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Mussel-Inspired Gold Hollow Superparticles for Photothermal Therapy Ye Tian, Shun Shen, Jiachun Feng, Xingguo Jiang, and Wuli Yang* Photothermal therapy (PTT), owing to its advantages over conventional cancer therapies, including specific spatial and temporal selectivity and minimal invasiveness, has drawn intensive research and clinical interests in the past decade.[1] In the therapeutic treatment, electromagnetic irradiation, specifically near-infrared (NIR) light, is focused at the tumor sites and the hyperthermia generated from the absorbed energy “burns out” cancer cells.[2] Absorbance in the NIR region and photothermal conversion (PTC) efficiency are two parameters that determine the efficacy of a PTC agent.[3] NIR region is a transparency window for biological tissues, leading to the deep penetration and low detriment of NIR light.[4] Recently, the PTC effects of many NIR-absorbing nanomaterials, organic and inorganic, are revealed, such as porphysome, polypyrrole (PPy) nanoparticles, graphene nanosheets (GNSs), single-walled carbon nanotubes (SWCNTs), and Cu2-xS nanocrystals.[5] Among them, gold nanostructures with various sizes and shapes have demonstrated extraordinary promise as PTC agents due to the efficient conversion of optical energy into heat based on localized surface plasmon resonance (LSPR).[6–13] However, their simplest family member, spherical gold nanoparticles (GNPs), lacks an absorption in the NIR region and thus their photothermal effect is limited.[14] Inspired by gold nanoshells and hollow nanospheres, whose LSPR can be red-shifted to the NIR region by tuning the cavity diameter and shell thickness,[15,16] Nie and co-workers[17] successfully solved the problem by the assembly of GNPs into vesicular superparticles with the assistance of amphiphilic block copolymers. Owing to the short interparticle distance in the obtained structures and the consequent plasmatic coupling effect, the superparticles exhibit an optical properties very different from the individual nanoparticles:[18,19] an LSPR peak is observed in the NIR region, making the obtained gold superparticles an effective PTC agent. Polydopamine (PDA) is a synthetic polymer mimicking the naturally occurring melanin with many striking physicochemical properties, including paramagnetism, electrical Y. Tian, Prof. J. Feng, Prof. W. Yang State Key Laboratory of Molecular Engineering of Polymers Department of Macromolecular Science Fudan University Shanghai 200433, P.R. China E-mail: [email protected] Dr. S. Shen, Prof. X. Jiang Key Laboratory of Smart Drug Delivery Ministry of Education & PLA School of Pharmacy Fudan University Shanghai 201203, P.R. China
DOI: 10.1002/adhm.201400787
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conductivity, adhesive property, and redox activities.[20,21] Last year, Lu and co-workers revealed the excellent photothermal effect of PDA,[22] which had emerged as a promising PTC agent with good biocompatibility since then. Also, the features as multifunctional coatings and the chemical reactivity of PDA have been well investigated,[20,23] and the deep understanding of its characters has already been employed in the assembly of nanometer-scale materials.[24] In our previous work, a mussel-inspired cross-linking strategy was developed to prepare graphene oxide papers (GOPs) that employed multiamino poly(ethylene imine) (PEI) to cross-link the PDA-capped graphene oxide sheets (PGOs).[25] Based on the strategy, in the present work, we further develop an assembly approach to preparing gold hollow superparticles (GHSPs) with enhanced photothermal effect. The approach (schemed in Figure 1a) includes the PDA coating of GNPs and the assembly of PDAcapped GNPs (PGPs) that utilizes their reaction with aminegroup-containing PEI and methoxy-poly(ethylene glycol) amine (mPEG-NH2). Strong plasmatic coupling effect is realized in our GHSP system when the GNPs are drawn near in the mussel-inspired cross-linking process and strong absorption in the NIR region is observed. Taking advantage of the light harvesting and photothermal contribution of PDA, the GHSPs exhibit outstanding PTC effect, even better than those of PGPs and pure PDA nanoparticles at an equivalent mass concentration. After in vivo administration, the GHSPs are able to ablate cancer with localized NIR laser exposure and show no longterm systemic toxicity, which make them of great promise as a PTC agent for cancer therapeutic applications. GNPs used in the present work are prepared with a citrate reduction method reported previously[26] and have an average diameter of 25 nm. For the PDA coating of GNPs, dopamine is self-polymerized on the surfaces of GNPs in a Tris-Cl buffer (10 × 10−3 M, pH 8.5).[27] (Tris is tris(hydroxymethyl)aminomethane, IUPAC name 2-amino-2-hydroxymethyl-propane1,3-diol.) Figure 1b presents the ultraviolet–visible (UV–vis) spectra of GNPs and PGPs, and both of them exhibit a characteristic extinction peak around 522 nm due to LSPR of 25 nm GNPs. The slight red-shift for the plasmon peak is due to the increase of refractive index around GNPs after PDA coating.[28] Besides, PGPs show obvious absorbance at long wavelength (600–1000 nm), which is mainly attributed to the light harvest contribution of the PDA coatings,[29] as pure PDA nanoparticles have obvious absorbance in this spectral region (the UV–vis spectrum of PDA nanoparticle dispersion is shown in Figure S1, Supporting Information). In the assembly process, the mixture of PEI and mPEG-NH2 is added to a dispersion of the obtained PGPs and GHSPs are generated after a mild shaking of 24 h. It is revealed that the assembly is highly pH dependent. At a pH value of 6.0, fine
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Figure 1. Mussel-inspired assembly of gold nanoparticles. a) Schematic illustration of the architecture of a GHSP. The diagram is not drawn to scale. b) UV–vis spectra of GNPs, PGPs, and GHSPs. c) TEM images of the assembly results: i–v) present the assemblies obtained from various pH environments, while (vi), (vii), and (viii) demonstrate the influence of mPEG-NH2/PEI ratios; the inset image in iii) shows a broken superparticle to reveal the hollow interior of GHSPs. d) Zeta potential of PGPs at different pH values. e) A digital photograph of the suspensions of GNPs, PGPs, and GHSPs.
GHSPs are formed, whose transmission electron microscope (TEM) images (Figure 1c and Figure S2a in the Supporting Information) clearly show that the superparticles with hollow interiors consist of a layer of densely packed PGPs. The superparticles have a narrow size distribution with the average size of 240 nm (measured by dynamic scattering light, DLS, Figure S3, Supporting Information). When the pH value is higher than 6.0, the PGPs aggregate into nondispersible clusters in the assembly process, while in a more acidic environment (pH 4.0 and 5.0), nonuniform assemblies with a loosely packed morphology are formed. To investigate the formation mechanism of the superparticles, the zeta potential of PGPs at different pH values is measured and the results are shown in Figure 1d. It is suggested that when the pH value of the system decreases, the PGPs tend to lose their negative charge on the surface, and gain some positive charge at an even lower pH value.[30] The zeta potential
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gets close to zero at pH 6.0, and the decreased hydrophilicity of PGPs results in the attractive interparticle interactions.[31] Moreover, the reaction between PEI and PDA (the chemistry of the reaction is schemed in Figure 1a) leads to the crosslinking of the PGPs,[32] which further increases the trend for agglomeration. Meanwhile, during the organization of PGPs into superparticles, mPEG-NH2 molecules are grafted to the surface of the PGPs with a mechanism similar to that of PEI, and the PEG chains stabilize the structures. In this case, the PEI cross-linking and PEGlyation provide the driving force for the gradual aggregation and rearrangement of PGPs into hollow assemblies. This reaction-induced assembly process is similar to the self-assembly of amphiphilic block copolymers into vesicular architecture, in which the hydrophobic block acts as cross-linking sites while the hydrophilic block stabilizes the structure in the aqueous media.[33] Nevertheless, the environmental pH value not only affects the hydrophilicity of the PGPs
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50.6 °C at a concentration of 50 µg mL−1, while the temperature of PBS increases by only 2.9 °C. It has been well demonstrated that hyperthermia is able to kill cancer cells directly over 45 °C (ablation) or at 39–42 °C (mild hyperthermia).[36] We can infer that after the injection of GHSPs, the tumor tissues can be easily heated to over 45 °C in the irradiation and the cancer can be ablated in a short period of time. Next, we compare the PTC capability of GHSPs with some other PTC agents. Upon laser illumination for 500 s, the PGPs, GNRs (the TEM image and UV–vis spectrum is displayed in Figure S4, Supporting Information) and PDA nanoparticles (at an equivalent concentration: 50 µg mL−1) raise the temperature by 11.5 °C, 20.1 °C, and 36.3 °C, respectively (Figure 2b). The PTC efficiency (η) is calculated as follows:[37]
η = (hSΔTmax − Q s ) / I(1 − 10 − A )
(1)
τ s = m sCs / hS
(2)
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but also has a significant influence on the rate of cross-linking and PEGlyation reactions. It is well developed that the aqueous chemical equilibrium between catechol and quinone shifts toward quinone under alkaline conditions, conferring latent reactivity toward amine groups.[34] Owing to the higher reactivity of the PDA coating at high pH values, the PGPs are crosslinked rapidly and agglomeration is observed. On the contrary, in an acidic environment, the amine groups of PEI and mPEGNH2 are protonated and the reactions are inhibited, leading to the incomplete assembly. According to the mechanism proposed above, it is reasonable to hypothesize that the assembly is also related to the amount of PEI and mPEG-NH2. Therefore, we change the mPEG-NH2/PEI ratio (molar ratio) in the assembly process. It can be observed from the TEM images (Figure 1c) that in the absence of mPEG-NH2 (mPEG-NH2/PEI = 0:10), the PGPs aggregate and precipitate, which is attributed to the decrease of dispersion stability in the PEI cross-linking process. On the other hand, if more mPEG-NH2 is added to the system (mPEGNH2/PEI = 5:5), the dispersibility of PGPs raises, and the PGPs assemble into small solid clusters, whose extinction peak is very different from the hollow superparticles (Figure S2c, Supporting Information). As a boundary condition, when the mPEG-NH2/PEI ratio reaches 10:0 (without the adding of PEI), the assembly cannot occur. These results prove our hypothesis and the assembly is able to be regulated by changing the molar ratio of mPEG-NH2/PEI. The assembly of PGPs leads to short interparticle distances and the plasmon peak is broadened owing to a strong plasmon resonance coupling effect between adjacent GNPs.[16,35] The assembly process is accompanied by a color change of the dispersion from purple to brown (Figure 1e), and the LSPR peaks of the obtained GHSPs are located at 810 nm (Figure 1b). The strong plasmatic absorption peak located in the NIR region motivates us to explore their potential use in the PTT of cancer. GHSPs are dispersed in phosphate-buffered saline (PBS) at concentrations ranging from 20 to 100 µg mL−1, and then irradiated with an 808 nm laser at 2 W cm−2 for 500 s. Pure PBS is used as a negative control. The temperatures of all samples increase during the laser exposure, and the temperature increased more rapidly as the concentration increases (Figure 2a). After 500 s, the temperature of GHSPs dispersion increases by
where h is the heat transfer coefficient, S is the surface area of the container, ΔTmax is the temperature change of GHSPs sample at the maximum steady state, I is the laser power, A is the absorbance of GHSPs, Qs is the heat associated with the light absorbance of the solvent, τs is the sample-system time constant and ms and Cs are the mass (0.1 g) and heat capacity (4.2 J g−1) of the solvent. According to Equations (1) and (2), the η value of the GHSPs is determined to be 38% (Figure S5, Supporting Information), remarkably higher than those of PGPs (15%) and GNRs (22%), only slightly lower than that of PDA nanoparticles (40%, Figure S6a, Supporting Information). Other than the PTC efficiency, how strong the nanomaterials can convert photon energy also depends on the strength of light–matter interactions.[21] To determine how effective the PTC agents absorb light, their UV–vis spectra (at an equivalent concentration: 50 µg mL−1) are summarized in Figure S6b (Supporting Information). As indicated, GHSPs exhibit a much higher absorbance value at 808 nm than the others, indicating the strong plasmatic coupling effect induced by the assembly is the main contributor to the outstanding photothermal effect of GHSPs. Before studying the photothermal effect of GHSPs in vivo, we first ought to demonstrate their in vitro photothermal behavior in cell culture experiments, and human breast adenocarcinoma cell line (MCF-7 cells) is used as model. The cells are
Figure 2. Photothermal effect of GHSPs. a) Temperature elevation of PBS and GHSPs suspension with different concentrations as a function of irradiation time. b) The photothermal response of the aqueous dispersion of GHSPs, the building blocks (PGPs), PDA nanoparticles, and GNRs at an equivalent concentration (50 µg mL−1) with an NIR laser (808 nm, 2 W cm−2). Irradiation is continued for 500 s, and then the laser is turned off. PBS is used as a negative control.
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Figure 3. In vitro cell assays. a) Confocal images of calcein-AM (green, live cells) and PI (red, dead cells) co-stained MCF-7 cells after laser irradiation. White bar indicates 1 mm. b) Cell viability of MCF-7 cells after incubation with increased concentrations of GHSPs.
incubated with GHSPs for 30 min and irradiated by the 808-nm laser (2 W cm−2) for 5 min. After that, calcein acetoxymethyl ester (calcein-AM)/propidium iodide (PI) co-staining is carried out to differentiate live (green) and dead (red) cells under confocal fluorescence imaging (Figure 3a). While cells within the laser spot are completely destroyed by hyperthermia, those far away from the irradiation region survive. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is conducted (Figure 3b) to quantitatively evaluate the photothermal effect. After incubation for 24 h with increasing concentrations of GHSPs, no significant cytotoxicity is observed and even up to a concentration of 1 mg mL−1, the cell viability still remains approximately 92%. However, upon laser irradiation, significant growth inhibition of MCF-7 cells is hindered and only 8.9% cells remain viable at a GHSP concentration of 200 µg mL−1,
suggesting that GHSPs can effectively kill cancer cells with the assistance of laser exposure. It should be noted that combining the influences of PTC efficiency (η) and absorbance in the NIR region, the resultant photothermal effect of GHSPs is much better than those of other PTC agents (Figure 2b), and they are more effective in killing cancer cells in the in vitro photothermal experiment (Figure S7, Supporting Information). Being aware of the photothermal effect of GHSPs, we finally conduct in vivo PTT. Animal care and handing procedures were in agreement with the guidelines evaluated and approved by the ethics committee of Fudan University. Mice bearing MCF-7 xenografts are intratumorally injected with GHSPs (dosage 1.0 mg kg−1) and then exposed to an 808 nm laser at 2 W cm−2 for 5 min. Thermal imaging with an infrared thermal camera is used to monitor the efficacy of the treatment (Figure 4a).
Figure 4. In vivo PTT. a) Thermal images of MCF-7 tumor-bearing mice exposed to an 808 nm laser for 5 min after the injection of PBS or GHSPs. b) Heat curves of tumors upon laser irradiation as a function of irradiation time. Inset: H&E staining of tumor sections collected from PBS- (right) and GHSP-treated (left) mice after laser irradiation. c) In vivo effects of the PTT on the growth of MCF-7 tumor. ***, P < 0.001 vs PBS, PBS+laser and GHSPs. d) Morbidity-free survival rate of mice bearing MCF-7 tumors after treatment.
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Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This work was financially supported by National Science Foundation of China (Grant Nos. 51273047 and 51473037) and the “Shu Guang”
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As shown in Figure 4b, the local tumor temperature rapidly increases to 60 °C within 5 min and no significant temperature increase is observed in other body parts of the mice. Hematoxylin and eosin (H&E) staining of tumor slices is conducted for the tumor collected immediately after laser illumination and significant cancer-cell damage in the tumor is found (Figure 4b, Inset). To determine the antitumor effect, both tumor sizes (Figure 4c) and morbidity-free survival rate (Figure 4d) are recorded after treatment. The results show that the tumors treated with PBS, GHSPs only, and PBS plus laser irradiation grow rapidly, suggesting the tumor growth is not affected by the laser irradiation or GHSPs alone. A single injection of GHSPs plus laser illumination leads to a complete remission of MCF-7 tumors and no tumor recurrence is observed over a course of 30 days. Free of tumors, all the mice in this group survive without a single death, confirming the success of the PTT. After the treatment, the GHSPs leak into the circulation and taken up by the reticuloendothelial system (RES) subsequently, including the liver and the spleen.[38] This clearance route is proved by the biodistribution of GHSPs in the major viscera (heart, liver, spleen, lung, and kidneys) and tumor tissues, which is quantified by the Au element in the tissues (measured by inductively coupled plasma mass spectrometry, ICP-MS, Figure S8, Supporting Information). The blood chemistry and hematology analysis reveal that no obvious hepatic toxicity is induced by the therapy and all of the hematological parameters in the treated groups appear to be normal compared with the control group (Figure S9 and Table S1, Supporting Information). The body weight of mice, whose change is an indicator of systemic toxicity,[39] is simultaneously measured after receiving the PTT. The body weight of mice in GHSPs- and GHSPs-pluslaser-treated groups do not differ greatly from that of PBS, indicating that the superparticles do not exhibit severe systemic toxicity (Figure S10, Supporting Information). It is suggested that GHSPs, which possess efficient photothermal activity and low systemic toxicity, can act as a hopeful PTC agent for the PTT of cancer. In conclusion, we have developed an approach to assemble PGPs into hollow superparticles via the dopamine chemistry. The obtained GHSPs show a strong LSPR peak in the NIR region and high PTC efficiency, and can kill cancer cells with the assistance of laser irradiation. The GHSPs show good biocompatibility in vivo and can effectively ablate tumors in the PTT. The high performance of the superparticles offers a new generation of PTC agent and the mussel-inspired assembly approach could be extremely useful for the transfer of nanomaterial science to realistic technologies.
project (12SG07) supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation. Received: December 11, 2014 Revised: January 23, 2015 Published online:
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Mussel-Inspired Gold Hollow Superparticles for Photothermal Therapy Ye Tian, Shun Shen, Jiachun Feng, Xingguo Jiang, and Wuli Yang* Photothermal therapy (PTT), owing to its advantages over conventional cancer therapies, including specific spatial and temporal selectivity and minimal invasiveness, has drawn intensive research and clinical interests in the past decade.[1] In the therapeutic treatment, electromagnetic irradiation, specifically near-infrared (NIR) light, is focused at the tumor sites and the hyperthermia generated from the absorbed energy “burns out” cancer cells.[2] Absorbance in the NIR region and photothermal conversion (PTC) efficiency are two parameters that determine the efficacy of a PTC agent.[3] NIR region is a transparency window for biological tissues, leading to the deep penetration and low detriment of NIR light.[4] Recently, the PTC effects of many NIR-absorbing nanomaterials, organic and inorganic, are revealed, such as porphysome, polypyrrole (PPy) nanoparticles, graphene nanosheets (GNSs), single-walled carbon nanotubes (SWCNTs), and Cu2-xS nanocrystals.[5] Among them, gold nanostructures with various sizes and shapes have demonstrated extraordinary promise as PTC agents due to the efficient conversion of optical energy into heat based on localized surface plasmon resonance (LSPR).[6–13] However, their simplest family member, spherical gold nanoparticles (GNPs), lacks an absorption in the NIR region and thus their photothermal effect is limited.[14] Inspired by gold nanoshells and hollow nanospheres, whose LSPR can be red-shifted to the NIR region by tuning the cavity diameter and shell thickness,[15,16] Nie and co-workers[17] successfully solved the problem by the assembly of GNPs into vesicular superparticles with the assistance of amphiphilic block copolymers. Owing to the short interparticle distance in the obtained structures and the consequent plasmatic coupling effect, the superparticles exhibit an optical properties very different from the individual nanoparticles:[18,19] an LSPR peak is observed in the NIR region, making the obtained gold superparticles an effective PTC agent. Polydopamine (PDA) is a synthetic polymer mimicking the naturally occurring melanin with many striking physicochemical properties, including paramagnetism, electrical Y. Tian, Prof. J. Feng, Prof. W. Yang State Key Laboratory of Molecular Engineering of Polymers Department of Macromolecular Science Fudan University Shanghai 200433, P.R. China E-mail: [email protected] Dr. S. Shen, Prof. X. Jiang Key Laboratory of Smart Drug Delivery Ministry of Education & PLA School of Pharmacy Fudan University Shanghai 201203, P.R. China
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conductivity, adhesive property, and redox activities.[20,21] Last year, Lu and co-workers revealed the excellent photothermal effect of PDA,[22] which had emerged as a promising PTC agent with good biocompatibility since then. Also, the features as multifunctional coatings and the chemical reactivity of PDA have been well investigated,[20,23] and the deep understanding of its characters has already been employed in the assembly of nanometer-scale materials.[24] In our previous work, a mussel-inspired cross-linking strategy was developed to prepare graphene oxide papers (GOPs) that employed multiamino poly(ethylene imine) (PEI) to cross-link the PDA-capped graphene oxide sheets (PGOs).[25] Based on the strategy, in the present work, we further develop an assembly approach to preparing gold hollow superparticles (GHSPs) with enhanced photothermal effect. The approach (schemed in Figure 1a) includes the PDA coating of GNPs and the assembly of PDAcapped GNPs (PGPs) that utilizes their reaction with aminegroup-containing PEI and methoxy-poly(ethylene glycol) amine (mPEG-NH2). Strong plasmatic coupling effect is realized in our GHSP system when the GNPs are drawn near in the mussel-inspired cross-linking process and strong absorption in the NIR region is observed. Taking advantage of the light harvesting and photothermal contribution of PDA, the GHSPs exhibit outstanding PTC effect, even better than those of PGPs and pure PDA nanoparticles at an equivalent mass concentration. After in vivo administration, the GHSPs are able to ablate cancer with localized NIR laser exposure and show no longterm systemic toxicity, which make them of great promise as a PTC agent for cancer therapeutic applications. GNPs used in the present work are prepared with a citrate reduction method reported previously[26] and have an average diameter of 25 nm. For the PDA coating of GNPs, dopamine is self-polymerized on the surfaces of GNPs in a Tris-Cl buffer (10 × 10−3 M, pH 8.5).[27] (Tris is tris(hydroxymethyl)aminomethane, IUPAC name 2-amino-2-hydroxymethyl-propane1,3-diol.) Figure 1b presents the ultraviolet–visible (UV–vis) spectra of GNPs and PGPs, and both of them exhibit a characteristic extinction peak around 522 nm due to LSPR of 25 nm GNPs. The slight red-shift for the plasmon peak is due to the increase of refractive index around GNPs after PDA coating.[28] Besides, PGPs show obvious absorbance at long wavelength (600–1000 nm), which is mainly attributed to the light harvest contribution of the PDA coatings,[29] as pure PDA nanoparticles have obvious absorbance in this spectral region (the UV–vis spectrum of PDA nanoparticle dispersion is shown in Figure S1, Supporting Information). In the assembly process, the mixture of PEI and mPEG-NH2 is added to a dispersion of the obtained PGPs and GHSPs are generated after a mild shaking of 24 h. It is revealed that the assembly is highly pH dependent. At a pH value of 6.0, fine
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Figure 1. Mussel-inspired assembly of gold nanoparticles. a) Schematic illustration of the architecture of a GHSP. The diagram is not drawn to scale. b) UV–vis spectra of GNPs, PGPs, and GHSPs. c) TEM images of the assembly results: i–v) present the assemblies obtained from various pH environments, while (vi), (vii), and (viii) demonstrate the influence of mPEG-NH2/PEI ratios; the inset image in iii) shows a broken superparticle to reveal the hollow interior of GHSPs. d) Zeta potential of PGPs at different pH values. e) A digital photograph of the suspensions of GNPs, PGPs, and GHSPs.
GHSPs are formed, whose transmission electron microscope (TEM) images (Figure 1c and Figure S2a in the Supporting Information) clearly show that the superparticles with hollow interiors consist of a layer of densely packed PGPs. The superparticles have a narrow size distribution with the average size of 240 nm (measured by dynamic scattering light, DLS, Figure S3, Supporting Information). When the pH value is higher than 6.0, the PGPs aggregate into nondispersible clusters in the assembly process, while in a more acidic environment (pH 4.0 and 5.0), nonuniform assemblies with a loosely packed morphology are formed. To investigate the formation mechanism of the superparticles, the zeta potential of PGPs at different pH values is measured and the results are shown in Figure 1d. It is suggested that when the pH value of the system decreases, the PGPs tend to lose their negative charge on the surface, and gain some positive charge at an even lower pH value.[30] The zeta potential
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gets close to zero at pH 6.0, and the decreased hydrophilicity of PGPs results in the attractive interparticle interactions.[31] Moreover, the reaction between PEI and PDA (the chemistry of the reaction is schemed in Figure 1a) leads to the crosslinking of the PGPs,[32] which further increases the trend for agglomeration. Meanwhile, during the organization of PGPs into superparticles, mPEG-NH2 molecules are grafted to the surface of the PGPs with a mechanism similar to that of PEI, and the PEG chains stabilize the structures. In this case, the PEI cross-linking and PEGlyation provide the driving force for the gradual aggregation and rearrangement of PGPs into hollow assemblies. This reaction-induced assembly process is similar to the self-assembly of amphiphilic block copolymers into vesicular architecture, in which the hydrophobic block acts as cross-linking sites while the hydrophilic block stabilizes the structure in the aqueous media.[33] Nevertheless, the environmental pH value not only affects the hydrophilicity of the PGPs
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50.6 °C at a concentration of 50 µg mL−1, while the temperature of PBS increases by only 2.9 °C. It has been well demonstrated that hyperthermia is able to kill cancer cells directly over 45 °C (ablation) or at 39–42 °C (mild hyperthermia).[36] We can infer that after the injection of GHSPs, the tumor tissues can be easily heated to over 45 °C in the irradiation and the cancer can be ablated in a short period of time. Next, we compare the PTC capability of GHSPs with some other PTC agents. Upon laser illumination for 500 s, the PGPs, GNRs (the TEM image and UV–vis spectrum is displayed in Figure S4, Supporting Information) and PDA nanoparticles (at an equivalent concentration: 50 µg mL−1) raise the temperature by 11.5 °C, 20.1 °C, and 36.3 °C, respectively (Figure 2b). The PTC efficiency (η) is calculated as follows:[37]
η = (hSΔTmax − Q s ) / I(1 − 10 − A )
(1)
τ s = m sCs / hS
(2)
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but also has a significant influence on the rate of cross-linking and PEGlyation reactions. It is well developed that the aqueous chemical equilibrium between catechol and quinone shifts toward quinone under alkaline conditions, conferring latent reactivity toward amine groups.[34] Owing to the higher reactivity of the PDA coating at high pH values, the PGPs are crosslinked rapidly and agglomeration is observed. On the contrary, in an acidic environment, the amine groups of PEI and mPEGNH2 are protonated and the reactions are inhibited, leading to the incomplete assembly. According to the mechanism proposed above, it is reasonable to hypothesize that the assembly is also related to the amount of PEI and mPEG-NH2. Therefore, we change the mPEG-NH2/PEI ratio (molar ratio) in the assembly process. It can be observed from the TEM images (Figure 1c) that in the absence of mPEG-NH2 (mPEG-NH2/PEI = 0:10), the PGPs aggregate and precipitate, which is attributed to the decrease of dispersion stability in the PEI cross-linking process. On the other hand, if more mPEG-NH2 is added to the system (mPEGNH2/PEI = 5:5), the dispersibility of PGPs raises, and the PGPs assemble into small solid clusters, whose extinction peak is very different from the hollow superparticles (Figure S2c, Supporting Information). As a boundary condition, when the mPEG-NH2/PEI ratio reaches 10:0 (without the adding of PEI), the assembly cannot occur. These results prove our hypothesis and the assembly is able to be regulated by changing the molar ratio of mPEG-NH2/PEI. The assembly of PGPs leads to short interparticle distances and the plasmon peak is broadened owing to a strong plasmon resonance coupling effect between adjacent GNPs.[16,35] The assembly process is accompanied by a color change of the dispersion from purple to brown (Figure 1e), and the LSPR peaks of the obtained GHSPs are located at 810 nm (Figure 1b). The strong plasmatic absorption peak located in the NIR region motivates us to explore their potential use in the PTT of cancer. GHSPs are dispersed in phosphate-buffered saline (PBS) at concentrations ranging from 20 to 100 µg mL−1, and then irradiated with an 808 nm laser at 2 W cm−2 for 500 s. Pure PBS is used as a negative control. The temperatures of all samples increase during the laser exposure, and the temperature increased more rapidly as the concentration increases (Figure 2a). After 500 s, the temperature of GHSPs dispersion increases by
where h is the heat transfer coefficient, S is the surface area of the container, ΔTmax is the temperature change of GHSPs sample at the maximum steady state, I is the laser power, A is the absorbance of GHSPs, Qs is the heat associated with the light absorbance of the solvent, τs is the sample-system time constant and ms and Cs are the mass (0.1 g) and heat capacity (4.2 J g−1) of the solvent. According to Equations (1) and (2), the η value of the GHSPs is determined to be 38% (Figure S5, Supporting Information), remarkably higher than those of PGPs (15%) and GNRs (22%), only slightly lower than that of PDA nanoparticles (40%, Figure S6a, Supporting Information). Other than the PTC efficiency, how strong the nanomaterials can convert photon energy also depends on the strength of light–matter interactions.[21] To determine how effective the PTC agents absorb light, their UV–vis spectra (at an equivalent concentration: 50 µg mL−1) are summarized in Figure S6b (Supporting Information). As indicated, GHSPs exhibit a much higher absorbance value at 808 nm than the others, indicating the strong plasmatic coupling effect induced by the assembly is the main contributor to the outstanding photothermal effect of GHSPs. Before studying the photothermal effect of GHSPs in vivo, we first ought to demonstrate their in vitro photothermal behavior in cell culture experiments, and human breast adenocarcinoma cell line (MCF-7 cells) is used as model. The cells are
Figure 2. Photothermal effect of GHSPs. a) Temperature elevation of PBS and GHSPs suspension with different concentrations as a function of irradiation time. b) The photothermal response of the aqueous dispersion of GHSPs, the building blocks (PGPs), PDA nanoparticles, and GNRs at an equivalent concentration (50 µg mL−1) with an NIR laser (808 nm, 2 W cm−2). Irradiation is continued for 500 s, and then the laser is turned off. PBS is used as a negative control.
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Figure 3. In vitro cell assays. a) Confocal images of calcein-AM (green, live cells) and PI (red, dead cells) co-stained MCF-7 cells after laser irradiation. White bar indicates 1 mm. b) Cell viability of MCF-7 cells after incubation with increased concentrations of GHSPs.
incubated with GHSPs for 30 min and irradiated by the 808-nm laser (2 W cm−2) for 5 min. After that, calcein acetoxymethyl ester (calcein-AM)/propidium iodide (PI) co-staining is carried out to differentiate live (green) and dead (red) cells under confocal fluorescence imaging (Figure 3a). While cells within the laser spot are completely destroyed by hyperthermia, those far away from the irradiation region survive. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is conducted (Figure 3b) to quantitatively evaluate the photothermal effect. After incubation for 24 h with increasing concentrations of GHSPs, no significant cytotoxicity is observed and even up to a concentration of 1 mg mL−1, the cell viability still remains approximately 92%. However, upon laser irradiation, significant growth inhibition of MCF-7 cells is hindered and only 8.9% cells remain viable at a GHSP concentration of 200 µg mL−1,
suggesting that GHSPs can effectively kill cancer cells with the assistance of laser exposure. It should be noted that combining the influences of PTC efficiency (η) and absorbance in the NIR region, the resultant photothermal effect of GHSPs is much better than those of other PTC agents (Figure 2b), and they are more effective in killing cancer cells in the in vitro photothermal experiment (Figure S7, Supporting Information). Being aware of the photothermal effect of GHSPs, we finally conduct in vivo PTT. Animal care and handing procedures were in agreement with the guidelines evaluated and approved by the ethics committee of Fudan University. Mice bearing MCF-7 xenografts are intratumorally injected with GHSPs (dosage 1.0 mg kg−1) and then exposed to an 808 nm laser at 2 W cm−2 for 5 min. Thermal imaging with an infrared thermal camera is used to monitor the efficacy of the treatment (Figure 4a).
Figure 4. In vivo PTT. a) Thermal images of MCF-7 tumor-bearing mice exposed to an 808 nm laser for 5 min after the injection of PBS or GHSPs. b) Heat curves of tumors upon laser irradiation as a function of irradiation time. Inset: H&E staining of tumor sections collected from PBS- (right) and GHSP-treated (left) mice after laser irradiation. c) In vivo effects of the PTT on the growth of MCF-7 tumor. ***, P < 0.001 vs PBS, PBS+laser and GHSPs. d) Morbidity-free survival rate of mice bearing MCF-7 tumors after treatment.
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Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This work was financially supported by National Science Foundation of China (Grant Nos. 51273047 and 51473037) and the “Shu Guang”
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As shown in Figure 4b, the local tumor temperature rapidly increases to 60 °C within 5 min and no significant temperature increase is observed in other body parts of the mice. Hematoxylin and eosin (H&E) staining of tumor slices is conducted for the tumor collected immediately after laser illumination and significant cancer-cell damage in the tumor is found (Figure 4b, Inset). To determine the antitumor effect, both tumor sizes (Figure 4c) and morbidity-free survival rate (Figure 4d) are recorded after treatment. The results show that the tumors treated with PBS, GHSPs only, and PBS plus laser irradiation grow rapidly, suggesting the tumor growth is not affected by the laser irradiation or GHSPs alone. A single injection of GHSPs plus laser illumination leads to a complete remission of MCF-7 tumors and no tumor recurrence is observed over a course of 30 days. Free of tumors, all the mice in this group survive without a single death, confirming the success of the PTT. After the treatment, the GHSPs leak into the circulation and taken up by the reticuloendothelial system (RES) subsequently, including the liver and the spleen.[38] This clearance route is proved by the biodistribution of GHSPs in the major viscera (heart, liver, spleen, lung, and kidneys) and tumor tissues, which is quantified by the Au element in the tissues (measured by inductively coupled plasma mass spectrometry, ICP-MS, Figure S8, Supporting Information). The blood chemistry and hematology analysis reveal that no obvious hepatic toxicity is induced by the therapy and all of the hematological parameters in the treated groups appear to be normal compared with the control group (Figure S9 and Table S1, Supporting Information). The body weight of mice, whose change is an indicator of systemic toxicity,[39] is simultaneously measured after receiving the PTT. The body weight of mice in GHSPs- and GHSPs-pluslaser-treated groups do not differ greatly from that of PBS, indicating that the superparticles do not exhibit severe systemic toxicity (Figure S10, Supporting Information). It is suggested that GHSPs, which possess efficient photothermal activity and low systemic toxicity, can act as a hopeful PTC agent for the PTT of cancer. In conclusion, we have developed an approach to assemble PGPs into hollow superparticles via the dopamine chemistry. The obtained GHSPs show a strong LSPR peak in the NIR region and high PTC efficiency, and can kill cancer cells with the assistance of laser irradiation. The GHSPs show good biocompatibility in vivo and can effectively ablate tumors in the PTT. The high performance of the superparticles offers a new generation of PTC agent and the mussel-inspired assembly approach could be extremely useful for the transfer of nanomaterial science to realistic technologies.
project (12SG07) supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation. Received: December 11, 2014 Revised: January 23, 2015 Published online:
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