparticles of this size remain in circulation for a long period of time, and efficiently accumulate in tumor tissues via enhanced permeability and retention (EPR) [32][33][34].

Results and Discussion
Nanomaterials 2017, 7, 160 3 of 8 in PBS) was added and cells were incubated for 4 h in the dark. The medium was then replaced with 100 mL of DMSO and the absorbance was monitored using a microplate reader at 550 nm. The cells treated with the same procedure without adding MTT were used as the background control. The cell viability experiment was repeated three times and four parallel wells were used for each group. Figure 1 shows the procedure of making PDA-PEG-CP nanoconstructs. PDA-NPs were made by oxidation and polymerization of dopamine in an alkaline solution, where the color of the solution rapidly turned to yellow (oxidation) and gradually changed to dark (polymerization). Figure 2a Figure 3a shows vis-NIR spectra of PDA-NPs, where a strong absorption in the near infrared wavelength region can be found at 800 nm (inset). The photothermal effect of PDA NPs of various concentrations under 808 nm laser radiation is tested with PBS as a negative control. Figure 3b shows the temperature change profile of 0.2 mL PBS containing different concentrations of PDA-NPs, revealing the concentration-dependent photothermal effect. The temperature increased with the irradiation time for all the samples, and a higher concentration of PDA NPs leads to a more rapidly increasing temperature (Figure 3b). Take 200 μg/mL of PDA NPs for example; after 500 s of irradiation, the temperature increased by 29.6 °C, while the negative control (PBS) increased only 4.6 °C under the same condition. The rapid temperature change is due to the relatively high absorption coefficient (7.3 × 10 8 M −1 cm −1 ) and photothermal efficiency (about 40%) at 808 nm of PDA NPs [26].  in PBS) was added and cells were incubated for 4 h in the dark. The medium was then replaced with 100 mL of DMSO and the absorbance was monitored using a microplate reader at 550 nm. The cells treated with the same procedure without adding MTT were used as the background control. The cell viability experiment was repeated three times and four parallel wells were used for each group. Figure 1 shows the procedure of making PDA-PEG-CP nanoconstructs. PDA-NPs were made by oxidation and polymerization of dopamine in an alkaline solution, where the color of the solution rapidly turned to yellow (oxidation) and gradually changed to dark (polymerization). Figure 2a Figure 3a shows vis-NIR spectra of PDA-NPs, where a strong absorption in the near infrared wavelength region can be found at 800 nm (inset). The photothermal effect of PDA NPs of various concentrations under 808 nm laser radiation is tested with PBS as a negative control. Figure 3b shows the temperature change profile of 0.2 mL PBS containing different concentrations of PDA-NPs, revealing the concentration-dependent photothermal effect. The temperature increased with the irradiation time for all the samples, and a higher concentration of PDA NPs leads to a more rapidly increasing temperature (Figure 3b). Take 200 μg/mL of PDA NPs for example; after 500 s of irradiation, the temperature increased by 29.6 °C, while the negative control (PBS) increased only 4.6 °C under the same condition. The rapid temperature change is due to the relatively high absorption coefficient (7.3 × 10 8 M −1 cm −1 ) and photothermal efficiency (about 40%) at 808 nm of PDA NPs [26].   Figure 3b shows the temperature change profile of 0.2 mL PBS containing different concentrations of PDA-NPs, revealing the concentration-dependent photothermal effect. The temperature increased with the irradiation time for all the samples, and a higher concentration of PDA NPs leads to a more rapidly increasing temperature (Figure 3b). Take 200 µg/mL of PDA NPs for example; after 500 s of irradiation, the temperature increased by 29.6 • C, while the negative control (PBS) increased only 4.6 • C under the same condition. The rapid temperature change is due to the relatively high absorption coefficient (7.3 × 10 8 M −1 cm −1 ) and photothermal efficiency (about 40%) at 808 nm of PDA NPs [26]. PDA-NPs were modified with mPEG-SH to enhance their circulation in physiological condition and facilitate their accumulation at tumor sites [35][36][37]. A Michael addition reaction was used to conjugate the thiol of PEG and α,β-unsaturated carbonyl on PDA [38]. The zeta potential of PDA decreased from −35.4 to −6.9 mV after PEGylation, confirming the successful conjugation of PEG on the PDA-NPs.

Results and Discussion
With abundant PDA groups on the surface, PDA-NPs can deliver the drug through electrostatic interaction, coordination and π-π interactions [39]. Cisplatin, a widely used anticancer drug was loaded onto the PEG-PDA NPs via the interaction between the platinum atom of cisplatin and catechol groups of PDA, and the nanoparticles were denoted as PEG-PDA-CP. The morphology and size of the NPs did not show obvious change between PDA and PDA-PEG-CP NPs, as seen in Figure  2b,c. The amount of cisplatin in the PEG-PDA NPs was measured to be 20% (m/m) using the OPDA method [31].
To study the release profile of cisplatin, PDA-PEG-CP solutions were suspended in PBS of different pH at 37 °C. The released cisplatin was quantified with an established method [31]. The formation of the complex of o-phenylenediamine (OPDA) and cisplatin was achieved by incubating OPDA and cisplatin in DMF spiked phosphate buffer at 100 °C for 10 min. The resulting solution showed a maximum absorbance at 705 nm, while neither OPDA nor cisplatin alone showed absorbance at 705 nm after the same treatment (Figure 4b). With the increasing concentration of cisplatin, the absorbance peak of the complex raised accordingly (Figure 4c), which is consistent with a previous report [31]. As a result, there is a good linear relationship between the absorbance intensity and concentration of 200 μL original cisplatin in the range of 0-100 μg/mL (Figure 4d). PDA-NPs were modified with mPEG-SH to enhance their circulation in physiological condition and facilitate their accumulation at tumor sites [35][36][37]. A Michael addition reaction was used to conjugate the thiol of PEG and α,β-unsaturated carbonyl on PDA [38]. The zeta potential of PDA decreased from −35.4 to −6.9 mV after PEGylation, confirming the successful conjugation of PEG on the PDA-NPs.
With abundant PDA groups on the surface, PDA-NPs can deliver the drug through electrostatic interaction, coordination and π-π interactions [39]. Cisplatin, a widely used anticancer drug was loaded onto the PEG-PDA NPs via the interaction between the platinum atom of cisplatin and catechol groups of PDA, and the nanoparticles were denoted as PEG-PDA-CP. The morphology and size of the NPs did not show obvious change between PDA and PDA-PEG-CP NPs, as seen in Figure 2b,c. The amount of cisplatin in the PEG-PDA NPs was measured to be 20% (m/m) using the OPDA method [31].
To study the release profile of cisplatin, PDA-PEG-CP solutions were suspended in PBS of different pH at 37 • C. The released cisplatin was quantified with an established method [31]. The formation of the complex of o-phenylenediamine (OPDA) and cisplatin was achieved by incubating OPDA and cisplatin in DMF spiked phosphate buffer at 100 • C for 10 min. The resulting solution showed a maximum absorbance at 705 nm, while neither OPDA nor cisplatin alone showed absorbance at 705 nm after the same treatment (Figure 4b). With the increasing concentration of cisplatin, the absorbance peak of the complex raised accordingly (Figure 4c), which is consistent with a previous report [31]. As a result, there is a good linear relationship between the absorbance intensity and concentration of 200 µL original cisplatin in the range of 0-100 µg/mL (Figure 4d). showed a maximum absorbance at 705 nm, while neither OPDA nor cisplatin alone showed absorbance at 705 nm after the same treatment (Figure 4b). With the increasing concentration of cisplatin, the absorbance peak of the complex raised accordingly (Figure 4c), which is consistent with a previous report [31]. As a result, there is a good linear relationship between the absorbance intensity and concentration of 200 μL original cisplatin in the range of 0-100 μg/mL (Figure 4d).  It is essential to trigger cisplatin release from the drug delivery vehicles at tumor sites, thus keeping the overall therapeutic efficacy of the packaged drugs [40]. To this end, the release profile of cisplatin from the same batch of PDA-PEG-CP was evaluated through dialysis against saline buffered solutions at acidic (pH 6.0) and physiological (pH 7.4) conditions at 37 °C to mimic the acidic environment of the tumor site and cellular endosomes [41]. Cisplatin showed burst release within the first 1 h at both pH values. As expected, it showed a faster release at pH 6.0 than at 7.4 throughout the remaining period ( Figure 5). After 72 h, 33.3% of the loaded cisplatin was released at pH 7.4; in comparison, at pH 6.0, about 45% cisplatin was released in the first 36 h and 55% release was obtained after 72 h. This acid-triggered drug release is due to the protonation of oxygen groups. In an acidic environment, H + would attack the lone electron pair of oxygen, leading to decomposing of drug-PDA complexes [21]. More importantly, at pH 6.0, it showed sustained release during the 72 h and a continuous release can be observed from 48 to 72 h. The acid-enhanced drug release would enhance cancer therapy due to the acidic environment of most tumor cells [42,43]. It is essential to trigger cisplatin release from the drug delivery vehicles at tumor sites, thus keeping the overall therapeutic efficacy of the packaged drugs [40]. To this end, the release profile of cisplatin from the same batch of PDA-PEG-CP was evaluated through dialysis against saline buffered solutions at acidic (pH 6.0) and physiological (pH 7.4) conditions at 37 • C to mimic the acidic environment of the tumor site and cellular endosomes [41]. Cisplatin showed burst release within the first 1 h at both pH values. As expected, it showed a faster release at pH 6.0 than at 7.4 throughout the remaining period ( Figure 5). After 72 h, 33.3% of the loaded cisplatin was released at pH 7.4; in comparison, at pH 6.0, about 45% cisplatin was released in the first 36 h and 55% release was obtained after 72 h. This acid-triggered drug release is due to the protonation of oxygen groups. In an acidic environment, H + would attack the lone electron pair of oxygen, leading to decomposing of drug-PDA complexes [21]. More importantly, at pH 6.0, it showed sustained release during the 72 h and a continuous release can be observed from 48 to 72 h. The acid-enhanced drug release would enhance cancer therapy due to the acidic environment of most tumor cells [42,43].
comparison, at pH 6.0, about 45% cisplatin was released in the first 36 h and 55% release was obtained after 72 h. This acid-triggered drug release is due to the protonation of oxygen groups. In an acidic environment, H + would attack the lone electron pair of oxygen, leading to decomposing of drug-PDA complexes [21]. More importantly, at pH 6.0, it showed sustained release during the 72 h and a continuous release can be observed from 48 to 72 h. The acid-enhanced drug release would enhance cancer therapy due to the acidic environment of most tumor cells [42,43]. HeLa cells were used to assess the cytotoxicity of the drug delivery system. No obvious toxicity was observed after cells were incubated with PDA-PEG up to 100 μg/mL for 24 h (black histogram in Figure 6a). However, after 808 nm laser irradiation, apparent cell apoptosis can be obtained especially when the PDA-PEG concentration exceeds 20 μg/mL (red histogram in Figure 6a). Up to 85% of cells were destroyed when incubated with 100 μg/mL of PDA-PEG under 808 nm laser irradiation. It indicates great potential in PDA-based photothermal therapy. To examine the chemotherapy effect, HeLa cells were used to assess the cytotoxicity of the drug delivery system. No obvious toxicity was observed after cells were incubated with PDA-PEG up to 100 µg/mL for 24 h (black histogram in Figure 6a). However, after 808 nm laser irradiation, apparent cell apoptosis can be obtained especially when the PDA-PEG concentration exceeds 20 µg/mL (red histogram in Figure 6a). Up to 85% of cells were destroyed when incubated with 100 µg/mL of PDA-PEG under 808 nm laser irradiation. It indicates great potential in PDA-based photothermal therapy. To examine the chemotherapy effect, PDA-PEG/CP at different concentrations of drug were incubated with HeLa cells for 24 h, then the cells were washed with PBS twice to remove non-internalized nanoparticles followed by 808 nm laser irradiation for 10 min. It was found that cell viability decreased with increasing PAD-PEG/CP concentration, and over 70% of cells were killed when incubated with 100 µg/mL of cisplatin loaded onto polydopamine nanoparticles (PDA NPs) ( Figure 6b). As expected, more cells were destroyed with laser irradiation after incubation with PDA-PEG-CP. Cell viability of combined therapy was found to be lower than the summation of photothermal and chemotherapy alone. The remarkably improved therapeutic effect may be attributed to the photothermal effect which not only kills cancer cells, but also effectively enhances the delivery and release of the drug into cells for improved chemotherapy. The results indicate a synergetic effect when chemotherapy and photothermal treatment are combined. PDA-PEG/CP at different concentrations of drug were incubated with HeLa cells for 24 h, then the cells were washed with PBS twice to remove non-internalized nanoparticles followed by 808 nm laser irradiation for 10 min. It was found that cell viability decreased with increasing PAD-PEG/CP concentration, and over 70% of cells were killed when incubated with 100 μg/mL of cisplatin loaded onto polydopamine nanoparticles (PDA NPs) ( Figure 6b). As expected, more cells were destroyed with laser irradiation after incubation with PDA-PEG-CP. Cell viability of combined therapy was found to be lower than the summation of photothermal and chemotherapy alone. The remarkably improved therapeutic effect may be attributed to the photothermal effect which not only kills cancer cells, but also effectively enhances the delivery and release of the drug into cells for improved chemotherapy. The results indicate a synergetic effect when chemotherapy and photothermal treatment are combined.

Conclusions
A new nanoplatform based on nature-inspired polydopamine nanoparticles (PDA NPs) was created for combined photothermal and chemotherapy in cancer treatment. PDA showed great biocompatibility and molecular loading property, and enhanced photothermal conversion efficiency. A controlled amount of cisplatin was loaded onto PDA-PEG nanoparticles by chelation between platinum and catechol groups on PDA in minutes. This PDA-PEG/CP shows pH-dependent drug

Conclusions
A new nanoplatform based on nature-inspired polydopamine nanoparticles (PDA NPs) was created for combined photothermal and chemotherapy in cancer treatment. PDA showed great biocompatibility and molecular loading property, and enhanced photothermal conversion efficiency. A controlled amount of cisplatin was loaded onto PDA-PEG nanoparticles by chelation between platinum and catechol groups on PDA in minutes. This PDA-PEG/CP shows pH-dependent drug release, excellent biocompatibility and a remarkable synergistic effect. The results suggest that the PDA-based nanoplatform shows great promise for clinical application.