Polymeric-gold nanohybrids for combined imaging and cancer therapy.

www.advhealthmat.de www.MaterialsViews.com

FULL PAPER

Polymeric-Gold Nanohybrids for Combined Imaging and Cancer Therapy Antonio Topete, Manuel Alatorre-Meda, Eva M. Villar-Alvarez, Susana Carregal-Romero, Silvia Barbosa, Wolfgang J. Parak,* Pablo Taboada,* and Víctor Mosquera In memoriam Dr. Antonio Topete Palomera

Here, the use of folic acid (FA)-functionalized, doxorubicin (DOXO)/superparamagnetic iron oxide nanoparticles (SPION)-loaded poly(lactic-co-glycolic acid) (PLGA)-Au porous shell nanoparticles (NPs) as potential nanoplatforms is reported for targeted multimodal chemo- and photothermal therapy combined with optical and magnetic resonance imaging in cancer. These polymeric-gold nanohybrids (PGNH) are produced by a seeded-growth method using chitosan as an electrostatic “glue” to attach Au seeds to DOXO/SPION-PLGA NPs. In order to determine their potential as theranostic nanoplatforms, their physicochemical properties, cellular uptake, and photothermal and chemotherapeutic efficiencies are tested in vitro using a human cervical cancer (HeLa) cell line. The present NPs show a near-infrared (NIR)-light-triggered release of cargo molecules under illumination and a great capacity to induce localized cell death in a well-focused region. The functionalization of the PGNH NPs with the targeting ligand FA improves their internalization efficiency and specificity. Furthermore, the possibility to guide the PGNH NPs to cancer cells by an external magnetic field is also proven in vitro, which additionally increases the cellular uptake and therapeutic efficiency.

Dr. A. Topete, Dr. M. Alatorre-Meda,[+] E. M. Villar-Alvarez, Dr. S. Barbosa, Prof. P. Taboada, Prof. V. Mosquera Grupo de Física de Coloides y Polímeros Universidad de Santiago de Compostela 15782 Santiago de Compostela, Spain E-mail: [email protected] Dr. S. Carregal-Romero,[++] Prof. W. J. Parak,[+++] Fachbereich Physik Philipps Universität Marburg, Renthof 7 35037 Marburg, Germany E-mail: [email protected] [+] Present address: 3B’s Research Group-Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark 48-909 Taipas, Guimarães, Portugal. ICVS/3B’s-PT Government Associate Laboratory, Braga, Guimarães, Portugal [++] Present address: Fundación Progreso y Salud, Junta Andalucía, Sevilla, Spain [+++]Present address: CIC Biomagune, San Sebastian, Spain

DOI: 10.1002/adhm.201400023

Adv. Healthcare Mater. 2014, DOI: 10.1002/adhm.201400023

1. Introduction

The outstanding physicochemical properties of nanomaterials and their clever combinations are allowing the development of multifunctional nanoplatforms for multimodal imaging,[1] simultaneous real-time diagnosis and therapy (theranostics),[1d,2] and multimodal therapy,[3] which holds promise to be in the near future an alternative to current, conventional diagnosis, and therapeutics for cancer, and are potentially offering new approaches for earlier detection and/or minimally invasive treatments.[4] One advantage of these new approaches is the easy incorporation of multidiagnostic and/or multitherapeutic functions into the same nanoscale complex, with the potential to simultaneously combine these features in clinical applications. This kind of biomedical nanosystems opens new windows to potentially overcome some of the different substantial impediments still unresolved in cancer therapeutics such as, for example, the substantial toxicity to normal tissues of drug doses required to completely eliminate tumors, the poor effectiveness of drug treatments against multidrug resistant cancer cells, the impossibility of early detection of very small tumors or blood circulating malignant cells, or the temporal separation of diagnostic and therapeutic clinical phases.[1c,1d,2a,2b] Hence, the simultaneous combination of several different therapies in a single NP to enhance malignant cell cytotoxicity with targeted drug delivery and diagnostic imaging capabilities appears as a very attractive choice to solve, at least partially, these issues. In this regard, a treatment that has shown promising results is thermo-chemotherapy.[5] Thermo-chemotherapy is the simultaneous application of a chemotherapeutic agent and hyperthermia (the therapeutic procedure used to raise the temperature of a body area affected by malignancy above 42–43 °C).[6] Despite the demonstrated enhanced cytotoxicity of some chemotherapeutic agents due to macroscopic temperature increases at low drug doses,[5c,7] temperature increments can affect cell structure and/or function, such as membranes, cytoskeleton or the synthesis of macromolecules, and DNA

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

wileyonlinelibrary.com

1

www.advhealthmat.de

FULL PAPER

www.MaterialsViews.com

repair; therefore, refined temperature control (both spatially and temporally) is required[6] in order to avoid undesirable side effects often caused due to non-targeted heating by conventional means (hot water baths, microwave, or ultrasound).[8] Recently, near-infrared (NIR) resonant nanomaterials such as gold (Au) nanoshells,[2c,2d,9] Au nanocages,[10] Au nanorods,[11] and single-walled carbon nanotubes[12] have shown a great ability to absorb NIR light and convert it into cytotoxic local heat able to be used for tumor-selective treatments. Hence, if heat and drugs could be simultaneously delivered by NPs at the tumor area, the therapeutic efficacy would be expected to be significantly improved with reduced side effects.[11d] To additionally minimize administrated doses and maximize therapeutic efficacy, molecules such as antibodies, peptides, or small ligands can be also attached to the surface of the former nanomaterials to provide improved targeted delivery through the recognition of overexpressed specific receptors on the surface of malignant cells[13]; nevertheless, different in vivo studies have pointed to such surface modifications only moderately enhance tumor/cell specificity and passive tumor targeting dominates,[14] so much more in-depth studies are still required in order to perfectly clarify the cell–NP interactions. Finally, to provide nanomaterials with diagnostic capabilities, the use of loaded/attached superparamagnetic iron oxide NPs (SPIONs) into composite NPs would allow their use as imaging T2 contrast agents in magnetic resonance imaging (MRI) to obtain highly resolved 3D images of tissues in living organisms.[15] Through SPIONs manipulation by an external magnetic field magnetically targeted drug delivery nanosystems for regulating drug release would be also expected.[16] Here, we report the use of folic acid (FA)-functionalized, doxorubicin (DOXO, a potent anticancer drug)/SPION-loaded poly(lactic-co-glycolic acid) (PLGA)-Au porous shell NPs for simultaneous targeted specific delivery of localized cytotoxic heat and chemotherapy in combination with optical and MRI diagnosis in human cervical cancer (HeLa) cells. Previously, Halas and co-workers[9b,17] synthesized Au nanoshells with a silica core, in which SPIONs and the fluorescent dye indocyanine green (ICG) were encapsulated in a silica layer surrounding the metal shell to be used as MRI and fluorescence imaging contrast agents. The therapeutic response of this nanohybrid was achieved through the NIR photothermal properties of the Au shell, and targeted delivery of the platform was pursued through the surface attachment of a human epidermal growth factor receptor 2 (HER2) antibody to target HER2 overexpressing cancer cells. A related system was developed by Hyeon and co-workers[18] but, in this case, SPIONs were directly embedded in the Au shell. This group also co-loaded SPIONs (or quantum dots, QDs) with DOXO inside PLGA NPs functionalized with FA ligands for simultaneous MRI (or fluorescence imaging) and targeted controlled delivery.[19] Dai and co-workers[20] obtained Au nanoshell micelles composed of a cholesteryl succinyl silane core loaded with DOXO and SPIONs to achieve the simultaneous combination of MRI, magnetic-targeted drug delivery, light-triggered drug release, and photothermal therapy. Yoo and co-workers[21] and Haam and co-workers[22] developed bare and antibody-functionalized DOXO-loaded PLGA-Au half and full nanoshells, respectively, to analyze the targeted and non-targeted simultaneous

2


cytotoxic combination provided by NIR-induced hyperthermia and released chemodrug on skin (A431), breast (SK-BR-3 and MCF7), and cervical (HeLa) cancer cells, and also for rheumatoid arthritis treatment. In this work, we present a polymeric-gold nanohybrid (PGNH) in which DOXO molecules and SPIONs are co-loaded to different extents inside the core of biocompatible and biodegradable PLGA NPs. Moreover, a complete porous gold layer is grown onto the surface of the polymeric NPs to provide NIRlight responsiveness to the PGNH NPs. FA attached to the surface of the Au layer allows PGNH NPs to be specifically delivered in vitro to FA receptors overexpressed on the membrane surface of a HeLa cancer cell line to promote their internalization by receptor-mediated endocytosis, increasing NP cell accumulation.[23] This accumulation is further enhanced when an external magnetic field is applied to the cultured cells by means of a magnetic-guiding effect.[16] Simultaneously, the presence of SPIONs increases the magnetic contrast of cancerous cells by MRI. Since these NPs are NIR-resonant, the simultaneous effect of targeted NIR-induced hyperthermia and chemotherapy results in larger cell toxicities at lower drug concentrations. In comparison with the pairwise combinations, a synergistic effect is observed. Thus, the importance of our report lies in the simultaneous successful combination of chemotherapy with hyperthermia, magnetic and ligand assisted-targeted delivery and high-resolution imaging fulfilled in a single hybrid nanoconstruct.

2. Results and Discussion 2.1. Preparation and Characterization of the Multifunctional Hybrid NPs Core–shell NPs composed of iron oxide coated with a layer of a different inorganic material such as gold are very interesting since the resulting nanostructures can combine the magnetic and optical properties of the constituent elements while reducing the toxicity of the iron oxide, which supports their application in theranostics.[24] To increase the effectiveness of these PGNH NPs, one must carefully control their physicochemical properties including size, shape, morphology, charge, and surface.[25] Here, different kinds of core–shell multifunctional polymeric-gold nanohybrids were prepared for comparison purposes: PLGA-Au NPs, PLGA-Au-FA NPs, DOXO/SPION-PLGAAu NPs, and DOXO/SPION-PLGA-Au-FA NPs, named in the following BNK-PGNH, BNK-PGNH-FA, DXSP-PGNH, and DXSP-PGNH-FA, respectively. The configuration of each nanohybrid is detailed in Figure S1 (Supporting Information). The synthesis of all these kinds of PGNH NPs was performed through a multistep procedure. PLGA NPs with (or without) the hydrophobic cargos (DOXO and SPIONs) were prepared by an oil-in-water (o/w) emulsion and subsequent solvent evaporation method, as previously described.[21d,26] In some experiments (NIR-triggered cargo release ones), the dye Nile Red (NR) was also used as a model hydrophobic drug (particles named as NR-PGNH, Figure S1e, Supporting Information). When using the anticancer drug DOXO, DOXO·HCl was converted to its




FULL PAPER Figure 1. Size distribution of DOXO/SPION-PLGA NPs as obtained by a) DLS (i.e., hydrodynamic diameter in solution) and b) TEM (i.e., the core diameter of the PLGA NPs in dried state). c) TEM and d) FEI-SEM images of PLGA NPs (i.e., the core diameter of the PLGA NPs in dried state). e,f) TEM images of DOXO/SPION-PLGA NPs. In e), one can observe embedded SPIONs in one individual PLGA NP as homogeneously distributed dark spots. DOXO/SPION-PLGA NPs were slightly heterogeneous, which could account for the broad UV–vis absorption peaks of PGNH NPs.

hydrophobic base form by addition of triethylamine[27] to favor its entrapment inside the hydrophobic polymeric matrix. With the same objective, oleic acid-stabilized SPIONs were synthesized by a coprecipitation method,[28] with an average inorganic core diameter of 9.8 ± 0.9 nm as measured by TEM (Figure S2a, Supporting Information). It is worth mentioning that the size of the resulting PGNH NPs is largely determined by the size of the primary PLGA NPs, and the type of embedded cargo only slightly changes the polymeric particle size. In this way, the molecular weight of the PLGA polymer as well as other external factors in the NP preparation procedure (polymer and stabilizer concentration, temperature, cargos concentration, ultrasonic power, and duration, etc.) can be tuned to reach the desired final PGNH NP size.[29]


A mixed dispersion of SPIONs, DOXO (or NR when corresponding), and PLGA in methylene chloride, as detailed in the Experimental Section, was poured into an aqueous solution of block copolymer F127, EO97PO69EO97 (where EO and PO are an oxyethlyene and an oxypropylene unit, respectively, and the subscripts denote the block lengths), used as an emulsifier and stabilizer, and the resulting solution underwent strong sonication. After solvent evaporation, DOXO/SPION-PLGA NPs displayed a mean hydrodynamic diameter of ≈122 ± 11 nm as measured by dynamic light scattering (DLS) (Figure 1a), which was consistent with TEM and field emission scanning electron microscopy (FE-SEM) images (Figure 1b–f). Differences in the obtained sizes between these techniques arise from the dehydration underwent by the samples during the preparation



3


FULL PAPER

PEG chains (Figure S5, Supporting Information) to bind the procedure for TEM and SEM measurements.[25] TEM images FA-overexpressed receptors on cell membrane surfaces. The of DOXO/SPION-PLGA NPs indicated that many SPIONs were number of SH-PEG5000-FA molecules conjugated to PGNH successfully embedded into the matrix of each PLGA NP. The successful incorporation of DOXO inside the PLGA NPs was NPs was calculated to be 1.78 × 106 molecules per PGNH. corroborated by the appearance of absorption and emission UV–vis spectra of DXSP-PGNH NPs at different stages of peaks at 490 and 591 nm, respectively, consistent with spectra preparation are shown in Figure 3a. The extinction spectrum of free DOXO (Figure S3, Supporting Information). of the blank PLGA NPs exhibited no obvious peak in the range from 400 to 800 nm (Figure 3a, plot 1) as a result of light scatThe number density of SPIONs and DOXO could be easily tering of the NPs.[31] The peak at ≈480 nm (Figure 3a, plot 2) tuned by varying the amount of SPIONs in the initial reaction mixture and the DOXO/PLGA molar ratio. In particular, when was attributed to the absorbance of DOXO corroborating its the mass ratio of DOXO to PLGA was 10 wt%, the maximum successful loading into the PLGA NPs. After Au-seed adsorpdrug encapsulation efficiency (EE) was ≈95%, and the loading tion, a plasmon resonance peak at ≈535 nm for Au NPs cluscapacity (LC) was 8.9 wt% in the presence of 5 wt% SPIONs (as tered at the surface of the PLGA NPs was present (Figure 3a, measured by inductively coupled plasma mass spectrometry, ICPplot 3), and the metal seeds were observed as densely distributed MS) compared with 73% EE and 3.2 wt% LC in their absence. dark spots by TEM (Figure 2a). The hydrodynamic diameter of This suggests a possible synergistic effect through additional PLGA-Au-seed NPs was evaluated to be ≈170 ± 15 nm by DLS hydrophobic interactions between DOXO molecules, oleic acid measurements. A new pronounced peak in the NIR range was (OA) surfactant on the SPIONs surfaces and PLGA chains.[20] observed in the UV–vis absorption spectra after reduction of an Au growth solution onto the surface of the attached Au-seeds To grow the porous Au layer, low-molecular-weight chitosan by ascorbic acid to give an Au shell layer (Figure 3a, plot 4). chains were first adsorbed onto the surface of the negatively The absorption maximum of this new peak ranged between charged PLGA NPs derived from the terminal carboxyl groups 750 and 900 nm depending on the thickness of the Au shell, of the PLGA chains (ζ-potential of ≈ −35.3 ± 4.8 mV)[30] by elecwhich was controlled through both ascorbic acid concentration trostatic interactions. The ζ-potential of the PLGA NPs was and volume of the added Au growth solution (Figure S6, Supincreased from −35.3 ± 3.2 to +60.4 ± 3.9 mV by increasing the porting Information). Furthermore, the plasmon peak was not amounts of the coated chitosan (Figure S4, Supporting Infordistorted nor broadened during the formation of the Au layer. mation), confirming the successful coating of chitosan on the surface of PLGA NPs. The hydrodynamic diameter changed The structure of the metallic nanoshell could be also changed from ≈140 ± 14 to ≈180 ± 17 nm after biopolymer coating, also from a relatively smooth surface to a spiky structure depending denoting the absence of any severe agglomeration due to the on the synthetic conditions, the latter resembling the classical electrostatic repulsions between the positively charged NPs structure of a virus capside (Figure 3c, inset). The virus capsideand the steric stabilization provided by the polyethylene oxide like structure of our PGNH NPs can be attributed to the use of chains surrounding the PLGA NPs. chitosan for the attachment of gold seeds. Chitosan has a double Subsequently, negatively charged Au seeds with a diameter of about 2–3 nm were adsorbed onto the surface of the chitosancoated PLGA NPs via electrostatic interactions. A ζ-potential of ≈ −11.4 ± 2.1 mV indicated the successful deposition of Au seeds onto the surface of polymeric NPs (Figure 2a). Finally, the attached Au nanoseeds were used to nucleate the growth of a porous gold nanoshell by reduction of HAuCl4 to bulk Au metal (Figure 2b–c). For PGNH NP stabilization, a heterobifunctional polyethyleneglycol chain, SH-PEG5000-OCH3 or SH-PEG5000-NH2 when corresponding, was used by exploiting the strong bond formation between sulfhydryls groups and metal gold surfaces. PGNH NPs settled due to the high density of the Au shell and the SPION-loaded PLGA core; nevertheless, after vortexing no agglomerates in solution were observed by visual inspection and DLS for at least 2 weeks after pegylation when stored in phosphate buffer saline (PBS) at 4 °C. To achieve targeted delivery of the nanoconstruct and enhance cellular accumulation inside cancerous cervical HeLa cells, Figure 2. a) TEM image of Au nanoseeds decorating DOXO/SPION-PLGA NPs; b) TEM and FA was conjugated to the amino groups of c) SEM images of DXSP-PGNH NPs.

4





FULL PAPER Figure 3. a) UV–vis absorption spectra of nanoplatforms at different stages of their growth process. The PLGA NP concentration was ≈4 × 1010 NP mL−1, while the final PGNH NP concentration was ≈1 × 109 NP mL−1. b) TEM images of PGNH NPs with a flat and c) a spiky surface mimicking a virus capside. d) SEM image of PGNH NPs showing their rough surface topology. e) High-magnification TEM images showing the multicrystalline and porous structure of the Au shell.

role in the growing mechanism: first, this biopolymer enables the attachment of negatively charged gold seeds to the polymeric particle core and contributes as a weak reducing agent in the redox reaction of HAuCl4 thanks to the high affinity of amine groups to Au; second, the scrolling and protrusion capacity of vicinal chitosan chains can triggered for additional agglomeration and growth coalescence of primary Au seeds, which can act as secondary nuclei to give larger crystals that configure the nanoshell spikes; that is, the chitosan chains can also act as a structure-directing agent supporting the growth of anisotropic gold shells.[32] The Au nanoshell also seemed to be dense, multicrystalline, and porous (Figure 3d–e), and it conveniently enables DOXO release through the gold pores (see below). Electron diffraction X-ray (EDX) analysis (Figure S7, Supporting Information) of non-functionalized bare PGNH NPs displayed Au and C peaks, the latter stemming from carbon atoms from the PLGA core, which corroborates the porous nature of the gold layer. In addition, SEM maps for each element distribution coincide, which provides an additional confirmation. The final hydrodynamic diameter for DXSP-PGNH-FA NPs was evaluated to be between ≈170 and 206 nm depending on the thickness of the Au shell by DLS measurements (Figure S8, Supporting Information), which is consistent with TEM images.

light-triggered release of drugs and photothermal therapy[33] at relatively low laser output energies due to the enhanced light absorption provided by their relatively large extinction crosssections.[34] Exposure of aqueous suspensions of PGNH NPs to NIR-continuous wave illumination (NIR-CW setup at 808 nm) resulted in a rapid elevation of solution temperatures, which is an important feature for selective treatment of solid tumors. To investigate the hyperthermic potential of PGNH NPs, first, the heat generated by NIR-CW illumination at a laser power of 2.5 W cm−2 over time (total illumination time = 10 min; total energy delivered = 188.6 J) on PGNH NP solutions of different concentrations was measured. No obvious temperature change was observed when deionized water was exposed to NIR light. Conversely, aqueous dispersions of PGNH NPs at concentrations of 1 × 108–1 × 1010 NPs mL−1 achieved temperature elevations of ≈6, 8, 11, 13, and 16 °C, respectively (Figure 4a). Hence, at concentrations above 5 × 108 NPs mL−1 samples can be easily heated up above 43 °C, which is sufficient to trigger apoptosis in tumor cells.[35] When changing the laser irradiation at a fixed NP concentration of 5 × 108 NPs mL−1, powers above 2.5 W cm−2 overcome the required temperature threshold for thermal ablation of cancer cells (Figure 4b). with temperature elevations of up to 40 °C, which can cause cargo release and cellular death (see below).

2.2. Temperature Elevation Induced by NIR Laser Irradiation

2.3. NIR Laser-Triggered Release of Cargo Inside Cells

The absorbance between 750 and 900 nm (Figure S6, Supporting Information) suggests that these hybrid NPs are suitable for NIR

In order to investigate whether NIR light excitation can affect and help to control cargo release from PGNH NPs, NR-PGNH




5

www.advhealthmat.de

FULL PAPER


Figure 4. Temperature increases under NIR illumination of PGNH NPs at: a) 1 × 108, 5 × 108, 1 × 109, 5 × 109, and 1 × 1010 NPs mL−1 at a fixed laser power of 2.5 W cm−2 (808 nm); b) at a fixed concentration of 5 × 108 NPs mL−1 and laser powers of 4, 6, 8, and 10 W cm−2 (808 nm). The solution temperature was set at 37 °C.

NPs where subjected to NIR-short pulsed illumination (NIRSPI setup: laser wavelength = 830 nm; spot area = 6 µm2; illumination time = 1 s). In principle, pulsed laser irradiation should allow a concentration of power density in small pulses, thus reducing the time exposure, which is advantageous for clinical practice. For this setup, the laser was coupled to a wide-field optical microscope and the beam focused through the microscope’s objective. As observed in Figure 5, large light-induced-temperature increases involved NR release from NR-PGNH NPs inside the cells after 24 h of NIR-SPI illumination, as seen by the onset of NR fluorescence; also, some cells are observed to undergo membrane blebbling, a generally accepted sign of cell death.[36] At this respect, when cells were illuminated with NIR-SPI with a power intensity of 0.3 mW µm−2 for 1 s (total energy delivered = 1.8 mJ), no damage or disruption of cells was observed but a diffusion of the NR dye. After NP internalization, NIRlight irradiation led to an enhanced cargo diffusion inside the cells as observed from the increased NR fluorescence intensity (Figure 5b,d), while it remained nearly unchanged in the absence of NIR irradiation (Figure 5a,c). All this suggests that cargo molecules were quickly released upon illumination, diffused into the cell cytosol (as concluded by the diffuse instead of granular fluorescence NR pattern),[37] and eventually might enter the cell nuclei, as in the case of DOXO known to interact with topoisomerase II to cause DNA cleavage and cytotoxicity.[38] When increasing the power light intensity to 0.6 mW µm−2 for 1 s (total energy delivered = 3.6 mJ), NR release inside the cells was greatly enhanced. However, irradiated cells are observed to change their morphology probably due to the localized temperature increase caused by NIR energy NPs absorption upon irradiation. Note that the energies reported here for local cargo

6


release with NIR-SPI are much lower than those required for heating of NPs bulk solutions with NIR-CW excitation. This might originated from the fact that internalized NPs cluster in endosomal/lysosomal compartments and, thus, the heating efficiency is amplified by plasmonic coupling[39] leading to a collective effect; by contrast, in the case of bulk illumination NPs are singly dispersed and, thus, heated individually. On the other hand, the morphology of PGNH NPs upon NIR-SPI excitation was studied by TEM. To do this, DXSPPGNH NPs were electrostatically attached to positively charged 5 µm SiO2 beads. TEM images of DXSP-PGNH/SiO2 assemblies were acquired before and after illumination with NIR light under NIR-SPI illumination conditions. Figure 6 shows images of irradiated NPs under different intensities. The structure of PGNH NPs was observed to be maintained up to power intensities of ≈0.3 mW µm−2 (Figure 6b). Above this threshold, fusion of the Au layer with subsequent loss of spikes (when present) and NP rupture was observed, for example, when illuminated at 0.6 mW µm−2 (Figure 6d–f), which is consistent with observations of single-cell in vitro NR release experiments. These data corroborated that the photostability of the present NPs was not well maintained under pulsed laser irradiation. Pulsed lasers can melt and reshape gold nanostructures to nanospheres even in picoseconds.[40] This irreversible shape transformation will dramatically deteriorate the heat generation ability.[40a] Hence, in view of the present observations, we decided to use continuous wave (CW) illumination to perform the analysis of the drug light-triggered release in vitro. Also, CW irradiation was used to carry out the in vitro cytotoxicity tests of DXSP-PGNH NPs, in this case, with the caution of keeping the total energy light input per cell similar to that used in the present pulsed laser-cargo release irradiation experiments (4.2 mJ per cell).




FULL PAPER

Figure 7 shows the characteristics of the DOXO release from bare DOXO/SPION PLGA (DXSP) NP and DXSP-PGNH NP solutions [in PBS buffer pH 7.4 with 10% (v/v) FBS] in the absence and presence of NIR-CW irradiation. Samples were irradiated repeatedly over a period of 5 min, followed by 1 h intervals with the laser turned off. We observed that the release profile of DXSPPGNH NPs follows a similar trend as that of DOXO/SPION PLGA NPs, that is, an initial burst release profile followed by a slower diffusion release phase. In the absence of irradiation, the release rate of DXSP-PGNH NPs was much slower than that of bare PLGA NPs. This behavior was ascribed to the presence of the Au layer but the drug concentrations released were still largely detectable, which also supported the assumption of the porous nature of DXSP-PGNH NPs. Conversely, a faster release was observed upon NIR irradiation of the DXSP-PGNH NP solution.[42] The DOXO released was 29.7% over the whole period in the absence of NIR laser irradiation. After the first 5 minNIR exposure, the percentage of released DOXO already steeply increased from 7.1% to 18.4%; after exposition to NIR light five times for 5 min, the accumulated released amount of DOXO over the whole process was about threefold greater than that in the absence of irradiation, ≈79.5%. This enhancement can be a consequence of both a faster diffusion of the cargo throughout the porous inorganic layer upon temperatures above the polymer Tg and a possible partial disruption of the DXSP-PGNH NP strucFigure 5. Confocal laser scanning microscopy images of HeLa cells containing Nile Red-loaded ture (Figure 7b). Moreover, it was noted that PGNH NPs. Rows (a) and (c) represent cells before illumination, and (b) and (d) cells 24 h after DOXO release was particularly enhanced NIR-SPI excitation at 0.3 mW μm−2 (b) and 0.6 mW μm−2 (d), respectively. The arrows indicate illuminated cells. Images in column (1) are transmitted light (differential interference con- immediately upon irradiation. However, the trast, DIC channel), column (2) shows merged images of DIC and fluorescence channels, and released amount of DOXO after each irradiacolumn (3) displays emission images of NR (λex= 510 nm, λem= 585 nm). Scale bars = 20 μm. tion cycle was progressively decreased due to the increased coverage of gold pores resulting from the melting effect of the metallic nanostructures[43] and the subsequent structural changes of 2.4. Triggered Release of DOXO by NIR Light some NPs. The PLGA copolymer that configures the reservoir for DOXO drug molecules showed a glass-transition temperature (Tg) of ≈45 °C,[41] which reduces the drug leakage during circula2.5. Magnetic Properties and In Vitro Magnetic-Resonance tion and releases the drug when needed, that is, in the vicinity Imaging of tumor. However, the incorporation of the Au layer allows an additional light-triggered drug release mechanism by applying Due to the presence of SPIONs in their composition, the preNIR laser irradiation, that is, the heat generated by DXSP-PGNH sent DXSP-PGNH NPs also possess robust magnetic propNPs under illumination is sufficiently high to overcome the erties, as observed when placing a magnet near the hybrid PLGA’s Tg inducing an enhanced release of the chemotheraNP solution (Figure S9, Supporting Information). The fielddependent magnetization curve of DXSP-PGNH NPs at 5 K peutic drug, as depicted by cell irradiation experiments (see showed hysteresis (Figure 8a), which is consistent with the Figure 5).




7


FULL PAPER

magnetic-Au-coated silica nanoshells.[9b] The Msat of the NPs was observed to be lower than that of free SPIONs (Figure S9c, Supporting Information) possibly due to the large diamagnetic mass of Au and PLGA contributing to the total mass of the NPs. Additionally, the presence of canted or noncollinear surface spins, which has been observed in ferromagnetic materials with substituted diamagnetic neighboring cations, may also contribute to a decrease in Msat since saturation of these spins usually requires higher magnetic fields.[9b] SPIONs have received great attention as T2 contrast agents due to their capability to shorten the spin–spin relaxation times, which results in a decrease in the MRI signal intensity and enables to differentiate between normal and abnormal tissues.[44] In addition, the specific weighted transverse-relaxivity (r2) values can be greatly increased by clustering SPIONs in reservoirs, which lead to higher saturation magnetizations than that of individual ones owing to the interactions between the assembled nanocrystals.[45] In order to evaluate the T2 contrast capabilities of the present hybrid NPs, DXSP-PGNH NPs with various Fe concentrations (as determined by ICP-MS) were investigated by T2weighted MRI at 9.4 T and 400 MHz. The signal intensity of the MR images decreased as the iron concentration increased, as expected for T2 contrast agents due to the shortening of the spin–spin relaxation time of water, as commented previously.[46] The specific relaxivity, r2, a measure of the change in spin–spin relaxation rate (T2−1) per unit Fe concentration, was determined to be 207 (× 10−3 M Fe)− 1 s− 1 for our DXSP-PGNH (Figure 8c). This high r2 value could be due to the large external magnetic field (9.4 T) applied to the NPs as well as their superior magnetic properties due to the enhanced magnetic interactions between clustered Figure 6. TEM images of DXSP-PGNH NPs supported on silica spheres of 5 μm size: a,c) before, and b,d) after NIR-SPI with a laser power of 0.3 and 0.6 mW μm−2, respectively. In SPIONs inside the polymeric matrix of the b), the spikes are smoothed but their shape is kept. d) At larger laser powers, a re-shaping from NPs. It is worth mentioning that at high a spiky morphology to a smoother sphere takes place. Also, e) detachment of some portions of magnetic field strengths, the two contrast the DXSP-PGNH NP surface and f) complete NP rupture was observed. agents approved by the US Food and Drug Administration (FDA), AMI-25 and Resovist, have considerably lower r2 values (160 and 151 (× 10−3M Fe)− 1s− 1, respectively) than the NPs synthesized thermal energy being insufficient to induce magnetic moment randomization. Thus, the NPs showed typical ferromagnetic here. Meanwhile, other types of hybrid NPs embedding clushysteresis loops with a remanence of 4.7 emu g−1 and a coertered SPIONs such as magnetic Au nanoshells, polymeric NPs, or hybrid fibers have been reported with relaxivities compacivity of 205 ± 6 Oe (Figure 8a). Conversely, the field-dependent rable to our DXSP-PGNH NPs.[9b,19,47] Hence, these data sugmagnetization curve of DXSP-PGNH NPs at 300 K shows no hysteresis (Figure 8b), which is consistent with superparagest that the present DXSP-PGNH NPs may be used in magmagnetic behavior arising from the embedded SPIONs. The netic imaging diagnosis, in which the distribution of the NPs saturation magnetization at 300 K, Msat, was determined to be depicted by MRI may be used to assist planning of chemo- and photothermal therapy and to predict the treatment outcome. 14.6 emu g−1, similar to that observed previously for

8





FULL PAPER

lysosomal structures. Upon illumination, light scattering from DXSP-PGNH NPs could be captured and used to determine the presence of NPs in vitro. Comparison of confocal microscopy images of HeLa cells incubated with non-functionalized DXSP-PGNH NPs and functionalized DXSP-PGNH-FA NPs showed a larger cellular uptake for the latter as denoted by the increased scattered light inside the cells[48] (denoted in green in Figure 9b–h by changing the color palette in the merged images shown). Figure 7. a) TEM image of DXSP-PGNH NPs after 10 min of NIR-CW, no substantial changes After 4 h of incubation with HeLa cells, in the structure of NPs were observed. The scale bar represents 200 nm. b) Release profile of only a relative small amount of non-funcDOXO from ( ) bare DOXO/SPION PLGA NPs and DXSP-PGNH NPs (•) in the absence ( ) tionalized NPs appeared to be inside the cells and presence of 2.5 W cm−2 NIR-CW irradiation at 808 nm. (Figure 9b–c); in contrast, FA-functionalized NPs were more numerous (a larger number of green spots inside cells, Figure 9d–e) and appeared to be spe2.6. Cellular Uptake and Intracellular Distribution cifically bound to the (outer) cell membrane and within intracellular vesicular compartments (Figure 10). This demonstrated Because targeted delivery to specific cells is essential to systhat the functionalized DXSP-PGNH-FA NPs are more easily temic cancer therapy, we exploited the overexpression of FA internalized by cells than the non-functionalized ones, presumreceptors on the cellular membrane of HeLa cells by attaching ably through a receptor-mediated endocytosis process.[49] On this ligand to the surface of DXSP-PGNH NPs. We investigated the cellular uptake of our hybrid NPs in vitro, their intracellular the other hand, NP uptake by cells was additionally enhanced distribution, and their cellular targeting efficacy through conupon application of an external magnetic field to cells by focal and optical microscopy, TEM, and MRI. placing a magnet below the culture wells, as observed by fluFrom optical microscopy images, it was possible to confirm orescence images (Figure 9f–g). Note that magnetic targeting that the present hybrid NPs are internalized inside the cells does not change the actual internalization process but simply (i.e., the dark black spots in Figure 9a), presumably in endo/ locally accumulates NPs, which results in a larger quantity of 䊏

ⵧ

Figure 8. Magnetization as a function of applied external magnetic field of DXSP-PGNH NPs at a) 5 and b) 300 K. c) T2−1 vs [Fe] for DXSP-PGNH NPs. Specific relaxivity, a measure of the MRI contrasting ability, was r2 = 207 (× 10−3 M Fe)− 1 s− 1.




9

www.advhealthmat.de

FULL PAPER


Figure 9. a) Optical microscopy image of HeLa cells with internalized DXSP-PGNH NPs. b,c) Confocal microscopy images of internalized DXSP-PGNH NPs; d,e) DXSP-PGNH-FA NPs; and f,g) DXSP-PGNH-FA NPs with magnetic targeting. Besides the plane parallel to the cell culture surface also crosssections through the planes perpendicular to the cell culture surface (as indicated by the dotted lines) are shown. The nuclei were stained with DAPI (blue channel, λexc = 355 nm), the actin cytoskeleton with BODIPY Phalloidin (red channel, λexc = 633 nm), and also light reflected from the PGNH NPs was recorded (green channel). h) 3D images of cells incubated with DXSP-PGNH-FA: i) DAPI channel, ii) reflected light by PGNH NPs, iii) BODIPY Phalloidin channel, iv) merged image of the three channels, and v) crop of one cell. The scale bars represent 20 μm.

internalized NPs. Our data demonstrate that the simultaneous use of folate and magnetic field exhibited a synergistic effect on NP targeting to HeLa cells. The NPs would remain in endosomal/lysosomal compartments and would not enter nuclei, as denoted by z-axis cross-sectional confocal microscopy and TEM images (Figure 9h,10f). Because our hybrid NPs are multifunctional, we were able to evaluate their enhanced uptake by TEM and MRI imaging. HeLa cells labeled with DXSP-PGNH NPs and resuspended in 1.6% (w/v) agarose solution clearly appear as hypointense spots in T2-MR images acquired with a 9.4-T MR scanner. Cells incubated with DXSP-PGNH-FA NPs (Figure 10d–e) were substantially darker than those incubated with the non-functionalized ones (Figure 10b–c).

10


On the other hand, TEM images (Figure 10f–h) showed that DXSP-PGNH-FA NPs were endocytosed and the NPs seemed to be accumulated within vesicles (see arrows). However, some NPs were found free in the cytoplasm, which suggested that a small fraction of NPs may escape from vesicles (Figure 10i), as also reported for other types of NPs.[50]

2.7. Chemo-Photothermal Cytotoxicity of Hybrid NPs In order to obtain a qualitative demonstration of the specificity and the enhanced localized cell cytotoxicity of our hybrid NPs, a cell viability assay in the presence and absence of irradiation was performed by staining viable cells with calcein




FULL PAPER

indicating that the light-exposed cells were dead (Figure 11c), whereas those not exposed were mostly alive (green cells in the inset of Figure 11c). For comparison, cells containing BNK-PGNH-FA NPs without loaded DOXO were also illuminated under the same conditions to elucidate whether the cell death was induced only through NIR-induced hyperthermia or by the combined effects of DOXO chemotoxicity and NIR-CW illumination. As shown in Figure 11d, many of the cells still appeared viable. Therefore, it is possible to conclude that the combined chemo- and phototherapy was more cytotoxic under the present conditions than chemo- or phototreatments alone. This increased rate of dead cells could be originated from the combination of i) an enhanced DOXO toxicity upon temperature elevation under NIR irradiation,[5c] ii) the larger sensitivity to heat of cells exposed to DOXO, iii) and/or to intracellular protein denaturation, which inhibits normal cellular growth and proliferation and possible induced some cell membrane damages.[51]

2.8. In Vitro Cell Growth Inhibition We further quantitatively evaluated the multidimensional therapeutic potential of the present hybrid NPs based on the combination of localized hyperthermia under NIR-CW laser irradiation, DOXO chemotherapy, and FA/ magnetic targeting by means of the crystal violet assay.[52] The crystal violet assays were used, provided that DOXO can usually cause interferences with formazan crystals when using the standard (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) [53] Figure 10. T2-weighted MR image of HeLa cells. a) Control sample with unlabeled 1 × 106 cells; (MTT) assay. Cell viabilities (CV) of HeLa cells treated b,d) 1 × 105; and c,e) 1 × 106 cells labeled with DXSP-PGNH NPs and DXSP-PGNH-FA NPs, respectively. f) TEM image showing cellular uptake of NPs. In g), the encapsulation of NPs with BNK-PGNH NPs at different NP conceninside endocytosic vesicles is observed. h) Image of DXSP-PGNH NPs entering the cells by trations in the absence of NIR irradiation were endocytosis and subsequent formation of vesicles. i) TEM image showing DXSP-PGNH NPs almost negligibly as observed in Figure 12a inside and outside a broken vesicle upon light irradiation. (orange column), confirming that these NPs can be considered as biocompatible. DXSPPGNH NPs in the absence of irradiation possessed a slightly acetoxymethyl ester (calcein AM) and dead cells with propidium larger toxicity than the former ones, which depended on NP iodide (PI) in order to verify the chemo-photothermal effect of concentration (Figure 12a, magenta column), that is, on the DXSP-PGNH-FA NPs. loaded DOXO concentration inside the hybrid NPs and its subFigure 11 shows fluorescence microscope images of HeLa sequent release. This toxicity could result from the diffusion cells incubated with DXSP-PGNH-FA NPs for 24 h. The of the drug through the pores of the metal layer. Nevertheless, estimated amount of DOXO contained inside these hybrid this reduction in CV was not very significant provided that the NPs was ≈1.3 × 10−6 M. In the absence of NIR irradiation, amount of DOXO released was too low to inhibit tumor cell cells (Figure 11a,b) showed a vivid green color over the entire proliferation to large extents in the time scale of the experiarea analyzed, suggesting that the NPs alone did not induce ment. In addition, some cell toxicity might be also originated cell death because of the low DOXO dose and the slow drug from the leakage of small amounts of nonencapsulated DOXO release. In contrast, after NIR-CW exposure (2.5 W cm−2 for released along the FA functionalization process, and by DOXO 10 min), a well-marked red-fluorescent region was present,




11

www.advhealthmat.de

FULL PAPER


concentrations of free DOXO, which resulted in lower viabilities compared with DXSPPGNH NPs containing the same amount of drug. A slight enhanced free DOXO cytotoxicity was observed under NIR irradiation (2.5 W cm−2 for 5 min). When exposing the cells to NIR laser illumination CVs in the presence of BNK-PGNH NPs and DXSPPGNH NPs decreased up to 67% and 39%, respectively. These larger cell mortalities appeared when the solution temperature raised above 43 °C upon irradiation, at which tumor cells are damaged probably by denaturation of intracellular proteins, as mentioned previously.[51] Notably, DXSP-PGNH NPs exhibited an additional cytotoxic effect [(CVBNK-PGNHs – CVDXSP-PGNHs)/CVBNK-PGNHs] of 42% when compared with BNK-PGNH NPs due to the remote release of DOXO, which was more important as the amount of DOXO increased in solution. The mechanism is speculated to be caused by the altered kinetics, permeability, and uptake of the chemotherapeutic agent during the heating process.[11d] The photothermal effect of the Au nanolayer can partially melt the core of the polymeric matrix, releasing a larger amount of DOXO from DXSP-PGNH NPs. Figure 11. Confocal images of nonilluminated HeLa cells containing a) DXSP-PGNH-FA NPs On the other hand, the presence of the and b) BNK-PGNH-FA NPs. HeLa cells containing c) DXSP-PGNH-FA NPs and d) BNK-PGNHtargeting ligand, FA, which favored an −2 FA NPs illuminated at 2.5 W cm for 10 min. The inset points to the laser-illuminated area. The black background color in c) stems from detachment of many dead cells after the washing enhanced NP accumulation inside the cells as shown above, led to a further decreased step with PBS. The scale bars in insets represent 5 mm. in the cell viability up to 31% after 24 h (Figure 12b). The targeting efficacy of FA was further corroborated by adding an excess of FA to cells prior the diffusion from some NPs with incomplete Au shells, which addition of DXSP-PGNH-FA NPs. In this case, cell viability was might be present in solution. This is further supported when similar to that observed for nonfunctionalized DXSP-PGNH comparing cytotoxicities of HeLa cells incubated with different

Figure 12. a) Cell growth inhibition of HeLa cells after incubation for 24 h in the presence of free DOXO in the absence (black) and presence (red) of NIR irradiation, 2.5 Wcm−2 for 5 min at 808 nm; BNK-PGNH NPs in the absence (orange) and presence of NIR irradiation (cyan); and DXSP-PGNH NPs in the absence (magenta) and presence of NIR irradiation (green). Different free DOXO and equivalent DOXO-loaded concentrations (13, 1.3, and 0.13 × 10−6 M) inside the hybrid NPs were tested (when corresponding). b) Cell growth inhibition of HeLa cells after incubation for 24 and 48 h in the presence of BNK-PGNH NPs (black) and DXSP-PGNH NPs (red) in the absence of NIR irradiation; BNK-PGNH NPs (orange), DXSP-PGNH NPs (cyan), DXSP-PGNH-FA NPs (magenta), DXSP-PGNH-FA NPs pretreated with 1 × 10−3 M free FA (yellow), and DXSP-PGNH-FA NPs + applied magnetic field (green) in the presence of NIR-CI (2.5 Wcm−2 for 5 min). The equivalent DOXO concentration in the case of DXSP-PGNH NPs and DXSP-PGNH-FA NPs was 1.3 × 10−6 M. Stars denoted statistically significant data (p < 0.05).

12





3. Conclusions We have synthesized polymeric core-coated Au nanoshells as a multifunctional platform to combine MR imaging, magnetic- and folic-acid-targeted drug delivery, NIR light-triggered drug release, and photothermal therapy simultaneously into one system. The resulting hybrid NPs displayed enhanced T2-weighted MRI properties and surface plasmon absorbance in the NIR region, thus, exhibiting a NIR-induced temperature elevation and a NIR light-triggered and stepwise release behavior of doxorubicin. The present hybrid NPs could be guided to the tumor area by the combination of ligand-induced and magnetic-assisted targeting [in addition to standard passive targeting via the enhanced permeation and retention (EPR) effect], which enhanced their targeting effectiveness as observed in vitro by microscopies. Cell viability tests revealed that the combination of chemotherapy, magnetic, and ligandinduced targeting, and NIR photothermal therapy through the use of DXSP-PGNH-FA NPs increased the likelihood of inducing cell death significantly showing a synergistic effect. This fact might potentially help to overcome resistance to chemotherapeutic agents, making it a promising approach for cancer therapy. In this regard, the permeability of tumor vessels and the sensitivity of tumor cells toward chemotherapeutics should be greatly enhanced by hyperthemia, holding the promise of improving drug efficacy. In addition, photothermal therapy may facilitate triggered and enhanced drug release from the multifunctional NPs, which is critical for achieving a high-effective drug concentration in the tumor. Future studies employing more sophisticated hybrid NPs should allow both spatially and temporally improved hyperthermia, opto-magnetic-resonance imaging contrast agents for in vivo monitoring of tissue pharmacokinetics and cell tracking, and to enable a perfect and on-demand control of drug release dynamics for completely efficient chemotherapy.


FULL PAPER

NPs. Furthermore, cell cytotoxicity was additionally increased when a magnetic field was applied to the cell culture for magnetic guiding of the NPs, achieving a mortality of 82%. The targeting-induced tumoricidal effect was calculated to be 20% ((CVDXSP-PGNHs – CVDXSP-PGNHs-FA)/CVBNK-PGNHs), and increased to 54% when magnetic targeting was also applied. After 24 h of additional incubation with the different hybrid NPs (48 h in total), the CVs were also examined revealing additional systemic cytotoxicity. CVs of HeLa cells treated with the functionalized NPs were additionally decreased due to the combined inhibitory effects of targeted hyperthermia and sustained DOXO release (Figure 12b). In the case of DXSPPGNH-FA, the CV was 20% with an enhanced cytotoxic effect of 23% if compared with nonfunctionalized DXSP-PGNH NPs. A cell toxicity of 93% was observed when an external magnetic field was applied; in this case, the targeting-induced tumoricidal effect was calculated to be 73% (CVDXSP-PGNHs – CVDXSP-PGNHs-FA+magnetic field)/CVBNK-PGNHs). All these results demonstrated the improved cellular uptake by the combined effect of FA and magnetic targeting, and the synergistic chemotherapeutic and Au-shell-driven hyperthermic efficacies of DXSP-PGNH-FA NPs.

4. Experimental Section Materials: Poly(D,L-lactide-co-glycolide) (PLGA) with 38–54 kDa molecular weight and 50:50 lactide–glycolide ratio, Pluronic F127, FeCl2, FeCl3, HAuCl3·3H2O, folic acid, N-(3-dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC), low-molecular-weight chitosan (LMW-chitosan, MW = 111 kDa), ascorbic acid, trisodium citrate dihydrate, sodium borohydride (NaBH4, 99%), crystal violet solution, and Nile Red (NR) were purchased from Sigma–Aldrich (St. Louis, MO, USA).N-hydroxysulfosuccinimide (sulfo-NHS) was purchased from ProteoChem (Cheyenne, USA). Calcein AM, propidium iodide, BODIPY Phalloidin, and ProLong Gold antifade reagent with DAPI were purchased from Invitrogen (Carlsbad, USA). Oleic acid with 90% purity was purchased from Alfa Aesar (Karlshrue, Germany). DOXO·HCl and Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, sodium pyruvate, and MEM nonessential amino acids (NEAA) were purchased from Fisher Scientific (Pittsburgh, USA). Polyethyleneglycol (PEG) molecules with different terminal groups (SH-PEG5000-NH2 and SH-PEG5000-OCH3) were purchased from Laysan Bio, Inc. (Arab, USA). Dialysis membrane tubing (molecular weight cutoffs ≈100–500 and ≈3500 Da) was purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA). All other chemicals and solvents were of reagent grade (purchased from Sigma– Aldrich). All glassware was washed with aqua regia and HF 5% (v/v) and extensively rinsed with water. Milli-Q water was used for preparation of aqueous solutions. Synthesis of SPIONs: Oleic acid-stabilized Fe3O4 SPIONs were synthesized by a coprecipitation method.[28,54] Briefly, aqueous solutions of 0.1 M of FeCl3 (30 mL) and FeCl2 (15 mL) prepared with N2 purgedwater were mixed; then, 3 mL of 5 M ammonia solution were added in small aliquots of 0.6 mL while stirring. A black precipitate was formed indicating the formation of SPIONs. After 20 min of stirring under N2 atmosphere, 56.4 mg of oleic acid was added to the SPIONs and the temperature was raised to 80 °C and kept for 30 min while stirring to evaporate the ammonia. The magnetic NPs were washed twice by centrifugation at 9000 rpm for 20 min to eliminate excess of oleic acid, the supernatant was discarded and the precipitate was lyophilized and stored at 4 °C. Preparation of DOXO/SPION-PLGA NPs: PLGA NPs containing SPIONs/DOXO or SPIONs/NR were prepared using a conventional emulsion-evaporation method with modifications. In a typical preparation, PLGA (25 mg) dissolved in a sealed vial containing 2 mL of methylene chloride (MC), was mixed with 2.5 mg of DOXO (previously converted to its hydrophobic base form by addition of triethylamine, as reported in the literature[27] or 2.5 mg of Nile Red, NR, when corresponding), and 2 mg of SPIONs (dispersed in MC) by sonication with a probe-type sonicator (20 kHz, Bandelin Sonopuls, Bandelin GmbH, Berlin, Germany) at 20 W for 10 min in an ice bath. DOXO (NR) concentration was measured by UV–vis spectroscopy with a calibration curve (Figure S10a–b). Then, this organic solution was added in a single step to a Pluronic F127 aqueous solution [50 mL, 1% (w/v)] and was immediately sonicated at 100 W for 15 min in an ice bath. The organic solvent was completely evaporated under mechanical stirring overnight, the dispersion was subsequently centrifuged twice at 9000 rpm for 30 min and 20 °C, the supernatant removed, and the final precipitate was redispersed in 25 mL of water (overall yield ≈90%). Synthesis of Citrate-Capped Au Nanoseeds: Citrate-capped gold seeds were prepared based on a method previously reported by Jana et al.[55] Briefly, 10 mL of 2.56 × 10−4 M tri-sodium citrate was mixed with 0.125 mL of 0.010 M HAuCl3·H2O. Next, 0.300 mL of 0.1 M ice cold NaBH4 solution was added all at once and mixed. The solution turned orange-pink immediately indicating NP formation. The average NP size (diameter of the inorganic Au core, not considering the organic coating) obtained from transmission electron microscopy (TEM) was 4 ± 2 nm. Preparation of DXSP-PGNH NPs: Au-citrated-capped nanoseeds were complexed to DOXO/SPION-PLGA NPs by means of electrostatic interactions between the positively charged chitosan on the surface of the NPs and negatively charged citrate-Au seeds. First, 5 mL of PLGA



13

www.advhealthmat.de

FULL PAPER

www.MaterialsViews.com NPs suspension was mixed with 0.25 mL of 1 wt% low-molecularweight-chitosan previously dissolved in 1% (v/v) acetic acid. After stirring for 4 h, NPs were centrifuged twice at 9000 rpm for 30 min to eliminate free and loosely adsorbed chitosan. Then, the positively charged NPs were mixed with 1 mL of Au nanoseed solution and stirred 4 h at ambient temperature. The sample was centrifuged at 7000 rpm for 15 min and 20 °C. The supernatant was discharged and the precipitate was redispersed in 5 mL of water (overall yield of ≈75%). To grow the gold nanoshells, 1 mL of the Au nanoseeddecorated DOXO/SPION-PLGA NPs was mixed with 20 mL of an Au growth solution (0.05 wt% K2CO3 and 0.02 wt% HAuCl3·3H2O stored for 24 h prior use) and stirred 1 min; subsequently, 50 μL of ascorbic acid (0.5 M) was added. Immediately after, the sample changed from clear to blue color, indicating the porous shell formation to give DOXO/SPION-PLGA NPs with a porous shell layer (DXSP-PGNH NPs). DXSP-PGNH NPs were left standing overnight for Ostwald rippening. Then, they were washed three times by centrifugation at 3500 rpm for 15 min and 20 °C, and finally redispersed by vortexing in water. High yields (≈95%) of DXSP-PGNH NPs were obtained with negligible generation of by-products such as small Au NPs. Despite some sedimentation due to the high density of gold layer and SPIONs cargo, DXSP-PGNH NPs presented no agglomeration in water, buffers or cell growth medium containing FBS, as observed by visual inspection and optical microscopy, and they could be easily redispersed for PEG stabilization and ligand-targeting attachment to become completely stable in aqueous solution. Conjugation of FA to DXSP-PGNH NPs: 5 mL of 11.33 × 10−3 M FA solution in 130 × 10−3 M NaHCO3 buffer (pH 7.0) was mixed with 5 mL of 34 × 10−3 M of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 5 mL of 34 × 10−3 M of N-hydroxisulfosuccinimide (sulfo-NHS), both dissolved in NaHCO3 buffer. This mixture was reacted for 20 min at ambient temperature and protected from light.[56] The activated FA was mixed with a 2.25 × 10−3 M solution of SH-PEG5000-NH2 dissolved in the same buffer ([FA]:[PEG] = 5). This was reacted 4 h at ambient temperature in the dark. After reaction, the SH-PEG5000-FA conjugate was dialyzed 24 h with a 3500 MWCO cellulose membrane against water to eliminate unreacted FA-NHS and by-products. DXSPPGNH NPs and BNK-PGNH NPs were pegylated with SH-PEG5000-FA in a straightforward process and the number of FA molecules attached to PGNH NPs was determined as detailed in the Supporting Information. Characterization of DOXO/SPION-PLGA and DXSP-PGNH NPs: NP sizes (i.e., hydrodynamic diameters) were obtained by dynamic light scattering (DLS) at 25 °C by means of an ALV-5000F (ALVGmbH, Germany) instrument with vertically polarized incident light (λ = 488 nm) supplied by a diode-pumped Nd:YAG solid-state laser (Coherent Inc., CA, USA) operated at 2 W, and combined with an ALV SP-86 digital correlator (sampling time 25 ns to 100 ms). NP sizes (i.e., diameters of cores in dried state) and morphologies were also acquired by transmission and scanning electron microscopy (TEM and SEM, respectively) by means of a Phillips CM-12 and a Carl Zeiss Libra 200 Fm Omega, and a FE-SEM ultra Plus electronic microscopes operating at 120 and 20 kV, respectively. Samples were prepared for analysis by evaporating a drop of the hybrid NP dispersion on a carboncoated copper grid without staining (TEM) or on a silicon wafer (SEM). ζ-potentials were measured with a Zetasizer Nano ZS (Malvern, UK) using disposable-folded capillary cells. Samples were measured in triplicate in PBS (pH 7) at 25 °C. Optical characterization of the NPs was carried out by UV–vis–NIR and fluorescence spectroscopies with a Cary Bio 100 spectrophotometer and a Cary Eclipse spectrofluorometer, respectively (Agilent Technologies, USA). The concentration of PGNH NPs was determined by viscometry and UV–vis spectroscopy (see Supporting Information). The DOXO loading capacity (LC) and encapsulation efficiency (EE) quantification process was determined by UV–vis and/or fluorescence spectroscopy (see Supporting Information) and the LC and EE of the SPIONs were assessed by ICP-MS in a Varian 820-MS equipment (Agilent Technologies, USA). Laser-Induced Temperature Increase with NIR-Continuous Wave Illumination (NIR-CW): Temperature increment tests were performed

14


with a continuous wave fiber-coupled diode laser source of 808 nm wavelength (50 W, Oclaro Inc, San Jose, CA, USA). The laser was powered by a Newport 5700-80-regulated laser diode driver (Newport Corporation, Irvine, CA, USA). A 200-μm-core optical fiber was used to transfer laser power from the laser unit to the target solution, and equipped with a lens telescope mounting accessory at the output, which allowed for tuning of the laser spot size in the range of 1–10 mm. The output power was independently calibrated using an optical power meter (Newport 1916-C) and laser spot size was previously measured with a laser beam profiler (Newport LBP-1-USB), which was placed at the same distance (8 cm) between the lens telescope output and the quartz cuvette (or the 96-well plate). For measuring the temperature change mediated by the present DXSP-PGNH NPs, 2 mL of DXSP-PGNH NPs were placed in a quartz cuvette and irradiated for 10 min intervals and/or at different power intensities. The temperature of samples was measured with a type J thermocouple inserted into the solution linked to a digital thermometer . In Vitro NIR-Laser Triggered Release of NR-PGNH NPs Cargo in Single Cells with Near-Infrared Short-Pulsed Illumination (NIR-SPI): In vitro lighttriggered release of cargo from NR-PGNH NPs was assayed with a widefield fluorescence microscope Axiovert 200M from Zeiss (Germany). The microscope was coupled with an 830-nm IR laser diode via fiber optics and a beam-coupling device (Rapp-Opto DL-830 CW laser, 130 mW maximum output) and fixed spot illumination coupling. The maximum light power reaching the sample image plane on top of the used 63x/1.4 oil immersion Plan-Apochromat objective was measured to be ≈30 mW (continuous output). The light energy was dispersed on a circular spot of ≈6 μm2. A tunable power supply output power of the laser could be varied smoothly from 0 to 30 mW effective light power on the sample plane. HeLa cells (1 × 105 per dish) were seeded and cultured for 24 h in 35 mm Ibidi μ-grid 500 dishes at standard culture conditions. Then, cells were washed with PBS and the medium was replaced with fresh medium containing NR-PGNH NPs (1 × 109 NPs mL−1) and incubated for 6 h. Subsequently, NP-containing cells were washed three times with PBS, and the medium containing NR-PGNH NPs was replaced with fresh medium and incubated for additional 18 h. The dishes were placed on the microscope stage within a controlled temperature and 5% CO2 atmosphere chamber. After localization and focusing, cells containing NR-PGNH NPs were illuminated for 1 s with a power of 0.3 or 0.6 mW μm−2 (= 1.8 mW/6 μm2 or 3.6 mW/6 μm2) (NIR-SPI), which correspond to laser fluences of 3 × 104 and 6 × 104 J cm−2 and total deposited energies of 1.8 and 3.6 mJ, respectively. These laser intensities cause negligible damage to cells as it was demonstrated previously.[57] Confocal microscopy images of NR-PGNH NPs-containing cells were acquired with a LSM 510 META confocal microscope from Zeiss (Germany) before and after illumination with the laser-microscope in order to observe the laser-triggered release of cargo from NR-PGNH NPs. NR was excited with a 488-nm argon laser and light emission was acquired with a LP 590-nm filter. Effect of NIR Light on DXSP-PGNH NP Shape (with NIR-SPI): In order to investigate the effect of laser illumination on the morphology of the NPs, 1 mL of SiO2 beads (5 μm) with positive surface charge was mixed with 1 mL of negatively charged DXSP-PGNH NPs for 15 min and centrifuged at 1500 rpm for 10 min to discard non-attached NPs. A drop of the SiO2/DXSP-PGNH NPs assembly was cast on a carbon-coated copper grid and left drying. TEM images of the assemblies placed in the center of the copper grid (easily detectable due to the large size of the SiO2/DXSP-PGNH NPs) were acquired and, then, the grid was placed on a microscope glass slide and illuminated with the laser-microscope tandem at the same powers previously used in release experiments. TEM images of the same SiO2/DXSP-PGNH NP assemblies previously analyzed were acquired after laser illumination. In this way, we were able to observe the effect of laser illumination on DXSP-PGNH NP morphology. NIR-Laser-Triggered Release of DOXO (with NIR-CI): Release kinetics of DOXO from DOXO/SPION-PLGA NPs and from DXSP-PGNH NPs was assessed in vitro at 37 °C in PBS buffer (pH 7.2). Briefly, 5 mL of DOXO/SPION-PLGA NPs or DXSP-PGNH NPs with known





echo time TE= 7.89 ms and repetition time TR = 100 ms, flip angle 20° and 2 averages. Magnetic susceptibility measurements were carried out with a SQUID magnetometer (Quantum Design MPMS5, San Diego, CA) at 5 and 300 K. Chemo-Photothermal Cytotoxicity (with NIR-CI): The chemophotothermal cytotoxic effect of nonfunctionalized, FA-functionalized, and magnetically guided FA-functionalized PGNH NPs was qualitatively assessed on HeLa cells. Cells (3 mL, 5 × 104 cells per well) were seeded in 6-well plates and grown for 24 h at standard culture conditions. Then, 200 μL (1 × 109 NPs mL−1) was added to cells. After 6 h of incubation, the NP-containing cells were washed three times with PBS pH 7.4 and illuminated with a laser (808 nm) at 2.5 W cm−2 for 5 min. Cells were incubated for further 18 h. Right before analysis with a confocal microscope (Leica Microsystems GmbH), cells were washed three times with PBS to eliminate any residue of serum esterase that could cause interference.[55] Then, 3 mL of PBS with 1 wt% of calcein AM (λex = 495 nm, λem = 515 nm) and 1 wt% of propidium iodide (λex = 495 nm, λem = 515 nm) were added and incubated for 10 min. In Vitro Cell Growth Cytotoxicity: Cytotoxicity induced by DXSP-PGNH NPs was determined by crystal violet staining[52,59] as the inhibition of cellular growth. To this end, HeLa cells with an optical confluence of 80%–90% were seeded into 96-well plates (100 μL, 1.5 × 104 cells per well) and grown for 24 h at standard culture conditions. Afterward, the DXSP-PGNH NPs and control samples were injected in the wells (100 μL) and incubated for 24 and 48 h. After incubation, the culture medium was discarded and the cells were washed with 10 × 10−3 M PBS, pH 7.4 several times. The cells were shaken at room temperature (300 rpm, 15 min) in the presence of 10 μL of a glutaraldehyde solution [11% (w/v) in water]. The solution was discarded and cells were washed three to four times with milli-Q water. The cells were then shaken at room temperature (300 rpm, 15 min) in the presence of 100 μL of a crystal violet solution [0.1% (w/v) in 200 × 10−3 M orthophosphoric acid, 200 × 10−3 M formic acid, and 200 × 10−3 M 2-N-morpholinoethanesulfonic acid (MES), pH 6]. The solution was discarded, and the cells were again washed three to four times with milli-Q water. Once washed, the cells were left for incubation at room temperature overnight for drying. Once dried, the cells were shaken at room temperature (300 rpm, 15 min) in the presence of 100 μL of acetic acid [10% (v/v) in water]. Immediately after, the absorbance of the resulting solution was measured with a Microplate Reader (FLUOstar Optima, BMG Labtech GmbH, Germany) operating at 595 nm. All experiments were triplicate carried out. The growth inhibition was quantified as: % Inhibition = 100 −

100 × OA TA

FULL PAPER

DOXO concentrations was placed in centrifuge tubes. Samples were maintained in a 37 °C bath with constant stirring. The samples were irradiated repeatedly over a period of 5 min, followed by 1 h intervals with the laser turned off. Each hour, 500 μL of sample was taken and centrifuged at 3500 rpm (DXSP-PGNH NPs) or at 5000 rpm (DOXO/ SPION-PLGA NPs) for 10 min at 20 °C. The supernatant was carefully extracted and its fluorescence at 594 nm was measured in a Cary Eclipse spectrophotometer (Agilent Technologies, Spain) for quantification of released DOXO with a calibration curve (Figure S10e,f, Supporting Information). After centrifugation and supernatant extraction, the settled DOXO/SPION-PLGA NPs and DXSP-PGNH NPs were resuspended with fresh buffer and returned to the original sample. Cellular Uptake by Confocal Laser Scanning Microscopy: DXSP-PGNH NP uptake was followed by confocal microscopy by seeding HeLa cells on poly-L-lysine-coated glass coverslips (12 × 12 mm2) placed inside 6-well plates (3 mL, 5 × 104 cells per well) and grown for 24 h at standard culture conditions [5% CO2 at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) FBS, 2 × 10−3 M L-glutamine, 1% penicillin/streptomycin, 1 × 10−3 M sodium pyruvate, and 0.1 × 10−3 M MEM nonessential amino acids (NEAA)]. Then, 200 μL (1 × 109 NPs mL−1) was added to cells. After 6 h of incubation, the NP-containing cells were washed three times with PBS pH 7.4 and then, fixed with paraformaldehyde 4% (w/v) for 10 min, washed with PBS, permeabilized with 0.2% (w/v) Triton X-100, and stained with BODIPY Phalloidin (Invitrogen). The cells were washed again with PBS, mounted on glass slides stained with ProLong Gold antifade DAPI (Invitrogen), and cured for 24 h at −20 °C. Samples were visualized with 20x and 63x objectives using a confocal spectral microscope Leica TCS-SP2 (Leica Microsystems GmbH, Heidelberg Mannheim, Germany), whereby the blue channel corresponds to DAPI (λex 355 nm), the red channel to BODIPY Phalloidin (λex 633 nm), and the green channel to DXSP-PGNH NPs as captured in reflection mode. Cellular Uptake by TEM: Uptake of DXSP-PGNH NPs by HeLa cells was also investigated by TEM. HeLa cells were seeded in 6-well plates (3 mL, 5 × 104 cells per well) and grown for 24 h at standard culture conditions. Then, 200 μL (1 × 109 NPs mL−1) was added to cells. After 6 h of incubation, the NP-containing cells were washed three times with PBS, trypsinized, and centrifuged at 1500 rpm for 4 min. Cell pellets were fixed with 500 μL of 2.5 wt% glutaraldehyde. The pellets were then included in an agar pellet, post-fixed with osmium tetraoxide in 0.1 M cacodylate buffer (1% (w/v)), and finally pelletized with Eponate (Ted Pella Inc, Redding, CA, USA). Ultra-thin cuts were obtained with an ultramicrotome (UltraCut S, Leica Microsystems GmbH) and were analyzed with TEM (JEOL JEM 1011, Japan). Cellular Uptake by Magnetic Resonance Imaging and Magnetic Susceptibility: Transverse relaxation times (T2) were measured at 9.4 T (400 MHz) with 440 mT m−1 gradients and 25 °C with a Bruker Biospin USR94/20 instrument (Ettlingen, Germany). MRI phantoms were constructed in 1.6% (w/v) agarose solutions (Sigma–Aldrich) following a previously published experimental protocol.[58] First, a 1.6% (w/v) agarose solution was heated and stirred at 80 °C until complete dissolution of the solid agarose. Then, while the system was fluid, an arrangement of seven centrifuge tubes (1.5 mL) was placed within the agarose and allowed to cool to room temperature. After solidification, the tubes were removed and an agarose mold with seven holes was obtained. For cell imaging, HeLa cells (2 × 106 cells) were seeded in 75 cm2 flasks and cultured for 24 h at standard condition. Then, the medium was replaced with fresh one containing a suitable amount of PGNH NPs (with or without FA) (1 × 109 NPs mL−1) and incubated for 6 h. Then, the medium with the NPs was removed and the cells were washed with PBS and incubated for 18 h. Cells were tripzinized and resuspended in 100 μL of PBS. In each hole of the phantom, a mixture of cells labeled with NPs and agarose 2% (w/v) was placed. After a resting period, 1.6% (w/v) agarose was added to the mold to seal the phantom holes. Imaging of the phantoms was performed acquiring a 3D-Gradient echo image (T2-weighting) with the following parameters: field of view: 80 mm × 80 mm × 40 mm, matrix size: 512 × 512 × 256 points giving a spatial resolution of 156 μm × 156 μm × 156 μm,

(1)

where OA and TA stand for the absorbance of studied samples and negative controls (cells in the absence of PGNH NPs), respectively. Statistical Analysis: All values shown are in mean ± SD unless otherwise stated. Analyses were done by unpaired Student’s t-test and considered significant at p < 0.05.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements The authors thank the Ministerio de Economía y Competitividad (MINECO) for research project MAT 2010-17336, the Xunta de Galicia for research grant CN2012/072, and the Fundación Ramón Areces for additional financial support. S.B. also thanks MINECO for her Ramon y Cajal fellowship. The authors also specially thank staff of Instituto de Ortopedia y Banco de tejidos Musculo-Esqueléticos of the Universidad



15

www.advhealthmat.de

FULL PAPER

www.MaterialsViews.com de Santiago de Compostela, and specially to Maite silva, for helpful assistance during in vitro cell experiments. Part of this work was also supported by HSFP (grant RGP0052/2012 to W.J.P). A.T. and M.A.-M. thank Mexico’s National Council of Science and Technology (CONACYT) for grants 203907 and 203732, respectively. Received: January 10, 2014 Revised: March 26, 2014 Published online:

[1] a) Z. Ali, A. Z. Abbasi, F. Zhang, P. Arosio, A. Lascialfari, M. F. Casula, A. Wenk, W. Kreyling, R. Plapper, M. Seidel, R. Niessner, J. Knöll, A. Seubert, W. J. Parak, Anal. Chem. 2011, 83, 2877; b) W. J. M. Mulder, A. W. Griffioen, G. J. Strijkers, D. P. Cormode, K. Nicolay, Z. A. Fayad, Nanomedicine 2007, 2, 307; c) D.-E. Lee, H. Koo, I.-C. Sun, J. H. Ryu, K. Kim, I. C. Kwon, Chem. Soc. Rev. 2012, 41, 2656; d) J. Kim, Y. Piao, T. Hyeon, Chem. Soc. Rev. 2009, 38, 372. [2] a) M. E. Caldorera-Moore, W. B. Liechty, N. A. Peppas, Acc. Chem. Res. 2011, 44, 1061; b) M. S. Bhojani, M. Van Dort, A. Rehemtulla, B. D. Ross, Mol. Pharm. 2010, 7, 1921; c) A. M. Gobin, M. H. Lee, N. J. Halas, W. D. James, R. A. Drezek, J. L. West, Nano Lett. 2007, 7, 1929; d) L. R. Hirsch, R. J. Stafford, J. A. Bankson, S. R. Sershen, B. Rivera, R. E. Price, J. D. Hazle, N. J. Halas, J. L. West, Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13549; e) H. Ke, J. Wang, Z. Dai, Y. Jin, E. Qu, Z. Xing, C. Guo, X. Yue, J. Liu, Angew. Chem. Int. Ed. 2011, 50, 3017; f) F. M. Kievit, M. Zhang, Adv. Mater. 2011, 23, H217; g) M. J. Sailor, J.- H. Park, Adv. Mater. 2012, 24, 3779. [3] a) H. Liu, D. Chen, L. Li, T. Liu, L. Tan, X. Wu, F. Tang, Angew. Chem Int. Ed. 2011, 50, 891; b) A. Ito, F. Matsuoka, H. Honda, T. Kobayashi, Cancer Immunol. Immunother. 2004, 53, 26; c) C.-M. J. Hu, L. Zhang, Biochem. Pharmacol. 2012, 83, 1104; d) M.-Y. Chang, A.-L. Shiau, Y.-H. Chen, C.-J. Chang, H. H. W. Chen, C.-L. Wu, Cancer Sci. 2008, 99, 1479; e) R. L. Atkinson, M. Zhang, P. Diagaradjane, S. Peddibhotla, A. Contreras, S. G. Hilsenbeck, W. A. Woodward, S. Krishnan, J. C. Chang, J. M. Rosen, Sci. Transl. Med. 2010, 2, 55. [4] a) D. Peer, J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit, R. Langer, Nat. Nanotechnol. 2007, 2, 751; b) S. Hearty, P. Leonard, R. O'Kennedy, Nat. Nanotechnol. 2010, 5, 9. [5] a) A. Heijden, C. Hulsbergen- Van de Kaa, J. A. Witjes, World J. Urol. 2007, 25, 303; b) O. Nativ, J. A. Witjes, K. Hendricksen, M. Cohen, D. Kedar, A. Sidi, R. Colombo, I. Leibovitch, J. Urol. 2009, 182, 1313; c) G. M. Hahn, J. Braun, I. Har-Kedar, Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 937. [6] P. Wust, B. Hildebrandt, G. Sreenivasa, B. Rau, J. Gellermann, H. Riess, R. Felix, P. M. Schlag, Lancet Oncol. 2002, 3, 487. [7] a) J. Braun, G. M. Hahn, Cancer Res. 1975, 35, 2921; b) S. Ning, G. M. Hahn, Cancer Res. 1991, 51, 5910; c) J. Overgaard, Cancer Res. 1976, 36, 3077; d) H. A. Johnson, M. Pavelec, J. Natl. Cancer Inst. 1973, 50, 903; e) C. D. Kowal, J. R. Bertino, Cancer Res. 1979, 39, 2285; f) P. Cherukuri, E. S. Glazer, S. A. Curley, Adv. Drug Delivery Rev. 2010, 62, 339; g) R. D. Issels, Eur. J. Cancer 2008, 44, 2546. [8] a) R. L. Manthe, S. P. Foy, N. Krishnamurthy, B. Sharma, V. Labhasetwar, Mol. Pharm. 2010, 7, 1880; b) H. P. Eikesdal, R. Bjerkvig, J. A. Raleigh, O. Mella, O. Dahl, Radiother. Oncol. 2001, 61, 313; c) B. K. Nelson, D. L. Conover, E. F. Krieg, D. L. Snyder, R. M. Edwards, Bioelectromagnetics 1997, 18, 349; d) C. J. Diederich, K. Hynynen, Ultrasound Med. Biol. 1999, 25, 871. [9] a) C. Loo, A. Lowery, N. Halas, J. West, R. Drezek, Nano Lett. 2005, 5, 709; b) R. Bardhan, W. Chen, C. Perez-Torres, M. Bartels, R. M. Huschka, L. L. Zhao, E. Morosan, R. G. Pautler, A. Joshi,

16


[10]

[11]

[12]

[13]

[14] [15]

[16] [17]

[18]

[19] [20] [21]

[22] [23]

[24] [25]

[26] [27] [28] [29]

N. J. Halas, Adv. Funct. Mater. 2009, 19, 3901; c) J. A. Schwartz, A. M. Shetty, R. E. Price, R. J. Stafford, J. C. Wang, R. K. Uthamanthil, K. Pham, R. J. McNichols, C. L. Coleman, J. D. Payne, Cancer Res. 2009, 69, 1659; d) S. J. Oldenburg, R. D. Averitt, S. L. Westcott, N. J. Halas, Chem. Phys. Lett. 1998, 288, 243. Y. Wang, K. C. L. Black, H. Luehmann, W. Li, Y. Zhang, X. Cai, D. Wan, S.-Y. Liu, M. Li, P. Kim, Z.-Y. Li, L. V. Wang, Y. Liu, Y. Xia, ACS Nano 2013, 7, 2068. a) T. K. Sau, C. J. Murphy, J. Am. Chem. Soc. 2004, 126, 8648; b) B. Nikoobakht, M. A. El-Sayed, Chem. Mater. 2003, 15, 1957; c) J. Pérez-Juste, I. Pastoriza-Santos, L. M. Liz-Marzán, P. Mulvaney, Coord. Chem. Rev. 2005, 249, 1870; d) T. S. Hauck, T. L. Jennings, T. Yatsenko, J. C. Kumaradas, W. C. W. Chan, Adv. Mater. 2008, 20, 3832; e) X. Huang, I. H. El-Sayed, W. Qian, M. A. El-Sayed, J. Am. Chem. Soc. 2006, 128, 2115. a) H. K. Moon, S. H. Lee, H. C. Choi, ACS Nano 2009, 3, 3707; b) A. Tan, S. Madani, J. Rajadas, G. Pastorin, A. Seifalian, J. Nanobiotechnol. 2012, 10, 34; c) F. Zhou, S. Wu, S. Song, W. R. Chen, D. E. Resasco, D. Xing, Biomaterials 2012, 33, 3235; d) Z. M. Markovic, L. M. Harhaji-Trajkovic, B. M. TodorovicMarkovic, D. P. Kepi , K. M. Arsikin, S. P. Jovanovi , A. C. Pantovic, M. D. Drami anin, V. S. Trajkovic, Biomaterials 2011, 32, 1121. a) O. C. Farokhzad, R. Langer, ACS Nano 2009, 3, 16; b) K. Riehemann, S. W. Schneider, T. A. Luger, B. Godin, M. Ferrari, H. Fuchs, Angew. Chem Int. Ed. 2009, 48, 872. E. I. Altınoglu, T. J. Russin, J. M. Kaiser, B. M. Barth, P. C. Eklund, M. Kester, J. H. Adair, ACS Nano 2008, 2, 2075. C. Alric, J. Taleb, G. L. Duc, C. Mandon, C. Billotey, A. L. MeurHerland, T. Brochard, F. Vocanson, M. Janier, P. Perriat, S. Roux, O. Tillement, J. Am. Chem. Soc. 2008, 130, 5908. P. del Pino, A. Munoz-Javier, D. Vlaskou, P. Rivera Gil, C. Plank, W. J. Parak, Nano Lett. 2010, 10, 3914. R. Bardhan, W. Chen, M. Bartels, C. Perez-Torres, M. F. Botero, R. W. McAninch, A. Contreras, R. Schiff, R. G. Pautler, N. J. Halas, A. Joshi, Nano Lett. 2010, 10, 4920. J. Kim, S. Park, J. E. Lee, S. M. Jin, J. H. Lee, I. S. Lee, I. Yang, J.-S. Kim, S. K. Kim, M.-H. Cho, T. Hyeon, Angew. Chem. Int. Ed. 2006, 45, 7754. J. Kim, J. E. Lee, S. H. Lee, J. H. Yu, J. H. Lee, T. G. Park, T. Hyeon, Adv. Mater. 2008, 20, 478. Y. Ma, X. Liang, S. Tong, G. Bao, Q. Ren, Z. Dai, Adv. Funct. Mater. 2013, 23, 815. a) S.-M. Lee, H. J. Kim, Y.-J. Ha, Y. N. Park, S.-K. Lee, Y.-B. Park, K.-H. Yoo, ACS Nano 2012, 7, 50; b) S.-M. Lee, H. Park, J.-W. Choi, Y. N. Park, C.-O. Yun, K.-H. Yoo, Angew. Chem. Int. Ed. 2011, 50, 7581; c) S.-M. Lee, H. Park, K.-H. Yoo, Adv. Mater. 2010, 22, 4049; d) H. Park, J. Yang, J. Lee, S. Haam, I.-H. Choi, K.-H. Yoo, ACS Nano 2009, 3, 2919. J. Yang, J. Lee, J. Kang, S. J. Oh, H.-J. Ko, J.-H. Son, K. Lee, J.-S. Suh, Y.-M. Huh, S. Haam, Adv. Mater. 2009, 21, 4339. a) M. W. Smith, M. Gumbleton, J. Drug Target. 2006, 14, 191; b) S. Mukherjee, R. N. Ghosh, F. R. Maxfield, Physiol. Rev. 1997, 77, 759. N. C. Bigall, W. J. Parak, D. Dorfs, Nano Today 2012, 7, 282. P. Rivera-Gil, D. Jimenez De Aberasturi, V. Wulf, B. Pelaz, P. Del Pino, Y. Zhao, J. M. De La Fuente, I. Ruiz De Larramendi, T. Rojo, X.-J. Liang, W. J. Parak, Acc. Chem. Res. 2012, 46, 743. H. Park, J. Yang, S. Seo, K. Kim, J. Suh, D. Kim, S. Haam, K.-H. Yoo, Small 2008, 4, 192. T. K. Jain, J. Richey, M. Strand, D. L. Leslie-Pelecky, C. A. Flask, V. Labhasetwar, Biomaterials 2008, 29, 4012. S. Li, Y. Ma, X. Yue, Z. Cao, Z. Dai, New J. Chem. 2009, 33, 2414. a) R. A. Jain, Biomaterials 2000, 21, 2475; b) K. S. Soppimath, T. M. Aminabhavi, A. R. Kulkarni, W. E. Rudzinski, J. Controlled




[31] [32] [33]

[34]

[35] [36]

[37] [38] [39] [40]

[41] [42]

[43] [44] [45]


[46] [47]

[48]

[49] [50] [51] [52]

[53] [54] [55] [56]

[57] [58]

[59]

FULL PAPER

[30]

Release 2001, 70, 1; c) M. Fraylich, W. Wang, K. Shakesheff, C. Alexander, B. Saunders, Langmuir 2008, 24, 7761; d) S. Acharya, S. K. Sahoo, Adv. Drug Delivery Rev. 2011, 63, 170. S. H. Kim, J. H. Jeong, K. W. Chun, T. G. Park, Langmuir 2005, 21, 8852. T. Ji, V. G. Lirtsman, Y. Avny, D. Davidov, Adv. Mater. 2001, 13, 1253. A. Topete, M. Alatorre-Meda, E. M. Villar-Alvarez, A. Cambón, S. Barbosa, P. Taboada, V. Mosquera, unpublished. X. Ji, R. Shao, A. M. Elliott, R. J. Stafford, E. Esparza-Coss, J. A. Bankson, G. Liang, Z.-P. Luo, K. Park, J. T. Markert, C. Li, J. Phys. Chem. C 2007, 111, 6245. M. P. Melancon, W. Lu, M. Zhong, M. Zhou, G. Liang, A. M. Elliott, J. D. Hazle, J. N. Myers, C. Li, R. Jason Stafford, Biomaterials 2011, 32, 7600. M. Paiva, D. J. Castro, M. Bublik, J. Sercarz, J. Investig. Med. 2006, 54, S381. a) L. Tong, Y. Zhao, T. B. Huff, M. N. Hansen, A. Wei, J. X. Cheng, Adv. Mater. 2007, 19, 3136; b) G. Bisker, D. Yeheskely-Hayon, L. Minai, D. Yelin, J. Controlled Release 2012, 162, 303. S. Carregal-Romero, M. Ochs, P. Rivera-Gil, C. Ganas, A. M. Pavlov, G. B. Sukhorukov, W. J. Parak, J. Controlled Release 2012, 159, 120. F. Zunino, G. Capranico, Anticancer Drug Des. 1990, 5, 307. C. Hrelescu, J. Stehr, M. Ringler, R. A. Sperling, W. J. Parak, T. A. Klar, J. Feldmann, J. Phys. Chem. C 2010, 114, 7401. a) H. Takahashi, T. Niidome, A. Nariai, Y. Niidome, S. Yamada, Nanotechnology 2006, 17, 4431; b) S. Link, C. Burda, B. Nikoobakht, M. A. El-Sayed, J. Phys. Chem. B 2000, 104, 6152. Sigma-ALDRICH, http://www.sigmaaldrich.com/catalog/product/ aldrich/739944?lang=de®ion=DE (accessed November 2012). a) Y. Zhong, C. Wang, L. Cheng, F. Meng, Z. Zhong, Z. Liu, Biomacromolecules 2013, 14, 2411; b) J. Liu, C. Detrembleur, B. Grignard, M.-C. De Pauw-Gillet, S. Mornet, M. Treguer-Delapierre, Y. Petit, C. Jérôme, E. Duguet, Chem. – Asian J. 2014, 9, 275. C. Guo, J. Wang, Z. Dai, Microchimica Acta 2011, 173, 375. R. Weissleder, K. Kelly, E. Y. Sun, T. Shtatland, L. Josephson, Nat. Biotechnol. 2005, 23, 1418. a) H. Ai, C. Flask, B. Weinberg, X. T. Shuai, M. D. Pagel, D. Farrell, J. Duerk, J. Gao, Adv. Mater. 2005, 17, 1949; b) J. Ge, Y. Hu, M. Biasini, W. P. Beyermann, Y. Yin, Angew. Chem. Int. Ed. 2007, 46, 4342; c) A. Z. Abbasi, L. A. Gutiérrez, L. L. del Mercato, F. Herranz, O. Chubykalo-Fesenko, S. Veintemillas-Verdaguer, W. J. Parak,

M. P. Morales, J. S. M. González, A. Hernando, P. de la Presa, J. Phys. Chem. C 2011, 115, 6257. R. Qiao, C. Yang, M. Gao, J. Mater. Chem. 2009, 19, 6274. a) J. Yang, C.-H. Lee, H.-J. Ko, J.-S. Suh, H.-G. Yoon, K. Lee, Y.-M. Huh, S. Haam, Angew. Chem. Int. Ed. 2007, 46, 8836; b) J. Juárez, A. Cambón, A. Topete, P. Taboada, V. Mosquera, Chem. Eur. J. 2011, 17, 7366; c) C. Niu, Z. Wang, G. Lu, T. M. Krupka, Y. Sun, Y. You, W. Song, H. Ran, P. Li, Y. Zheng, Biomaterials 2013, 34, 2307; d) N. Schleich, P. Sibret, P. Danhier, B. Ucakar, S. Laurent, R. N. Muller, C. Jérôme, B. Gallez, V. Préat, F. Danhier, Int. J. Pharm. 2013, 447, 94; e) K. Li, D. Ding, D. Huo, K.-Y. Pu, N. N. P. Thao, Y. Hu, Z. Li, B. Liu, Adv. Funct. Mater. 2012, 22, 3107. a) R. Bhattacharya, C. R. Patra, A. Earl, S. Wang, A. Katarya, L. Lu, J. N. Kizhakkedathu, M. J. Yaszemski, P. R. Greipp, D. Mukhopadhyay, P. Mukherjee, Nanomedicine 2007, 3, 224; b) V. Dixit, J. Van den Bossche, D. M. Sherman, D. H. Zhang, J. Zhai, E. Wang, Chem. Eur. J. 2009, 15, 9868. M. Colombo, S. Mazzucchelli, J. M. Montenegro, E. Galbiati, F. Corsi, W. J. Parak, D. Prosperi, Small 2012, 8, 1492. C. Brandenberger, C. Mühlfeld, Z. Ali, A.-G. Lenz, O. Schmid, W. J. Parak, P. Gehr, B. Rothen-Rutishauser, Small 2010, 6, 1669. D. Hanahan, R. A. Weinberg, Cell 2000, 100, 57. A. Cambon, A. Rey-Rico, D. Mistry, J. Brea, M. I. Loza, D. Attwood, S. Barbosa, C. Alvarez-Lorenzo, A. Concheiro, P. Taboada, V. Mosquera, Int. J. Pharm. 2013, 445, 47. J. Meerloo, G. L. Kaspers, J. Cloos, in Cancer Cell Culture, Vol. 731 (Ed: I. A. Cree), Humana Press, New York, USA 2011, p. 237. T. K. Jain, M. A. Morales, S. K. Sahoo, D. L. Leslie-Pelecky, V. Labhasetwar, Mol. Pharm. 2005, 2, 194. N. R. Jana, L. Gearheart, C. J. Murphy, J. Phys. Chem. B 2001, 105, 4065. A. Rollett, T. Reiter, P. Nogueira, M. Cardinale, A. Loureiro, A. Gomes, A. Cavaco-Paulo, A. Moreira, A. M. Carmo, G. M. Guebitz, Int. J. Pharm. 2012, 427, 460. A. Muñoz-Javier, P. del Pino, M. F. Bedard, D. Ho, A. G. Skirtach, G. B. Sukhorukov, C. Plank, W. J. Parak, Langmuir 2008, 24, 12517. J. Trekker, M. Hodenius, S. Soenen, W. Van Roy, M. De Cuyper, L. Lagae, U. Himmelreich, Proc. Intl. Soc. Mag. Reson. Med. 2012, 20, 4342. E. Vega-Avila, M. K. Pugsley, Proc. West. Pharmacol. Soc. 2011, 54, 10.



17

Combined therapy for inflammatory breast cancer.

Polydopamine Nanoparticles for Combined Chemo- and Photothermal Cancer Therapy.

Combined cisplatin and radiation therapy for advanced bladder cancer.

Radionuclide (131)I labeled reduced graphene oxide for nuclear imaging guided combined radio- and photothermal therapy of cancer.

CT imaging and photothermal therapy of cancer.

Combined Treatments with Photodynamic Therapy for Non-Melanoma Skin Cancer.

Multicomponent, Tumor-Homing Chitosan Nanoparticles for Cancer Imaging and Therapy.

Alkylphosphocholine analogs for broad-spectrum cancer imaging and therapy.

An activatable theranostic for targeted cancer therapy and imaging.

Multifunctional gold nanostars for molecular imaging and cancer therapy.

Combined modality therapy in esophageal cancer.

CT imaging.

Protein modified upconversion nanoparticles for imaging-guided combined photothermal and photodynamic therapy.

Theranostic nanoemulsions: codelivery of hydrophobic drug and hydrophilic imaging probe for cancer therapy and imaging.

Mussel-inspired plasmonic nanohybrids for light harvesting.

Protein-based photothermal theranostics for imaging-guided cancer therapy.

F-18 fluoromisonidazole for imaging tumor hypoxia: imaging the microenvironment for personalized cancer therapy.

Design and characterization of a combined OCT and wide field imaging falloposcope for ovarian cancer detection.

Fluorescent supramolecular micelles for imaging-guided cancer therapy.

Chitosan combined with molecular beacon for mir-155 detection and imaging in lung cancer.

Supraglottic laryngectomy for intermediate-stage cancer: U.T. M.D. Anderson Cancer Center experience with combined therapy.

Thyroid-stimulating hormone levels after radiotherapy and combined therapy for head and neck cancer.

A new combined therapy for functional organ preservation and survival in lateral oropharyngeal wall cancer.

Perspectives for the combined use of photodynamic therapy and hyperthermia in cancer patient.