Self-Assembled Polysaccharide-Diphenylalanine/Au Nanospheres for Photothermal Therapy and Photoacoustic Imaging.

Gold-based nanomaterials have attracted extensive interest for potential application in photothermal therapy (PTT) owing to their distinctive properties including high photothermal transduction, biocompatibility, and low cytotoxicity. Herein, assembled gold nanoparticle architecture-based photothermal conversion agents were synthesized by using polysaccharides (alginate dialdehyde, ADA) as both the cross-linker to induce self-assembly of diphenylalanine (FF) and the reducer for in situ reduction of Au3+ ions into Au nanoparticles (Au NPs). The extinction spectrum of the obtained self-assembled ADA-FF/Au nanospheres was finely modulated into a near-infrared region by controlling the growth of Au NPs inside the assemblies. The strong plasmonic coupling effect of the assembled Au NPs also leads to high photothermal conversion (η = 40%) of the ADA-FF/Au nanospheres, hence presenting good performance in PTT and photoacoustic imaging. This synthesis technique is promising to construct nanomaterials with desired functions for potential biomedical application by self-assembly of various nanocrystals in situ.

Introduction

Photothermal therapy (PTT), which “cooks” cancer cells using photothermal conversion agents (PTCAs) to convert optical energy to heat, has attracted lot of attention because of its spatiotemporal addressability and minimal invasiveness.[1] High photothermal transduction efficiency, absorbance in the near-infrared (NIR) region, and facial synthesis process are crucial for the further application of PTCAs in PTT.[2] Among these, the absorption in the NIR region is considered to be a key property of PTCAs because the NIR light can deeply penetrate into soft tissues on account of low absorption and scattering from blood, water, and tissue.[3]

Gold-based nanostructures have been developed as PTCAs because of their biocompatibility, low cytotoxicity, and their photothermal conversion capability,[4,5] Spherical gold nanoparticle (Au NP)-based nanomaterials, which can transfer the optical energy to heat relying on their localized surface plasmon resonance (LSPR) properties, have been extensively studied for PTT, however subjected to the low extinction in NIR.[6] Then, gold nanomaterials with various morphologies, including nanorods,[7,8] nanocages,[9] and nanoshells,[10] have been tried and proved to be effective as PTCAs. However, those gold-based nanomaterials suffer from radiation instability, poor repeatability in synthesis, and biotoxicity for future applications.[11−13] Au NP architectures self-assembled by Au NPs with polymers or biomacromolecules may overcome these disadvantages by tuning LSPR of Au NPs from visible to the NIR region while retaining the stability of the nanomaterials.[5,14,15] Recently, Nie et al.[16,17] successfully fabricated vesicular superparticles by assembly of Au NPs with the assistance of amphiphilic block copolymers, thus the extinction spectrum was effectively red-shifted to the NIR region because of the strong plasmon couplings between adjacent Au NPs. Furthermore, the ultrahigh surface plasmon also enhances the performance of the assembled gold nanomaterials in photoacoustic (PA) imaging.[18−20]

Generally, construction of self-assembled Au NP architectures consists of three steps, including synthesis of dispersed Au NPs, surface modification of Au NPs using functionalized polymers, and acquiring self-assembly architectures by cross-link or self-assembly with polymers.[14,21] In some cases, the obtained structures are coated by biocompatible polymers such as polyethylene glycol to guarantee the stabilization and biocompatibility of the products in the biotic environment.[22,23] Obviously, these tedious synthesis processes are time-consuming and the yield is relatively low.

Our previous work verified that alginate dialdehyde (ADA) can be used as both the cross-linker to induce self-assembly of FF and the in situ reducer of Au3+ ions into Au nanoparticles (Au NPs) to form gold-based nanospheres as nanodrug carriers in a very simple and high-yielding one-pot synthesis progress.[24,25] However, the Au NPs inside the nanospheres are quite discrete, which cannot modulate the absorption spectra of the assembled nanospheres to the NIR region. In this work, nucleation and growth of Au NPs inside the ADA–FF nanospheres are precisely controlled to regulate the distance between adjacent Au NPs. Thus, a plasmonic coupling effect was enhanced by the formation of abundant inter-nanoparticle junctions, causing the absorption peak of the ADA–FF/Au nanospheres shift from visible to NIR region. These ADA–FF/Au nanospheres present excellent PA response and enhanced photothermal conversion efficiency (η = 40%) upon 808 nm laser irradiation. The in vitro experiment indicates their good compatibility and photothermal treatment effect of ADA–FF/Au nanospheres.

Results and Discussion

Preparation and Characterization of ADA–FF/Au Nanospheres

ADA–FF/Au nanospheres are synthesized as shown in Scheme . When HAuCl4 solution was mixed with FF and ADA, the aldehyde groups of ADA and the amino groups of FF reacted and formed Schiff base covalent bonds between them, generating the ADA–FF unit. Meanwhile, the Au3+ irons are in situ reduced to Au NPs by aldehyde groups of ADA and attached to the polymer chain because of the formation of coordination bindings with ADA. Continuous injection of HAuCl4 is exploited to grow the gold seeds, hence forming ADA–FF/Au nanospheres. The interparticle distance between adjacent Au NPs can be finely controlled by adjusting the particle size of Au NPs.

Scheme 1

Schematic Illustration of the Synthesis Process of ADA–FF/Au Nanospheres and Their Applications in PTT and PA Imaging

Figures a and S1 show the morphological evolution of the ADA–FF/Au nanospheres during synthesis. It can be found that self-assembled ADA–FF/Au nanospheres containing small gold seeds are quickly formed when Au3+ is added into the solution. After continuous injection of diluted Au3+ solution for 15 min, the size of the Au NPs increases, whereas Au NPs are still discrete, and 60 min later, ADA–FF/Au nanospheres with assembled Au NPs inside are formed. Figure b shows, in this process, that the size of the Au NPs increases from 8 to 30 nm in ADA–FF/Au nanospheres, indicating the gradual growth of Au NPs with continuous dropping of Au3+ solution. The growth of Au NPs shortened the interparticle distances, which results in a red shift of the absorption spectra (Figure c) from visible to NIR region because a plasmonic coupling effect is enhanced between adjacent Au NPs. Meanwhile, a color change of the solution from pink to cyan can be observed during this process (Figure c inset). However, with the prolongation of reaction time, the LSPR absorption peak broadens, probably because of the nonuniform particle size distribution of Au NPs. It is observed that ADA–FF/Au nanospheres have an average hydrodynamic diameter of 253 nm and highly negative charged surface (−30 mV, Figure S2), indicating the presence of COO– groups of polymers on the surface of nanospheres, which gives the stability of ADA–FF/Au nanospheres in aqueous solutions. This could also be confirmed by the smooth surface of the nanospheres, as shown in the scanning electron microscopy (SEM) images of Figure S3. Energy-dispersive X-ray spectroscopy (EDXS) (Figure S4) and transmission electron microscopy (TEM) elemental mapping of ADA–FF/Au nanospheres (Figure d) indicate the compositions of C, N, O, and Au and most of Au NPs are located inside the nanospheres, causing the abundant internanoparticle junctions among Au NPs.

Figure 1

(a) Morphological evolution of the ADA–FF/Au nanospheres. (b) Effective diameters of Au NPs in ADA–FF/Au nanospheres. (c) Absorption spectra and color change (inset) of intermediate products during synthesis. (d) Elemental maps of ADA–FF/Au nanospheres.

Figure a shows the Fourier transform infrared spectroscopy (FTIR) spectra of FF, ADA, and ADA–FF/Au nanospheres. For ADA–FF/Au nanospheres, the peak at 3255 cm–1 attributed to the fact that the stretching vibration of −NH2 largely weakened when Schiff bonds (C=N) were formed between FF and ADA.[26] The peak at 1731 cm–1 belonging to the free aldehyde group of ADA disappears, which may relate to the large consumption of CHO in reducing Au3+ and the formation of Schiff bonds with FF.[27] The peak of amino I at 1604 cm–1 in FF shifts to 1619 cm–1 in ADA–FF/Au nanospheres because of the formation of C=N stretching bonds. The X-ray photoelectron spectroscopy (XPS) spectra of N 1s band (Figure b) also confirm the existence of Schiff bonds. The peak at 399 eV belongs to the C=N group and the peak located at 400.4 eV is attributed to the O=C–N groups.[28] The peaks located at 85.2 and 88.2 eV in the Au 4f band spectra (Figure c) belong to gold atoms, which illustrate the absence of other metallic compounds.[29]Figure d shows an emission peak centered at 306 nm in the fluorescent emission (FL) spectrum of FF, whereas a peak at 410 nm emerged for ADA–FF/Au nanospheres. This result indicates that FF may use J-aggregate arrangements in ADA–FF/Au nanospheres through the π–π interactions between aromatic groups. Another new peak at 460 nm suggests the formation of Schiff bonds between ADA and FF.[30,31]

Figure 2

(a) FTIR spectra of FF, ADA, and ADA–FF/Au nanospheres. XPS spectra of (b) N 1s and (c) Au 4f. (d) FL spectra of FF and ADA–FF/Au nanospheres.

In this synthesis process, it is revealed that the interparticle distance between adjacent Au NPs is of crucial importance for the plasmonic coupling effect, which enables the absorption range of ADA–FF/Au nanospheres broaden to the NIR region. The trick of precisely controlling the nucleation and growth of Au NPs depends on the concentration of Au3+ solution. Gold seeds generate under conditions of high chemical supersaturation while the growth stage proceeds under a much slower and milder reducing condition.[32−34] Herein, a high concentration of HAuCl4 was used first to quickly form gold seeds in the system. Then, a diluted HAuCl4 solution is slowly injected for the growth of seeds, decreasing secondary nucleation in the system. The reaction medium is another crucial factor for the synthesis of ADA-FF/Au nanospheres. In this work, ethanol is added to reduce the solubility ADA in the reaction medium, thus avoiding free ADA in water to generate dissociated Au NPs in the system.[35] After the formation of Au seeds, the ethanol solution of HAuCl4 was slowly injected into reaction system. Thus, Au3+ ions are prone to be reduced inside the nanospheres because there is almost no free ADA in the continuous phase. Figure a–e shows that with the increment of ethanol, the amount of free Au NPs gradually decrease and eventually disappear. Nevertheless, it can be observed that the Au NPs inside the nanospheres with 120 μL of ethanol (Figure e) are smaller and more homogeneous than that with 90 μL of ethanol (Figure d). The absorption spectrum (Figure f) shows an obvious red shift of the LSPR peak from the visible to the NIR region with the increase of ethanol dosage. However, ethanol cannot be added in a whole, which may cause a severe agglomeration possibly because free ADA chains are prone to precipitate out.

Figure 3

TEM images of ADA–FF/Au nanospheres with the addition of (a) 0, (b) 30, (c) 60, (d) 90, and (e) 120 μL of ethanol in solution and (f) their UV–vis absorption spectra.

Photothermal Effect and PA Imaging of ADA–FF/Au Nanospheres

Because of the strong plasmatic absorption in the NIR region, the photothermal effect of ADA–FF/Au nanospheres was measured to confirm their potential application in tumor treatment. Figure a illustrates that when irradiating with an 808 nm laser at 1.5 W/cm2, the temperature of ADA–FF/Au nanosphere aqueous dispersion with different concentrations (0–200 μg/mL) all increase more quickly as the concentration increases. After 5 min of irradiation, the temperature of ADA–FF/Au nanosphere aqueous dispersion with the concentration of 200 μg/mL ascents to 61.4 °C, while the temperature of water only elevates by 2.3 °C. Moreover, the temperature of ADA–FF/Au nanosphere aqueous dispersion increases more rapidly when increasing the laser power (0.5–2 W/cm2), while the concentration is fixed to 100 μg/mL, as shown in Figure b. The changes of temperature during irradiation can be directly observed by infrared photos (Figure c,d). After being heated under 2 W/cm2 laser irradiation for 15 min, the sample was cooled to room temperature. It no obvious change in TEM images and absorption spectra (Figure S5) has been found, showing the good stability of ADA–FF/Au nanospheres in PTT.

Figure 4

(a,b) Temperature elevation of ADA–FF/Au nanosphere aqueous dispersion. (c) Infrared thermal images of ADA–FF/Au nanosphere aqueous dispersion with different concentrations and (d) different irradiation time. (e) Temperature evaluation of the dispersion with laser on for 5 min and then turned off. (f) Time vs negative natural logarithm of the temperature during the cooling period.

A vital index, photothermal conversion efficiency (η), is measured to estimate the potential application of photothermal agents in PTT. According to the conversion model in previous work,[21] 100 μg/mL of ADA-FF/Au nanosphere aqueous dispersion was exploited as the representative. The temperature was recorded each 10 s and shown in picture (Figure e). The η of ADA–FF/Au nanospheres is calculated according to the formulas.

In eq , h is the heat-transfer coefficient, S is the container surface area, Tmax is the highest temperature, Tsurr is the ambient temperature, and Q0 is the heat generated by water and container under laser irradiation. In eq , P is the laser power, A808 is the absorption intensity of ADA–FF/Au nanospheres at 808 nm, m is the mass of the solution, CH is the heat capacity of water, and τs is the time constant.[15]Figure f shows that the constant (τs) is determined to be 226.3. Then, the photothermal transduction efficiency is calculated to be 40%, which is higher than that of gold nanorods (18%),[11] gold nanoshells (22%),[36] and gold nanoplates (29%),[37] indicating the strong plasmonic coupling effect of the Au NPs inside the nanospheres.

PA imaging is a valid way to detect deeper tissue signals in human body.[38] Emerging exogenous contrast agents can effectively transfer the light energy to a temperature rise in the tissue and produce ultrasonic waves through thermoelastic expansion.[39,40] Because the strong plasmonic coupling effect of ADA–FF/Au nanospheres caused absorption in the NIR region, it is reasonable to hypothesize that it could also be used as an agent for PA imaging.[41] The samples show strong bright signals in PA images compared with those without ADA–FF/Au nanospheres in water, and the signals become much brighter with the increase of concentration (Figure a). Moreover, Figure b confirms that the PA signal is a linear variation with the concentration of ADA–FF/Au nanospheres.

Figure 5

(a) PA images and (b) intensities of the dispersion irradiated with NIR laser.

As illustrated, ADA–FF/Au nanospheres exhibit good performances both in PTT and PA imaging because of their strong absorption in the NIR region and a good light-to-heat conversion. It could be concluded that shortening the distance between the adjacent Au NPs in gold-based nanospheres by precisely modulating the nucleation and growth of Au NPs enhances the plasmonic coupling effect.

Cytoxicity and in Vitro Photothermal Effect of ADA–FF/Au Nanospheres

To further confirm the potential application of ADA–FF/Au nanospheres in tumor therapy, cytoxicity in human tumor cells (4T1 cells) was measured by determining cellular viability using a CCK-8 assay. In the assessment, 4T1 cells were incubated for 24 h before use. Then, 0, 10, 50, 100, 150, and 200 μg/mL of ADA–FF/Au nanosphere aqueous dispersion were added into 96-hole plates to incubate with 4T1 cells for another 24 h. As shown in Figure a, the cell viability still maintains a high level (cell viability ≥ 95%) while concentration of ADA–FF/Au nanospheres reaches 200 μg/mL. Therefore, the result shows that ADA–FF/Au nanospheres have a good biocompatibility and can be tested in vitro for further researches.

Figure 6

(a) Cytotoxicity of ADA–FF/Au nanospheres. (b) Cell viabilities of 4T1 with and without 808 nm laser irradiation (2 W/cm2, 15 min). (c) Confocal microscopic images of 4T1 cells stained by calcein-AM/propidium iodide.

The cell viability of 4T1 cells with and without laser irradiation was compared to confirm the in vitro photothermal effect of ADA–FF/Au nanospheres. Different concentrations of ADA–FF/Au nanospheres were incubated with 4T1 cells for 4 h before irradiation. CCK-8 assay shows (Figure b) that with the increase of ADA–FF/Au nanosphere concentration, more than 95% of cells are still alive for every controlled group without irradiation, while after irradiating under NIR laser for 15 min, the cell viability remarkably decreases and becomes lower along with the increase of ADA–FF/Au nanosphere concentration. When the concentration reaches 200 μg/mL, merely 20% of cells are alive. Calcein-AM (green) and PI (red), which are able to differentiate live and dead cells, are also exploited to evaluate the cell viability (Figure c). The red fluorescence only appears when the cells are incubated with ADA–FF/Au nanospheres and irradiated by the laser, suggesting that this material enables to kill cancer cells in vitro PTT.

Conclusion

In summary, ADA–FF/Au nanospheres were synthesized by using a facial method with ADA as both the reducer for in situ reduction of Au3+ ions into Au NPs and the cross-linker to induce self-assembly of FF. The size of Au NPs in nanospheres was adjusted to shorten the distance between adjacent particles and lead to a red shift of the LSPR absorption into the NIR region. In general, the as-obtained ADA–FF/Au nanospheres have the following features: (i) good PTT effect and high photothermal conversion efficiency (η = 40%); (ii) simultaneous and sensitive PA imaging performance; and (iii) good biocompatibility. This unique Au-based nanomaterial is promising to further promote the practical applications of novel PTCAs for cancer therapeutic and diagnostic.

Experimental Section

Materials

Ethanol, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), diphenylalanine peptide (FF, 98%), gold chloride trihydrate (HAuCl4·3H2O, ≥99.9%), sodium alginate, and sodium periodate (NaIO4) were all purchased from Aladdin Chemical Reagent Co. Ltd.

Preparation of Polysaccharide–diphenylalanine/Au (ADA–FF/Au) Nanospheres

ADA was obtained by an oxidization reaction of sodium alginate.[42] Typically, FF (3.9 mg) dissolved in HFIP (130 μL) was mixed with 1 mL of ADA aqueous solution at 70 °C, followed by the addition of HAuCl4 solution (25 μL, 50 mM). When the color of the solution turned into pink, a HAuCl4 aqueous solution (6.67 mM, 1500 mL) mixed with a certain amount of (0, 30, 60, 90, and 120 μL) ethanol was injected into the solution with the a speed of 1.5 mL/h continuously to obtain ADA–FF/Au nanospheres. The reaction was continued for another 1 h after dropping of Au3+. Finally, the products were collected by centrifugation at 5000 rpm for 5 min and washed three times with deionized water and then stored at 5 °C for subsequent characterization.

Photothermal Effect and PA Imaging of ADA–FF/Au Nanospheres

The 0.30 mL of ADA-FF/Au nanosphere aqueous dispersion (50, 100, 150, and 200 μg/mL) was pipetted into a 1 mL centrifugation tube and exposed under a 808 nm laser radiation (FC-808-5000-MM/1–5000Mw, China) on a 0.66 cm spot with the power density of 1.5 W/cm2. Then, 0.30 mL of ADA–FF/Au nanosphere aqueous dispersion (100 μg/mL) was irradiated under the same condition at different power densities (0.5, 1.0, 1.5, and 2 W/cm2). NIR camera (FLIR-T62101, Sweden) was used to record the temperature of the solution each 10 s for 5 min.

To test the photothermal conversion efficiency of ADA–FF/Au nanospheres, 0.30 mL of ADA–FF/Au nanosphere (100 μg/mL) aqueous dispersion in centrifuged tubes was irradiated upon laser on 0.66 cm spot at a power density of 1.5 W/cm2 for 5 min and cooled down for another 5 min without laser irradiation. Real-time temperature of the solution was recorded in the same way as mentioned above.[43]

The PA signals of ADA–FF/Au nanospheres were obtained from an agarose gel phantom having different concentrations of ADA–FF/Au nanospheres (0, 38, 76, 153, 246, and 307 μg/mL) using a multimode PA imager (Vevo LAZR, America).

Cytoxicity Assay and in Vitro Photothermal Effect of ADA–FF/Au Nanospheres

The tumor cell viability assay was conducted using a Cell Counting Kit-8 (CCK-8). 4T1 cells were cultured in the standard cell media for 24 h. Then, ADA–FF/Au nanospheres with various concentrations (0, 10, 50, 100, 150, and 200 μg/mL) were incubated with cells in the 96-hole plates for 24 h. Next, 10 μL of CCK-8 solution with culture media was added and incubated with cells for another 2 h. Subsequently, the optical densities of the cells were analyzed at an absorbance of 450 nm, which was the exact wavelength for CCK-8 assay.[44] The cell viability was calculated according to the articles.[45]

To investigate the photothermal effect of the products, 4T1 cells with and without ADA–FF/Au nanospheres (0, 50, 100, 150, and 200 μg/mL) were incubated for 4 h and then exposed to an 808 nm NIR laser (2 W/cm2, 15 min). Next, a CCK-8 assay and a calcein-AM/PI test were employed to evaluate the cell viability. The CCK-8 assay was exploited in the same way as mentioned above and the calcein-AM/PI staining assay was conducted as follows. After irradiation, calcein-AM solution was used to stain the cells for 30 min, followed by washing cells with phosphate-buffered saline (PBS) for three times. Then, the PI solution was used to dye cells for another 10 min and PBS was used to wash cells for three times. After being treated with calcein-AM and PI, the cells were observed via a confocal microscope (Nikon C2+, Japan).[46]

Characterization

Morphologies of the products were observed using a transmission electron microscope (Tecnai G2 20 TWIN, America) at an acceleration voltage of 200 kV with a charge-coupled device (CCD) camera and a field emission SEM (Zeiss Ultra 55, Germany) with a CCD camera under a 20 kV voltage. The elemental composition and distribution of the product were characterized using a high-resolution TEM (HRTEM, Tecnai G2 F20 S-Twin, America) at an acceleration voltage of 200 kV and an energy-dispersive X-ray spectrometer (X-Max 80T, America). The UV–vis absorption spectrum was recorded by a UV–vis spectrophotometer (PerkinElmer Lambda 750, America) under ambient conditions. Fluorescent emission (FL) spectra were recorded using a PTI QM40 instrument (America). The particle sizes, size distribution, and zeta potential of the products were achieved by using a Nano-ZS90 (Malvern, England). The FTIR spectrum of the samples was conducted with powder-pressed KBr pellets using a Nicolet Nexus 470 instrument (America). XPS analysis was executed with a PHI 5000C instrument (America).