Polydopamine-Encapsulated Fe3O4 with an ... - ACS Publications

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Polydopamine-Encapsulated Fe3O4 with an Adsorbed HSP70 Inhibitor for Improved Photothermal Inactivation of Bacteria Dongdong Liu,†,‡ Liyi Ma,† Lidong Liu,† Lu Wang,† Yuxin Liu,† Qi Jia,† Quanwei Guo,† Ge Zhang,† and Jing Zhou*,† †

Department of Chemistry, Capital Normal University, Beijing 100048, People’s Republic of China College of Resource Environment, Tourism, Capital Normal University, Beijing 100048, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: Photothermal treatment, a new approach for inactivation of bacteria and pathogens that does not depend on traditional therapeutic approaches, has recently received much attention. In this study, a new type of nanoplatform (PDA@ Fe3O4 + PES) was fabricated by using polydopamine (PDA, a photothermal conversion agent) to encapsulate Fe3O4 (a magnetic nanoparticle) and support 2-phenylethynesulfonamide (PES, an inhibitor of heat shock protein 70 (HSP70)). Upon near-infrared light irradiation, the increased temperature weakens π−π and hydrogen bonding interactions, and PES is released from the PDA@Fe3O4 + PES. The released PES inhibits the function of HSP70, reducing bacterial tolerance to photothermal therapy and improving the therapeutic effect against infectious bacterial pathogens. After treatment, PDA@Fe3O4 + PES can be recovered using the magnetic property of the Fe3O4 cores. Consequently, PDA@Fe3O4 + PES possesses the potential to be a recyclable photothermal agent for enhanced photothermal bacterial inactivation without causing secondary pollution. KEYWORDS: photothermal, polydopamine, HSP70 inhibitor, bacteria, magnetic nanoparticles

1. INTRODUCTION Pathogens, including Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), are well-known as food contaminants that cause bacterial infectious diseases.1 Preventing outbreaks of pathogens is very important for their biological diversity and high infectivity.2 As the most effective therapeutic approach, antibiotics, such as penicillin, have a long period and wide application in infections treatment. However, frequent and excessive use of conventional antibiotics has caused extensive multidrug resistance in pathogens and bacteria.3 The difficulty of developing completely new antibiotics has led to new approaches such as photothermal treatment,4−9 and has received much attention in the field of infectious pathogens and bacteria inactivation.10−16 Upregulation of heat shock proteins (HSPs) has been proven to enable cells to acquire tolerance to particular stress.17 Heat shock protein 70 (HSP70), as a member of the HSP family and an ATP-dependent molecular chaperone, can be encoded by an evolutionarily conserved gene family that is widely found in organisms from bacteria to mammals. It plays a supporting role in polypeptide (or protein) folding and refolding, protein complex formation or aggregation, and protein transporting promotion. Also, as an important apoptotic regulator of signaling pathways, HSP70 can be induced by stress and accumulate in cells to provide protection from severe living conditions, such as overheating. The 2-phenylethynesulfona© 2016 American Chemical Society

mide (PES), a stable HSP70 inhibitor, has low cytotoxicity to living systems and can selectively interact with HSP70, which can interfere with the normal function of HSP70 in different kinds of cell signaling pathways.18−20 Our recent work has shown that PES can be released from the nanogel under nearinfrared (NIR) irradiation, which can further improve the therapeutic effect in the tumor-bearing mice model, and hence improve the photothermal therapy depth.21 The presence of the free PES is expected to reduce bacterial cells’ tolerance to heat, resulting in improved photothermal inactivation of the bacteria. Magnetic nanoparticles generate an induced magnetic dipole allowing selective control of location and motion with a magnetic field applied externally.22,23 Recently, magnetic nanoparticle-based nanoplatforms have been intensively developed for magnetic resonance imaging (MRI),24 magnetically guided drug delivery,25−27 hyperthermia treatment,28−30 and selective separation or targeting.31−34 Moreover, magnetic nanoparticle-based antibacterial materials can be recovered and reused,35 avoiding secondary pollution. In this work, a novel nanoplatform (PDA@Fe3O4 + PES) was fabricated by loading PES onto the surface of polydopReceived: July 5, 2016 Accepted: September 1, 2016 Published: September 1, 2016 24455

DOI: 10.1021/acsami.6b08119 ACS Appl. Mater. Interfaces 2016, 8, 24455−24462

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ACS Applied Materials & Interfaces

Figure 1. TEM images of (a) Fe3O4 and (b) PDA@Fe3O4, scale bar: 50 nm. (c) The size characterization and (d) the hydrodynamic diameter of PDA@Fe3O4.

gently stirred for another 12 h at RT. The PES-loaded PDA@ Fe3O4 (PDA@Fe3O4 + PES) was collected by centrifugation (12 000 rpm, 3 min) from the dispersion. With the help of the ninhydrin method, the amount of loaded PES was calculated. By means of the given computing method, the entrapment efficiency and loading content were accounted for.

amine-coated Fe3O4 nanoparticles. Upon near-infrared irradiation, PES is released from the PDA@Fe3O4 + PES, enabling inhibition of HSP70 and reduction of bacterial tolerance to heat. The ablation effect on bacteria, recovery properties, and toxicity of the nanoplatform were evaluated.

2. EXPERIMENTAL SECTION 2.1. Preparation of PDA@Fe3O4 + PES Nanoplatform. 2.1.1. Preparation of Fe3O4 Nanoparticles (Fe3O4). The Fe3O4 nanoparticles (Fe3O4) were prepared via a polyol progress as previously reported.24 In a typical procedure, sodium acetate trihydrate (2.0 g), trisodium citrate (0.5 g), and FeCl3·6H2O (1.1 g) were added into ethylene glycol (33 mL) with stirring until the solution is clear. Then, a sealed Teflon-lined autoclave was used for the hydrothermal treatment of the obtained solution at 200 °C for 10 h. After that, the system in the sealed autoclave was allowed to naturally cool down to room temperature (RT). The brownish-black precipitates were collected after having been washed with ethanol and deionized water to obtain Fe3O4. 2.1.2. Preparation of PDA-Encapsulated Fe3O4 Nanocomposites (PDA@Fe3O4). According to a typical procedure, the obtained Fe3O4 (7 mg) was scattered in 40 mL of 10 mM Tris-HCl buffer solution (pH = 8.5) by sonication, and 32 mg of FeCl3·6H2O and 40 mg of dopamine hydrochloride were then dissolved into the above suspension under generous stirring. After stirring at RT for another 4 h, polydopamine (PDA)-encapsulated Fe3O4 nanocomposites (PDA@Fe3O4) were prepared successfully. The obtained products were centrifugated and washed to remove the unreacted reagents. 2.1.3. Preparation of PES-Loaded PDA@Fe3O4 Nanocomposites (PDA@Fe3O4 + PES). To study the adsorption properties of PES onto the PDA@Fe3O4, PES loading experiments were performed at RT. Typically, PES solution (1.25 mL, 1.2 mg mL−1) and PDA@Fe3O4 (2.5 mL, 1.0 mg mL−1) were mixed in PBS buffer (pH = 7.4) under stirring and

encapsulation efficiency weight of PES in PDA@Fe3O4 + PES = initial weight of PES

loading content =

(1)

weight of PES in PDA@Fe3O4 + PES weight of PDA@Fe3O4 + PES (2)

2.2. PES Release Studies. To determine the property of PES release, PDA@Fe3O4 + PES solution (1.5 mL, 0.53 mg mL−1) was treated with a water bath to simulate the condition of NIR irradiation. After having been treated with different kinds of hydrothermal pretreatment (37, 40, 50, and 60 °C for 1 h), the supernatant was then collected by centrifugation at 12 800 rpm for 4 min, respectively. With the advantage of the amino group in PES, the PES release amount was verified by the ninhydrin method. 2.3. Photothermal Inactivation of Bacteria. Bacteria (S. aureus and E. coli) were transferred to PBS buffer (pH = 7.4); PDA@Fe3O4 (100 μL, 500 μg mL−1) and PDA@Fe3O4 + PES (100 μL, 500 μg mL−1) solutions were cultured at 37 °C for 24 h under gentle rotation, respectively. Sterile water was used to dilute the bacteria until 105 cfu mL−1. The acquired solution was exposed to the irradiation of a 785 nm laser for 300 s (0.5 W cm−2). After that, the spread plate method was used to place the solution into a solid medium. After being cultured for 24 h, the bacteria colony number was calculated. A blank group and material only group (PDA@Fe3O4 + PES) were set as control. 24456


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ACS Applied Materials & Interfaces 2.4. Separation Efficiency Measurement. The concentrations of PDA@Fe3O4 + PES that remained in supernatant solutions were calculated by ultraviolet−visible−near-infrared (UV−vis−NIR) absorption spectra. Then, PDA@Fe3O4 + PES removal efficiency (R) was determined according to the following equations.36

R = V (C0 − Ce)/m

Scheme 1. (a) Scheme of the Preparation of PDA@Fe3O4 + PES with (b) Enhanced Photothermal Inactivation Effect on Bacteria and Potential Recovery for Recycle Usage

(3)

C0 and Ce represent the concentrations of PDA@Fe3O4 + PES in aqueous solution before and after the separation, separately; m represents the mass of PDA@Fe3O4 + PES, and V represents the aqueous solution volume. 2.5. Recycling Property Study. Bacteria (S. aureus and E. coli) were transferred to PDA@Fe3O4 + PES solution (100 μL, 500 μg mL−1) and cultured at 37 °C with middle speed of rotation, respectively. After 24 h, the bacterial suspension was diluted with PBS buffer (pH = 7.4) until 105 cfu mL−1. Photothermal inactivation experiments were then carried out by exposing the obtained mixture to the irradiation of a 785 nm laser for 300 s (0.5 W cm−2). After inactivation, PDA@Fe3O4 solution was attracted to the wall of the tube by a magnetic field, and the supernatant was removed. The separated PDA@ Fe3O4 was cleaned by pure water for several times and retested in the following antibacterial application. The photothermal inactivation effect on bacteria of each circle was evaluated by the spread plate method and by counting the bacteria colony number after photothermal inactivation.

PDA, PDA@Fe3O4 is expected to kill bacteria by absorption of NIR light and production of cytotoxic heat within minutes. Hence, the absorption and photothermal properties of PDA@ Fe3O4 were studied. The UV−vis−NIR spectrum of PDA@ Fe3O4 solution exhibited broad absorbance in the 500−900 nm region (Figure 2c), showing higher NIR absorbance compared with Fe3O4. A PDA@Fe3O4 solution (0.25 mg mL−1) was also exposed to a 785 nm laser (0.5 W cm−2) to determine the photothermal properties of the nanoplatform. As shown in Figure 3a, the photothermal images changed as the time goes on, and indicated that the temperature of the PDA@Fe3O4 solution came up to 47 °C from 25.7 °C within 800 s (Figure 3b). These results demonstrated that PDA@Fe3O4 has an efficient photoheat conversion ability under substantial irradiation of NIR. Upon the same NIR irradiation, the temperature rose higher as the concentration of PDA@Fe3O4 increased (Figure 2d). Therefore, the temperature of the PDA@Fe3O4 solution will continuously increase as the concentration of the solution increased. In addition, the photothermal conversion efficiency of the PDA@Fe3O4 was ∼57% (Figure 3b,c). Further, under NIR irradiation for five on/ off cycles, PDA@Fe3O4 demonstrated excellent photostability (Figure 4a). The absorption study of PDA@Fe3O4 also suggested an excellent photostability (Figure 4b). Above all, with excellent photothermal conversion efficiency and stability, PDA@Fe3O4 has the potential to be used in the domain of bioapplication as a coupling agent for photothermal ablation. 3.3. Measurement of Magnetic Properties. As a consequence of the Fe3O4 cores, with a nearby magnetic field, the PDA@Fe3O4 solution became clear, and the nanocomposites could be attracted to the bottle wall within 5 min (Figure 4c, inset, and Figure S2). Also, when the magnetic field was removed, the PDA@Fe3O4 could be redispersed in water after gentle shaking. To explore the magnetic properties of PDA@Fe3O4, the magnetization curve was determined using a vibrating sample magnetometer (VSM). The magnetic hysteresis curve suggested the superparamagnetic behavior of

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization. Uniform Fe3O4 cores were prepared using a modified polyol method24 and showed good dispersibility in water as a result of citrate capping groups on the nanoparticle surface. As the transmission electron microscopy (TEM) image reveals, the Fe3O4 cores exhibited a nearly spherical shape when dropped on the copper grid. Narrow particle size distribution can be observed to be ∼140 nm (Figure 1a). Polydopamine (PDA)-encapsulated Fe3O4 nanocomposites (PDA@Fe3O4) were synthesized by dispersion of Fe3O4 in dopamine hydrochloride solution (pH = 8.5) with gentle stirring at RT for 4 h (Scheme 1). A TEM image showed that PDA@Fe3O4 was successfully synthesized with a 5−10 nm PDA shell. (Figure 1b,c). The hydrodynamic diameter of the PDA@Fe3O4 was measured to be ∼230 nm (Figure 1d), which is obtained by dynamic light scattering (DLS) data. Compared with the Fourier transform infrared spectrum (FTIR) of Fe3O4, an absorption peak at 1290 cm−1 was present in Fe3O4@PDA and PDA spectra (Figure 2a). As the X-ray photoelectron spectroscopy (XPS) demonstrated, the obtained nanoplatforms (PDA@Fe3O4) were composed by Fe, O, N, and possibly H elements (Figure S1a). Likewise, on the observation of the thermogravimetric analysis (TGA) analysis, weight loss behavior of PDA@Fe3O4 demonstrated that PDA@ Fe3O4 has been synthesized successfully (Figure S1b). These results demonstrated that the Fe3O4 cores were well-coated with PDA. The zeta-potential of PDA@Fe3O4 in aqueous solution (pH = 7.0) was −30 mV (Figure 2b), indicating their high dispersibility in aqueous solution. 3.2. Measurement of Photothermal Properties. Polydopamine (PDA) coatings can be easily formed on many different kinds of substrates, giving excellent biocompatibility, and causing no immune response.37,38 Moreover, for its ability of converting NIR to heat energy, PDA has the prospect to be a photothermal agent for therapeutic use.21,39,40 With its layer of 24457


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Figure 2. (a) FTIR spectra of Fe3O4, PDA, and PDA@Fe3O4. (b) The zeta-potential of PDA@Fe3O4 solution (pH = 7.0). (c) UV−vis−NIR spectra of the Fe3O4 and PDA@Fe3O4 solution. (d) Temperature variation of different concentrations of PDA@Fe3O4 solution vs irradiation time.

Figure 3. (a) Photothermal images for the PDA@Fe3O4 solution (0.25 mg mL−1) with different irradiation time (785 nm, 0.5 W cm−2, 0−15 min). (b) Photothermal temperature change curve of the PDA@Fe3O4 solution (on−off period). (c) Cooling time vs −ln θ obtained from part b.

PDA@Fe3O4, for displaying no evident remanence or coercivity at 300 K (Figure 4c). The saturation magnetization value of PDA@Fe3O4 was 56.2 emu g−1, which was strong enough to facilitate their recovery with the help of a magnet. What is worth mentioning is that the presence of the PDA shell did not strongly weaken the magnetic properties of Fe3O4 (Figure S3), since the coated PDA on the Fe3O4 cores was only 5−10 nm (Figure 1a,b). These results clearly demonstrated that PDA@ Fe3O4 had excellent magnetic properties and prospects for practical use in selective separation. 3.4. Toxicity Studies. Biocompatibility is a serious concern for any nanoplatform which is designed to be used in the biological field. Methylthiazolyltetrazolium (MTT) assays on the human colon cancer cell line (HCT116) cells were

implemented to determine the toxicity of PDA@Fe3O4. As shown in Figure 4d, it can be seen that the PDA@Fe3O4 caused no significant apoptosis of HCT116 cells which were cultured with different concentrations of the nanoparticles (0−1.0 mg mL−1) for 12 or 24 h. These findings suggest that, thanks to the advantages of low toxicity and high biocompatibility, PDA@ Fe3O4 nanoparticles showed a bright future for potential bioapplication. 3.5. HSP70 Inhibitor Loading and Laser-Controlled Release. The functional groups on PDA including catechol, amine, and imine enhanced the special abilities of the PDAfunctionalized nanoparticles for absorption and separation.41,42 The PDA-functionalized nanoparticles have already been exploited as reusable adsorbents of lanthanum(III) and copper 24458


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Figure 4. (a) On−off temperature curve of the PDA@Fe3O4 solution (0.25 mg mL−1) under the irradiation of a 785 nm laser (0.5 W cm−2). (b) The UV−vis−NIR spectra of PDA@Fe3O4 solution under different NIR irradiated conditions. (c) Magnetic hysteresis curves of PDA@Fe3O4 at 300 K. Inset: digital image of PDA@Fe3O4 solution alongside a magnet. (d) HCT116 cell viability with PDA@Fe3O4 (0, 0.2, 0.4, 0.6, 0.8, 1.0 mg mL−1) treatment for 12 and 24 h.

Figure 5. (a, c) Photographs and (b, d) bacteria viability of the colonies of (a, b) E. coli and (c, d) S. aureus under different incubated conditions. Note: *p < 0.05, **p < 0.01, ***p < 0.001.

ions, for separation of organic mixed dyes, and for adsorption of lysozyme.36,43−47 The small molecule HSP70 inhibitor, 2phenylethynesulfonamide (PES), could break the normal function of HSP70 in different kinds of cell signaling pathways which would cause the cells to be less tolerant to external injuries, such as overheating. Here, PES was loaded onto the PDA@Fe3O4 via π−π stacking and/or hydrogen bonding to form a nanoplatform which can be abbreviated as PDA@Fe3O4

+ PES (Scheme 1A). The adsorption and desorption capacities of PDA@Fe3O4 + PES were further examined. On the basis of detection of the amino group on PES using the ninhydrin method, the PES encapsulation efficiency came up to 97.27% by calculation, and the loading content was measured to be 36.85%. The PDA@Fe3O4 + PES nanocomposite was dissolved in PBS buffer (pH = 7.4), 5% glucose solution, 0.9% NaCl solution, and serum solution. After the sample rested at room 24459


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Figure 6. Recyclable antibacterial study of PDA@Fe3O4 + PES nanocomposite. (a) Schematic illustration of the recyclable antibacterial application of PDA@Fe3O4 + PES. (b) Photo image of PDA@Fe3O4 + PES treated bacteria suspension (left) and the separation of PDA@Fe3O4 + PES in the bacteria suspension with a magnetic field (right). Bacterial mortality of (c) E. coli and (d) S. aureus after the photothermal inactivation of continuous three-times-recycled PDA@Fe3O4 + PES.

NIR against E. coli. Under consistent conditions, the ablation effect on S. aureus (Figure 5c and Figure S6) identified with those on E. coli. The introduction of PES enhanced the inactivation effect of PDA@Fe3O4 on bacteria. The Kruskal− Wallis test and SPSS 16.0 IBM software were introduced to evaluate the significant differences among these groups and analyzed the data. As for statistical significance, P-values 0.05) effects of PDA@Fe3O4 + PES, PBS + NIR, or PDA@Fe3O4 + NIR group on E. coli. Nevertheless, a confidence level of 99.9% (P < 0.001) confirmed the significant difference between the PDA@Fe3O4 + PES + NIR group and the blank group, which was revealed by the Kruskal−Wallis test. Similar results were observed for S. aureus (Figure 5c,d). Pvalue