A Versatile Carbonic Anhydrase IX Targeting Ligand-Functionalized

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A Versatile Carbonic Anhydrase IX Targeting Ligand Functionalized Porous Silicon Nanoplatform for Dual Hypoxia Cancer Therapy and Imaging Agne Janoniene, Zehua Liu, Lina Baranauskiene, Ermei Mäkilä, Ming Ma, Jarno Salonen, Jouni Hirvonen, Honbgo Zhang, Vilma Petrikaite, and Hélder A. Santos ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b04038 • Publication Date (Web): 06 Apr 2017 Downloaded from http://pubs.acs.org on April 7, 2017

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A Versatile Carbonic Anhydrase IX Targeting Ligand Functionalized Porous Silicon Nanoplatform for Dual Hypoxia Cancer Therapy and Imaging Agne Janoniene 1,2,‡, Zehua Liu 2,‡, Lina Baranauskiene 1, Ermei Mäkilä 3, Ming Ma 4, Jarno Salonen 3, Jouni Hirvonen 2, Hongbo Zhang 2,5,†,*, Vilma Petrikaite 1,6,*, Hélder A. Santos 2,* 1

Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius

University, LT-10257 Vilnius, Lithuania 2

Division of Pharmaceutical Chemistry and Technology, Drug Research Program, Faculty of

Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland 3

Laboratory of Industrial Physics, Department of Physics, University of Turku, FI-20014 Turku,

Finland 4

Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China

5

Department of Pharmaceutical Science, Åbo Akademi University, FI-20520 Turku, Finland

6

Department of Drug chemistry, Faculty of Pharmacy, Lithuanian University of Health

Sciences, LT-44307 Kaunas, Lithuania

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KEYWORDS: Porous silicon particles; carbonic anhydrase IX; hypoxia targeting; drug resistance; FRET.

ABSTRACT. Hypoxia occurs in most solid tumors and it has been shown to be an independent prognostic indicator of a poor clinical outcome for patients with various cancers. Therefore, constructing a nanosystem specifically targeting cancer cells under hypoxia conditions is a promising approach for cancer therapy. Herein, we develop a porous silicon (PSi) based nanosystem for targeted cancer therapy. VD11-4-2, a novel inhibitor for carbonic anhydrase IX (CA IX) is anchored on PSi particles (VD-PSi). As CA IX is mainly expressed on the cancer cell membrane under hypoxia condition, this nanocomplex inherits a strong affinity towards hypoxic human breast adenocarcinoma (MCF-7) cells, thus a better killing efficiency for the hypoxiainduced drug resistance cancer cell is observed. Furthermore, the release of doxorubicin (DOX) from VD-PSi showed pH dependence, which is possibly due to the hydrogen-bonding interaction between DOX and VD11-4-2. Fluorescence resonance energy transfer effect between DOX and VD11-4-2 is observed and applied for monitoring the DOX release intracellularly. Protein inhibition and binding assays showed that VD-PSi binds and inhibits CA IX. Overall, we developed a novel nanosystem inheriting several advantageous properties, which has great potential for targeted treatment of cancer cells under hypoxic condition.

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1. INTRODUCTION Hypoxia, a pathological condition in which tissue is deprived of an adequate supply of oxygen, is one of the hallmarks for solid tumor.1,2 In particular, hypoxia is a negative factor for cancer therapy as it contributes to chemoresistance, radioresistance, angiogenesis, invasiveness, and metastasis.3,4,5 Therefore, using agents that specifically target hypoxia-related proteins and further applying them as ligands in nanoplatforms for cancer cells targeting and effective hypoxia induced resistant cancer treatment are expected to rise as a new generation of anticancer therapy.6,7,8 Representative approaches for hypoxia-targeted cancer therapies are based on carbonic anhydrase IX (CA IX), a protein located on the outer cell membrane of cancer cells, which is expressed when hypoxia occurs in the tumor and is rarely expressed in normal tissues.9,10 Carbonic anhydrases (CA) is a family of metalloenzymes which has 15 different isoforms in human body for catalyzing the reaction where carbon dioxide and water are converted into bicarbonate ion and proton.11,12 Isoform CA IX is associated with some of the cancer phenomena, like the cell migration from primary tumor sites and chemo/radio therapy resistance.13,14,15 Therefore, CA IX is a desirable protein for tumor targeting, because of both its convenient location on the outer membrane of the cancer cells and also for its exclusive expression under hypoxia condition.16,17 Recently, we have synthesized a new compound, VD114-2,18 a sulfonamidic compound that possesses a high affinity (about 50 pM) and excellent selectivity towards CA IX.19 Hence, it is a promising candidate ligand to be applied in nanoplatform field for targeted cancer therapy. Among all the particles, porous silicon (PSi) particles are favorable drug carriers, because of their biocompatibility,20,21,22 advanced pharmacokinetic properties,23 low inflammatory, versatile

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surface functional groups24 and large, feasible drug loading capability. 25,26 Nevertheless, several disadvantages of bare PSi particles need to be properly solved, such as the phenomenon of premature release due to PSi’s particles large (usually larger than 8 nm) and easily accessible pores when it is loaded with small hydrophilic molecules, as well as insufficient cellular interaction and uptake, and lacking of targeting ability. Therefore, surface modification of PSi particles is demanded to solve these disadvantages and to move towards a more preferable drug delivery system.27,28,29 Upon such consideration, we designed and constructed a PSi-based CA IX targeting nanocomplex (VD-PSi) with a focus on whether this new compound, VD11-4-2, can be exploited to improve the hypoxia cancer cell targeting ability of the nanosystems, thus partly solving the hypoxia-induced drug resistance. VD11-4-2 is conjugated on PSi particles through a N,N'-carbonyldiimidazole (CDI) catalyzed reaction using functionalized polyethylene glycol (NH2-PEG-NHBoc) as a linking agent. Doxorubicin (DOX) is used as an anticancer drug to load into the nanocomplex. We hypothesize that comparing to unmodified PSi particles, this newly developed nanocarrier system possesses a specific hypoxia-induced targeting ability towards MCF-7 cells, with improved DOX loading and controlled releasing ability. VD11-4-2 also has a notable fluorescence, and thus, can be further used for bio-imaging. Förster resonance energy transfer (FRET) between VD11-4-2 and DOX is direct and sensitive system to monitor DOX release from the nanoplatform within the cells. This is the first time VD11-4-2 is used as a targeting agent for cancer cell treatment. Here, we report the preparation, characterization, and in vitro evaluation of this nanosystem in cancer cells.

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2. MATERIALS AND METHODS 2.1. Materials and Cell Culturing. N,N'-carbonyldiimidazole (CDI), N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), HEPES, NH2-PEG-NH-Di-tert-butyl pyrocarbonate (Boc) and all other compounds were bought from Sigma-Aldrich (St. Louis, MO, USA). Doxorubicin was purchased from TCI (Tokyo, Japan). Compound VD11-4-2 was synthesized and kindly supplied by Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University. Phosphate buffered saline (PBS, pH 7.4 10×), Hanks’ balanced salt solution (HBSS, 10×), Dulbecco’s Modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin (2.5%), sodium pyruvate, nonessential amino acids (NEAA, 100×), L-glutamine (100×) and penicillin-streptomycin (PEST, 100×) were purchased from HyClone (Waltham, USA). The human breast cancer cell MCF-7 was cultured in DMEM supplemented with 10% FBS, 1% NEAA, 1% L-glutamine, and 1% PEST (100 IU mL-1) in flasks and incubated at 37°C in a humidified atmosphere (95%) and 5% CO2.

2.2.Preparation of VD-PSi. The fabrication of undecylenic acid-modified thermally hydrocarbonized PSi (UnTHCPSi) particles has been described elsewhere.20,30 More detailed information can be found in Supporting Information (SI). Surface carboxylic acid groups are utilized to graft PEG and further conjugate VD11-4-2 as the ligand to increase the targeting ability of particles. Motivation behind the selection of PEG linker NH2-PEG-NH-Boc can be found in SI. The synthesis of VD11-4-2 is described elsewhere.18,19 The as-prepared UnTHCPSi particles (1 mg) was dispersed of anhydrous N,Ndimethylformamide (DMF) (1 mL). After the addition of EDC (8 µL) and of NHS (6 mg), the

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solution was stirred for 2 h to activate the carboxylic acid groups to NHS ester. Then 5 mg of NH2-PEG-NH-Boc was dissolved in 1 mL of anhydrous DMF and mixed with the solution to react for 24 h. The Boc-NH-PEG-UnTHCPSi was thoroughly washed for 3 times with DMF, then re-dispersed in 3 mL of CH2Cl2 (DCM) containing 1.8 mL of trifluoroacetic acid (TFA). The solution was stirred for 3 h to remove the Boc protection and expose the amine group. Then the NH2-PEG-UnTHCPSi particles were washed with saturated NaHCO3, EtOH and DCM once with each, and finally re-dispersed in 1 mL of toluene. Dry toluene (2 mL), VD11-4-2 (1 mg), and CDI (0.4 mg) were added to a 10 mL round-bottom flask fitted with a dry argon (Ar) inlet and magnetic stirrer and heated to 60 °C with stirring for 3 h. Then as-prepared NH2-PEG-UnTHCPSi was added dropwise.31 The solution was left to stir at 60 °C for overnight to get the final VD11-4-2-PEG-UnTHCPSi (further described as VD-PSi). Particles was washed 3 times with ethanol and store in it.

2.3. Physicochemical Characterization. Hydrodynamic diameter (Z-average) and zeta (ζ)potential measurements of the particles were carried out using a Zetasizer Nano ZS (Malvern Instruments, UK) at 25 °C, pH=7.0. Attenuated total reflectance Fourier transform infrared (ATR−FTIR) spectroscopy was used to analyze the chemical composition of the UnTHCPSi particles before and after conjugation with the VD11-4-2. All samples were screened at room temperature by a Bruker VERTEX 70 series FTIR spectrometer (Bruker Optics, Germany) equipped to a horizontal ATR sampling accessory (MIRacle, Pike Technology, Inc.). The spectra were recorded between 4000 and 500 cm-1 with a 2 cm-1 resolution.

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Transmission electron microscopy (TEM, Jeol JEM-1400, Jeol Ltd., Japan) was used to study the shape of the particles. The EDX spectrum was measured with an Oxford INCA 350 EDX (Oxford, UK) microanalysis system connected to the Hitachi S-4800 (SEM, Hitachi, Japan). The element distribution was monitored with EDX mapping. Quantitative conjugating efficacy was measured by fluorescence intensity with Varioskan Flash Multimode Reader (Thermo Fisher Scientific, USA). Detailed protocol is described in SI.

2.4. Human Plasma Stability Studies. To determine the impact of the VD11-4-2 conjugation on the stability of the PSi particles, the UnTHCPSi and VD-PSi are incubated with human plasma at 37 ± 1 °C for 2 h. Samples were withdrawn at different time intervals and diluted several times with water before the average particle size and average ζ-potential measurements (Zetasizer Nano ZS, Malvern Instruments, UK). Anonymous donor human plasma was obtained from the Finnish Red Cross Blood Service.32

2.5. DOX Loading and Releasing Studies. A certain amount of VD-PSi and PSi particles containing 1 mg PSi was dispersed into 2 mL PBS buffer at pH value of 7.4, and then 3 mg of DOX were added to stir overnight. After the drug loading, the particles were collected by centrifugation and prepared for the release study. In vitro release of DOX from VD-PSi was conducted using 6.7 mM PBS buffer, its pH value was adjusted by NaOH or HCl to 7.4 and 5.0, separately. The dissolution tests were performed at 37 °C under sink conditions33 by immersing 50 µg of each particles in 2 mL of buffer dissolution media using a shaking method at a shaking speed rate of 200 rpm. 100 µL of samples were withdrawn from each dissolution test at different time points. The collected samples were centrifuged at 16,000 g for 5 min and analyzed by

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fluorescence intensity (λex = 490 nm, λem = 600 nm), and the obtained particles were redispersed in an equal volume of fresh release medium to continue the drug release tests. UV-Vis spectrum was used to confirm the loading of DOX into VD-PSi. Free DOX and free VD11-4-2 (dissolved in DMSO at the concentration of 10 mg/mL as stock solution) were dissolved in 6.7 mM PBS buffer at the concentration of 40 µg/mL, 3 mL of DOX loaded VD-PSi (DOX@VD-PSi) which contains 600 µg PSi particles was also prepared. Their UV-spectra were acquired by full-wavelength scanning with UV-1600PC spectrophotometer (VWR, USA).

2.6. Fluorescence Resonance Energy Transfer (FRET) Effect Between VD11-4-2 and DOX. VD11-4-2 at the concentration of 12 µg/mL (0.027 mM) was prepared in 6.7 mM PBS buffer and an increasing molar ratios of DOX was serially added (0, 0.5, 1, 2, 4, 8, 16). After each addition of DOX, the solution was mixed by vortexing, and then the fluorescence spectrum of the solution is measured using a Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek, USA) with an excitation wavelength of 360 nm and a recorded emission range of 400−700 nm. Same procedure was used for all following experiments. Then increasing molar ratios of VD11-4-2 was added to a fixed concentration of DOX. Afterwards, 5 mg/mL DOX@VD-PSi and VD-PSi were also prepared, and their corresponding fluorescence spectra were recorded. For the evaluation of pH influence in FRET, different types of buffers (75 mM) were used for VD11-4-2 and increasing concentrations of DOX solutions prepared: acetate (pH 4.97), MES (pH 5.31), HEPES (pH 7.42). Temperature points used were 25 °C and 37 °C. NaCl with the concentration of 0.02 M and 0.1 M were used in compounds solution preparation for ionic strength measurements.

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2.7. Binding to CA IX and Catalytic Activity Inhibition. Fluorescent thermal shift assay (FTSA) was carried out to determine protein-ligand binding and was described in details in SI. Stopped-flow kinetic CO2 hydration assay was performed using Applied Photophysics SX.18MV-R stopped-flow spectrometer. Reaction velocities were measured by recording the absorbance of phenol-red indicator (30 µm, λ = 557 nm).34 Saturated CO2 solution was prepared by bubbling the gas in Milli-Q water at 25 °C for 1 h. Serial dilutions with 16 concentrations of VD11-4-2 compound; peg-PSi and VD-PSi particles were prepared and used for testing inhibition. Protein CA IX preparation and purification were reported previously.19 Samples containing 20 µM CA IX, 0 to 2 µM ligand (or 0 to 75 µM of particles), 20 mM HEPES with 100 mM NaCl and 60 µm phenol-red at pH 7.5, 10 % FBS, and 2.5 % ethanol, were incubated for 1 h at 25 °C and then used for stopped-flow experiments, where equal volumes of the protein sample and CO2 saturated water were mixed and the reaction was observed. Sample without protein (spontaneous CO2 hydration) and sample of protein without any inhibitor were used as zero and full activity controls, respectively. Kd values were determined using the Moririson model for tight binding inhibitors.35,36

2.8. Medium pH changes In vitro. Cells were seeded in 24 well plate (100000 cells per well) in normoxia and in hypoxia and left to attach overnight. Then, solutions of particles (100 µg ml1) and compound VD11-4-2 (6.4 µg ml-1 as compound IC50)19 were added to the medium followed by 48 h of incubation. The medium from the cells was collected and the pH was measured by SevenCompact™ pH/Ion meter S220 (Mettler Toledo, Spain).

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2.9. Immunofluorescence. MCF-7 cells were grown on glass coverslips and incubated under normoxic and hypoxic conditions. After 48 h, cells were fixed with cold methanol. Non-specific binding was blocked by incubation with PBS containing 2% bovine serum albumin (BSA) and 2% FBS. Cells were incubated with CA IX-specific monoclonal antibody (MAb) M75 (BioScience, Slovakia) at a concentration of 1:100 for 1 h, then incubated with goat anti-mouse AlexaFluor 488-conjugated IgG antibody (Life Technologies, USA) for 1 h. DAPI (Thermo Fisher, USA) was used to stain nucleus. Immunofluorescence of the cells was imaged with a The Invitrogen™ EVOS™ FL Auto Imaging System (Thermo Fisher Scientific, USA). Program ImageJ was used for the quantitative evaluation of fluorescence intensity of FITC labelled secondary CA IX antibody in three hypoxic (59 cells in total) and normoxic (53 cells in total) images. Average value per pixel was presented.

2.10. In vitro Cytotoxicity Study. The cytotoxicity of the different formulation groups were evaluated by an ATP-based luminescent cell viability assay, as described elsewhere.37 Briefly, MCF-7 cells were seeded in 96-well plates at the density of 0.3×104 cells/well and allowed to attach overnight. Then, the cell culture medium was replaced by 100 µL of fully supplemented medium containing different concentrations of DOX@VD-PSi (PSi particles concentration 10, 25, 50, and 100 µg/mL), DOX@PSi, extra free VD11-4-2 (10 µg/mL) + DOX@VD-PSi, free DOX, free VD11-4-2 (dissolved in DMSO as a stock solution) + free DOX (21 µg/mL DOX and 1.2 µg/mL VD11-4-2 equivalent at the PSi concentration of 100 µg/mL) and PSi particles with corresponding concentration. After 48 h incubation, the number of living cells was determined by the ATP-luminescent based cell viability kit (CellTiter-Glo®, Promega, USA), according to the manufacture’s protocol. Each experiment was performed at least in triplicate. MCF-7 cells

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cultured in growth medium without particles or with 1% of Triton X-100 or different concentration of DMSO served as negative, positive and DMSO controls, respectively. The luminescence was measured with a Varioskan Flashfluorometer (Thermo Fisher Scientific, USA). The hypoxic conditions were induced using cobalt chloride (CoCl2)38 at the concentration of 240 µM.39 After the cells attached medium with CoCl2 is added to the cell culture flasks and incubated for 48 h at 37 ℃ in a humidified atmosphere (95%) and 5% CO2 with changing the medium every 24 h. The added particles were also dispersed in medium containing CoCl2. The following studies were performed using the same experimental setup, as described above.

2.11. Fluorescence Confocal Microscope Studies. Suspensions of MCF-7 cells (1×105 cells/well) are seeded in Lab-Tek Chamber Slides (Thermo Fisher Scientific, USA). After 24 h of cell attachment to the wells, the cells were cultured with CoCl2 containing medium for 24 h to create hypoxia condition, as described above. Afterwards, the cells were rinsed three times with 1× HBSS (pH 7.4). Then, DOX@VD-PSi (200 µL; 100 µg/mL PSi particles concentration) were added into the wells. Following with 3 h and 24 h incubation, the samples were removed and the wells were washed three times with 1× HBSS (pH 7.4). Then CellMask DeepRedTM (Life Technologies, USA) was used to stain the cell membranes by incubating it with the cells at 37 °C for 3 min. Then, the cells were washed twice with the buffer, and fixed with 2.5% glutaraldehyde at room temperature for 20 min. The cells were again washed twice with the buffer, and stored in 2 mL of it.

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2.12. Flow cytometry. Suspensions of MCF-7 cells (2.0 ml per well) were seeded in 6-well plates at the concentration of 2.5×105 cells mL-1. After 24 h of cell attachment to the wells, the cells were further incubated with CoCl2 containing medium to create the hypoxia condition. Then, the cells were first rinsed three times with 1× HBSS (pH 7.4) and then DOX loaded particles (1 mL, 100 µg ml-1) were incubated with the cells for 3 h. Then, the samples were removed and the wells were washed five times with 1× HBSS (pH 7.4), and the cells were harvested immediately or subsequently cultured in fresh medium for another 3 h. After washing with 1× HBSS (pH 7.4), the cells were harvested and fixed with 2.5% glutaraldehyde in 1× 6.7 mM PBS (pH 7.4) for 30 min at room temperature. Exactly 10000 events were collected on a LSRII flow cytometer (BD Biosciences, USA) with a laser excitation wavelength of 488 nm using FACS Diva software to measure the fluorescence signal of DOX or on a LSR Fortessa flow cytometer (BD Biosciences, USA) with a laser excitation wavelength of 405 nm, using FACS Diva software to measure the fluorescence signal of VD11-4-2.

2.13. Statistical analysis. All the experiments were done at least in triplicate independent measurements and the obtained values are reported as mean ± standard deviation (s.d.). Student’s t-test was used to statistically evaluate the obtained values with the level of significance set at probabilities of **p < 0.01 and ***p < 0.005.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of the Nanoplatform. VD11-4-2 has a high affinity and selectivity towards CA IX protein, which is likely to be considered as a cancer marker as it is barely expressed in normal tissues.40 Therefore, VD11-4-2 possesses potential to be applied as a novel targeting ligand for anticancer drug delivery. UnTHCPSi particles were used in this study

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and prepared as described elsewhere,20,41 with a surface area of 251 ± 16 m2/g, a total pore volume of 0.69 ± 0.02 cm3/g, and an average pore diameter of 11.1 ± 0.4 nm. The hydrodynamic size (Z-average) of the particles was 184 ± 1 nm with a polydispersity index (PdI) of 0.13 ± 0.02. The zeta (ζ)-potential value of the particles was −24 ± 1 mV (Figure S1) due to the negatively charged carboxylic groups present at the particle’s surface. In the present study, PSi particles functionalized with carboxyl group (UnTHCPSi) were conjugated with the amine functionalized polyethylene glycol using EDC/NHS coupling29,42 and CDI based chemical reaction31 was used for conjugating VD11-4-2 to PEG modified PSi particles (Figure 1).

Figure 1. Reagents and conditions of the synthesis of VD-PSi. (i) CDI, toluene, 60 °C, 3 h; (ii) EDC, NHS, NH2-PEG-NHBoc, DMF, overnight; (iii) TFA, DCM, 2 h; (iv) CDI, toluene, 60 °C, 3 h.

Changes of the particles size and ζ-potential were measured during and after the reactions (Figure S1). After the conjugation of PEG and the de-protection of Boc, the ζ-potential changes dramatically from −30 mV to +19 mV due to the amine groups on the surface, and after the

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further conjugation with VD11-4-2, the ζ-potential changes back to −20 mV. TEM imaging of the particles showed their monodispersity before and after the modification (Figure S2). FTIR spectra of the samples during each modification step were measured and shown in Figure 2. The spectra of the bare particles shows the typical absorption bands for thermally hydrocarbonized PSi around 1032 cm-1 and the carbonyl stretching band at 1720 cm-1 resulting from the functionalization to provide carboxylic acid termination. After the conjugation of PEG, new bands at 1205 cm-1 and 1140 cm-1 were observed to be νas(C-N-C) and νas(N-C=O). Also, the reaction between UnTHCPSi with PEG led to the formation of amide, making the band of ν(C=O) shift from 1720 cm-1 to 1650 cm-1.43 All these findings suggest that PEG was successfully anchored on the particles surface. After the further modification of NH2-PEGUnTHCPSi with VD11-4-2, a new band appeared in 1260 cm-1, which is the typical band for aryl-fluorine.44 All these results confirmed the successful modification of VD11-4-2 on UnTHCPSi particles.42,45

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Figure 2. FTIR spectra of bare PSi (UNTHCPSi(UN)), PEG conjugated PSi (UN-PEG), VD-PSi (UN-PEG-VD11), VD11-4-2 (VD11) and Boc-NH-PEG-NH2 (PEG).

Energy-dispersive X-ray spectroscopy (EDX) mapping was also carried out to verify the presence of VD11-4-2 on the surface of VD-PSi (Figure 3). After the modification, EDX mapping spectra clearly show that fluoride (F) elements (existing only in VD11-4-2) were localized on the particles with good dispersity, which was another strong evidence for the successful and well dispersible conjugation of VD11-4-2 on the surface of VD-PSi particles. Overall, this confirmed that the conjugations on the PSi particles surface were successful at each step.

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Figure 3. (a) TEM image of VD-PSi. (b) Si element mapping. (c) F element mapping.

3.2. Stability Studies. Ideally, nanocarriers injected into the bloodstream should show minimal interactions with plasma proteins in order to avoid agglomeration and rapid clearance by the macrophages.46 The stability tests were conducted in physiological conditions, where particles were incubated in human plasma (at 37 °C for 2 h).32 Results showed that when the PSi particles was conjugated with PEG and VD11-4-2, it exhibited a lower variation in size and PdI compared to the pure PSi particles, indicating less interactions with the human plasma proteins (Figure 4). This is mainly due to the low aqueous stability of UnTHCPSi particles, because of its undecylenic acid modification, but it could be partly solved by the surface modification with PEG, typically used as a stealth agent.47,48

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Figure 4. (a) Impact of the human plasma on the particle size, (b) polydispersity index (PdI), and (c) ζ-potential for both the PSi and the VD-PSi particles within 2 h incubation. The results were

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calculated from the light scattering measurements data as a function of time at 37 °C. Values denote the mean ± s.d. (n = 3).

3.3. In Vitro Drug Loading and Release Studies. DOX was used as a model drug to load into VD-PSi (DOX@VD-PSi), and then loading and release tests in different pH values were performed. Bare PSi particles was used as a control. Interestingly, the loading degree of DOX into the particles had increased from 10.1 ± 0.1% in bare PSi, to 21.4 ± 0.1% in VD-PSi. Furthermore, when DOX was loaded into bare PSi particles, due to the large and easily accessible pores, most of the drug released within 1 h at both pH values (Figure 5a).49 However, in the VD-PSi’s case, DOX release showed clear pH responsive tendency, where at the pH value of 7.4 within 1 h only 30.6 ± 12.6 % DOX was released, while the release rate was as high as 87.2 ± 14.2 % within 1 h at the pH value of 5.0 (Figure 5a). We hypothesized that the possible explanation for the enhanced loading efficacy and the pH sensitive releasing trend was the formation of hydrogen bonds between VD11-4-2 and DOX. Under neutral condition (pH = 7.4), hydrogen bonding could be formed between ─OH, ─NH2, ─C=O within VD11-4-2 and ─OH, ─C─O─C, ─C=O, ─NH2 within DOX. However, under the acidic environment, the H+ in the solution competed with the hydrogen-bond-forming groups and then weakened the above mentioned hydrogen-bonding interactions.50

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Figure 5. (a) In vitro DOX release curves of DOX@PSi and DOX@VD-PSi at pH 5.0 and pH 7.4 PBS buffer at 37 °C within 24 h. Data is shown as mean ± s.d. (n = 3). (b) UV-vis absorption spectra of DOX, VD11-4-2, and DOX@VD-PSi in aqueous solution indicating the hydrogen bond between VD11-4-2 and DOX.

In order to confirm the previous hypothesis, we conducted UV-Vis experiments. The successful conjugation of VD11-4-2 and the stacking of DOX into VD-PSi were confirmed from the spectrum of the DOX@VD-PSi nanohybrid solution, as it clearly shows the characteristic absorption peaks of both VD11-4-2 and DOX (Figure 5b). Noteworthy, comparing to free DOX, λmax of DOX within DOX@VD-PSi had an obvious red shift from 490 nm to 530 nm. Moreover, a red shift for VD11-4-2 was also observed in the corresponding spectra (357 nm vs. 375 nm). The explanation for this is generally accepted to be the ground-state electron donor-acceptor (X─H┄Y) interactions between VD11-4-2 and DOX, whereas for the band which corresponds to the X─H stretch shifts to lower frequency (red shift) is one of the main characteristic features for hydrogen bond formation.51,52

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Overall, when loaded into VD-PSi, DOX showed both an enhanced loading efficacy comparing to bare PSi and also inherited a pH-responsive release trend, where the low extent of premature release of DOX at pH 7.4 would be predicted to reduce the adverse effects in healthy tissues upon particles administration in vivo.

3.4. FRET Effect Between VD11-4-2 and DOX. Here, we found that VD11-4-2 is a fluorescent compound, with the maximum emission wavelength at 500 nm when excited with 360 nm laser, while the λex of DOX is 490 nm (λem is 600 nm) (Figure S3).53 This distinctive fluorescence feature of VD11-4-2 gives us the opportunity to assemble a system resulting in the formation of a FRET complex: a donor VD11-4-2 energy transfer to the acceptor DOX, where the fluorescence of VD11-4-2 is quenched as a result of DOX absorbance. In order to evaluate if such FRET system occurs, it was first measured for the free compounds. Fluorescence spectroscopy was used to monitor the interaction between DOX and VD11-4-2 at a series of mole ratios. Sequential decreases in the fluorescence emission spectrum of VD11-4-2 were observed when a fixed 12 µg ml-1 concentration (regarding to the VD11-4-2 conjugation efficiency to PSi particles of 1.2 %) of VD11-4-2 was incubated with an increasing molar ratio of DOX (1:0; 1:0.5; 1:1; 1:2; 1:4; 1:8) (Figure 6a). This result suggests that DOX causes energy transfer from VD11-4-2 to DOX, which diminishes the fluorescence of VD11-4-2. Afterwards, increasing molar ratios of VD11-4-2 (1:0; 1:0.5; 1:1; 1:2; 1:4; 1:8) was added to a fixed concentration of DOX (12 µg ml-1) and the fluorescence intensity of DOX (550−600 nm) increased with increasing concentration of VD11-4-2, which further proved the FRET effect (Figure 6b). When the ratio was above 1:8, the fluorescence intensity of DOX was decreased, which was probably caused by the high concentration induced self-quenching (Figure 6c).54

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FRET effect in VD-PSi and DOX@VD-PSi systems was then investigated. The same phenomenon was observed within the nanocomplex, and when the DOX was stacked into VDPSi (in this case the molar ratio between VD11-4-2 and DOX was 1:12), the fluorescence of VD11-4-2 could be barely seen, and only DOX fluorescence was noticeable at about 600 nm. The result coincides with the observation of free compounds fluorescence (Figure 6d). A clear blue shift for VD11-4-2 emission spectrum was observed as the λmax-em changed from 490 nm to 470 nm, which could be because of ester bond formation through the hydroxyl group in VD-PSi. Carbonyl is a strong electron withdrawing group and decreases the electron density of VD11-4-2 which further leads to the blue shift of its emission spectrum.55,56

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Figure 6. (a) Fluorescence spectra of free VD11-4-2 (0.027 mM) with an increasing molar ratios (0, 1, 2, 4, 8) of VD11-4-2 at a λex = 360 nm. VD11-4-2 fluorescence intensity (at 500 nm) reduces with an increasing molar ratio of DOX. (b) Fluorescence spectra of free DOX (0.021 mM) with an increasing molar ratio of VD11-4-2. DOX fluorescence intensity increased with increasing concentration of VD11-4-2 (λex = 360 nm) (c) Fluorescence intensity change of DOX as well as VD11-4-2 when adding DOX into a fixed concentration of VD11-4-2. (d) VD-PSi and DOX@VD-PSi at the PSi concentration of 5 mg ml-1 at a λex = 360 nm.

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The influence of temperature, ionic strength and pH to the fluorescence intensity were determined to evaluate whether FRET effect would occur in various physiological conditions. To determine whether the pH would influence FRET effect, different buffers (MES pH 5.31, acetate pH 4.97; HEPES pH 7.42) were used in the preparation of solution. Results showed that neither pH nor higher temperature (37°C) reduced the fluorescence intensity (Figure S4). The ionic strength (0.1 M NaCl) also did not strongly influence the FRET effect (Figure S5). These results showed that various physiological conditions would not interfere with the FRET effect between DOX and VD11-4-2.

3.5. Particles Binding and Inhibition to Protein. As discussed above, VD11-4-2 is a selective compound and binds strongly to CA IX.19 To verify that the conjugation to the particles does not interfere with the compound ability to bind to the target protein CA IX, fluorescent thermal shift assay (FTSA),57,58 was carried out. FTSA confirmed very tight binding: upon addition of up to 0.3 mg ml-1 VD-PSi (equivalent to ca. 24 µM of attached VD11-4-2) protein Tm shift of >12 °C was observed (Figure S6a). Analysis of ligand dosing curves (Figure S6b) resulted in Kd for VD-PSi equal to 0.14 nM (calculated according to total immobilized compound) compared 0.05 nM for free VD11-4-2. This minor difference could be caused by part of immobilized compound being inaccessible to the protein, as this is supported by the presence of double transitions (corresponding to free and ligand-bound protein) in FTSA raw data, specific for tightly binding ligands in conditions when the concentration of available ligand was lower than protein concentration used.59 Using CA IX concentration 4 µM, double transitions were observed in samples with 40 and 59 µg ml-1 of VD-PSi (which correspond to 3.2 and 4.7 µM of immobilized compound), while only transition of ligand-bound was observed when 89 µg

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ml-1 of VD-PSi (7.1 µM of immobilized VD11-4-2) was added. Therefore, the amount of protein accessible immobilized VD11-4-2 was lower than 100 %, but higher than 56 %. The capability of VD-PSi to inhibit CA IX catalytic activity was evaluated and compared to free compound VD11-4-2, using stopped-flow CO2 hydration method. The experiments were established in the presence of FBS to reduce possible non-specific protein adsorption and inhibition by the particles. VD11-4-2 remained effective in inhibiting CA IX after its attachment to the particles (Figure 7). VD-PSi inhibited CA IX catalytic activity, they showed slightly higher Kd value compared to free VD11-4-2: 4 nM compared to 0.5 nM. This difference is the result of possible inaccessibility of portion of immobilized compound, as discussed above. Only very weak inhibition of CA IX catalytic activity was observed with highest tested PEG-PSi concentrations, which was probably due to unspecific enzyme adsorption.60

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Figure 7. CA IX protein inhibition determined by the stopped-flow kinetic CO2 hydration assay. Black: VD11-4-2; red: VD-PSi; and blue: PEG-PSi particles (PSi modified with PEG). Top axis corresponds to the concentration of the particles (PEG-PSi and VD-PSi) and the bottom axis to the concentration of VD11-4-2. Concentration of VD11-4-2 in VD-PSi was equal to its conjugation efficiency (100 µg ml-1 VD-PSi contains 1.2 µg ml-1 VD11-4-2). Data points correspond to the relative inhibition of a CA as a function of the total added compound concentration. The lines were fit according to the Morrison model.35,36

CAIX is one of the proteins that maintain neutral pH of the cell under hypoxia by producing bicarbonate from carbon dioxide,61 and the excess of proton ions after the reaction acidifies the cell environment.62 The pH changes of cell medium were monitored to evaluate the VD-PSi inhibition of CA IX (Figure S7). The pH of the medium in hypoxia turned to slightly acidic comparing to the normoxia group (7.5±1.5% vs. 7.8±2.5%, ***p < 0.005) after 48 h of

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incubation. Under hypoxia condition all the groups (VD11-4-2, VD-PSi, and PSi) affected the pH of the medium. VD-PSi was most potent in stopping cell acidification when comparing with control (7.8±8.9% vs. 7.5±1.5%, *p < 0.05). The VD-PSi effect on extracellular pH changes coincides with the results of protein binding and inhibition: as VD-PSi occupies CA IX active center, the protein function is inhibited and this is followed by the reduced amount of protons. VD-PSi stopped medium acidification more than bare PSi under hypoxia (7.8±8.9% vs. 7.7±4.1%, *p < 0.05). The influence of bare PSi on the changes of extracellular pH can be explained by the non-specific binding to the protein and its function inhibition.60 No statistically significant changes of medium pH were observed between groups where cells were incubated under normoxia.

3.6. In vitro Viability Study and Targeting Ability of VD-PSi. Immunofluorescence analysis was carried out to both quantitatively and qualitatively verify the exclusive expression of CA IX in MCF-7 cells under hypoxic condition (Figure 8a−c, Figure S8b). The fluorescence intensity of fluorescein isothiocyanate (FITC) labelled secondary CA IX antibody shows a significant enhancement of CA IX expression under hypoxic condition in comparison with normoxic condition (8.5±1.6 vs. 0.6±0.4, **p < 0.01). The result coincide the literature.63 These results confirm that CA IX can be applied as a target for hypoxia tumor treatment. ATP-luminescent based cell viability experiments were conducted to validate the enhanced anticancer efficiency and targeting ability of VD-PSi. Firstly, particles (PSi and VD-PSi) biocompatibility and VD11-4-2 cytotoxicity for cancer cells was determined (Figure S8). Results showed that both particles (PSi and VD-PSi) had a very slight dose dependent cytotoxic effect, and VD11-4-2 was nontoxic for cells (at the concentrations 0.12-1.2 µg ml-1). When

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applied to the cells at normoxic conditions, DOX@VD-PSi did not show an increased killing efficacy compared to bare DOX@PSi, as can be seen in Figure 8d. The cancer cell viability was actually found to be higher than the bare DOX@PSi at highest concentration (35.6±1.4% vs. 4.6±0.4%, ***p < 0.005). This can be because of the lower release rate of DOX from DOX@VD-PSi at physiological conditions compared to the release of DOX from DOX@PSi, as shown by the in vitro drug release studies (Figure 5 a). Moreover, the higher viability for the cells after PEG modification of the PSi particles can be explained by the weaker cellular interaction of VD-PSi with the cells due to the presence of PEG. Furthermore, CA IX, which is the specific receptor for VD11-4-2, is majorly expressed under hypoxia condition64 and this explains why there was no targeting ability for VD-PSi. Overall, in normoxic conditions, VD-PSi did not target the cells and it even inhibited the drug release, which may probably diminish the side effects of DOX (Figure 8d). Parallel experiments were conducted under hypoxic conditions, which usually occur in solid tumors.62 Compared to normoxic conditions, a clear drug resistance was observed in all the groups. For example, at the highest concentration of free DOX, the cancer cell viability changed from 15.1 ± 1.3% to 56.9 ± 4.3% (***p < 0.005), similar tendency was also observed in other groups. Previous papers suggested that hypoxic conditions can generate several types of drug resistance in many cancer cell lines.65,66,67 The use of the CA IX inhibitor at the inhibiting concentrations could thus enhance the anticancer drug effect by the tumor microenvironment pH change.68 Amongst all the groups tested, VD-PSi showed the highest killing ratio (DOX@VDPSi vs. DOX@PSi, 34.2 ± 2.7% vs. 55.9 ± 7.5%, **p < 0.01; DOX@VD-PSi vs. DOX, 34.2 ± 2.7% vs. 56.9 ± 4.3%, ***p < 0.005) and proved the increased anticancer effect of DOX@VDPSi under hypoxic conditions. Moreover, we postulated that free VD11-4-2 would bind to the

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CA IX protein existing on the cell membrane, which would further weaken the affinity between VD-PSi and the cell. The confirmation can be seen in Figure 8e, where extra free VD11-4-2 truly promoted the cell viability (DOX@VD-PSi vs. free VD11-4-2 + DOX@VD-PSi, 34.2 ± 2.7% vs. 47.4 ± 2.1%, ***p < 0.005), and the tendency was generally the same as with bare PSi (free VD11-4-2 + DOX@VD-PSi vs. DOX@PSi, 47.4 ± 2.1% vs. 55.9 ± 7.5%, p = 0.13, no significance). All these results confirmed the superior affinity and specificity of VD11-4-2 towards CA IX in hypoxic conditions, which further provided the exclusive cancer targeting ability of VD-PSi.

Figure 8. Immunofluorescence images showing the MCF-7 cells expression of CA IX under (a) hypoxic and (b) normoxic conditions. Nucleus (blue fluorescence) was stained with DAPI and CA IX (green) was stained with primary antibody against CA IX and secondary antibody

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conjugated with FITC. Scale bars indicate 50 µm. (c) Quantitative fluorescence intensity differences of stained CA IX between hypoxia and normoxia. (d) Normoxia and (e) hypoxia cell viability results of MCF-7 breast cancer cells treated with different concentrations of DOX in different formulations for 48 h evaluated by an ATP-based luminescent cell viability assay. All the data sets were compared with the controls (MCF-7 cells cultured in growth medium without particles or with 1% of Triton X-100 were negative and positive controls respectively). The concentration indicates the amount of PSi particles tested; for the other compounds the concentration was equivalent to the corresponding concentration within VD-PSi (from 0.12 to 1.2 µg/mL for VD11-4-2 and 2.14 to 21.4 µg/mL for DOX). Concentration for free VD11-4-2 was 10 µg/mL. Data is shown as mean ± s.d. (n ≥ 3).

3.7. Intracellular Uptake and DOX Release from VD-PSi. Confocal laser scanning microscopy experiments were conducted to confirm our hypothesis about using FRET effect between VD11-4-2 and DOX to monitor the drug release. While DOX is inside the particles, 360 nm wavelength excites VD11-4-2 and the energy from VD11-4-2 transfers to DOX, thus DOX fluorescence signal (~600 nm) is observed with no notable VD11-4-2 fluorescence (~500 nm). After DOX is released from the particles, and the distance between DOX and VD11-4-2 is larger than the typical limiting distance of FRET (about 10 nm)69, the fluorescence signal of VD11-4-2 is observed. After 3 h incubation with DOX@VD-PSi, no significant signal from VD11-4-2 was observed; however, the signal of DOX could be clearly seen in the sample (Figure 9a). It should be noted that the major signal of DOX did not evenly spread into the nucleus of the cells, but presented itself as dots and can be observed in the cytoplasm, which means particles were taken-up by the

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targeted cancer cells. At this stage, VD-11-4-2 was in the fluorescence “OFF” state as DOX was still loaded inside the particles and not yet released. After 24 h incubation (Figure 9a), DOX signal was accumulated in the nucleus, suggesting that DOX had already been released as a free drug.70 In addition, a clear signal from VD-PSi was also observed. With the releasing of DOX and its penetration into the nucleus, it was less likely to have interaction with VD11-4-2. All these results suggest that VD-PSi has also the potential to be applied as an imaging agent for intracellular drug delivery.

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Figure 9. (a) Confocal microscope images of DOX@VD-PSi interacting with MCF-7 breast cancer cells after incubation at 37 °C for 3 h and 24 h under hypoxic conditions. Blue: VD-PSi; green: DOX; red: cell membranes stained with CellMask DeepRed. All the scale bars indicate 20 µm. (b) Flow cytometry mean fluorescence intensity of VD11-4-2 (left) and DOX (right) after incubation and washing (0 h), and after additional 3 h incubation (3 h). Error bars represent mean ± s.d. (n = 3).

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To evaluate the intracellular DOX concentration flow cytometry experiments were conducted. First, we incubated the cells with particles for 3 h, after which the cells were washed thoroughly with HBSS for 5 times to remove the free particles. Then, the cells were collected immediately to check the fluorescence signal or further incubated with fresh medium for another 3 h. As it can be seen in the Figure 9b, after 3 more hours of incubation, the fluorescence signal of VD11-4-2 has a significant increase, while the difference of DOX signal is not so obvious. This is consistent with previous FRET investigation by confocal microscopy: after DOX is released from the vehicle, the FRET pair is broken and followed by the increase of VD11-4-2 signal and unchanged DOX signal.

4. CONCLUSIONS In this study, a novel compound VD11-4-2 is used for dual-function as cancer cell targeting agent and applied for the first time for surface modification of PSi particles for imaging. After conjugating of PSi particles with VD11-4-2, this newly constructed nanosystem possesses several important preeminent features as compared to the un-modified particles, including: (1) DOX loading efficacy is enhanced in VD-PSi, which also exhibits a pH dependent release manner; (2) A donor−acceptor model FRET between VD11-4-2 and DOX is observed in this system and applied in the drug release monitoring; and (3) it possesses a specific hypoxiainduced targeting ability towards MCF-7 cells, which further enhances the anticancer efficiency and also partly solves the hypoxia-induced drug resistance. Protein CA IX is expressed in numerous cancer cells under hypoxic conditions and we expect our nanosystem would be effective agent fighting with various solid tumors with hypoxia conditions inside of them. Our

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results suggest this nanoplatform as a highly promising candidate for targeted delivery applications, and this study is a solid step towards further in vivo investigations.

Supporting information: Additional experimental details (Fabrication of PSi, The selection of PEG linker, Quantitative Conjugating Efficacy, Fluorescent Thermal Shift Assay), particles size and zeta potential changes, TEM images, VD11-4-2 and DOX fluorescence spectra; FRET spectra, Protein binding and inhibition, pH changes, cell viability results.

Corresponding Authors * Hongbo Zhang, [email protected] * Vilma Petrikaite, [email protected] * Hélder A. Santos, [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. ACKNOWLEDGMENT A. Janoniene and Z. Liu contributed equally to this work. Prof. H. Zhang acknowledges Jane and Aatos Erkko Foundation (Grant No. 4704010) for financial support. Prof. H. A. Santos acknowledges financial support from the Academy of Finland (decision nos. 252215 and 281300), the University of Helsinki Research Funds, the Biocentrum Helsinki, Sigrid Jusélius Foundation (decision no. 4704580), and the European Research Council under the European

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Union’s Seventh Framework Programme (FP/2007–2013; Grant no. 310892). The authors thank the Electron Microscopy Unit and the Flow Cytometry Unit of the Institute of Biotechnology, University of Helsinki, for providing the necessary laboratory facilities and assistance.

REFERENCES (1)

Wilson, W. R.; Hay, M. P. Targeting Hypoxia in Cancer Therapy. Nat. Rev. Cancer 2011, 11, 393–410.

(2)

Brahimi-Horn, M. C.; Chiche, J.; Pouyssegur, J. Hypoxia and Cancer. J. Mol. Med. 2007, 85, 1301–1307.

(3)

Cosse, J.-P.; Michiels, C. Tumour Hypoxia Affects the Responsiveness of Cancer Cells to Chemotherapy and Promotes Cancer Progression. Anti-Cancer Agents Med. Chem. 2008, 8 (7), 790–797.

(4)

Pouysségur, J.; Dayan, F.; Mazure, N. M. Hypoxia Signalling in Cancer and Approaches to Enforce Tumour Regression. Nature 2006, 441, 437–443.

(5)

Pettersen, E. O.; Ebbesen, P.; Gieling, R. G.; Williams, K. J.; Dubois, L.; Lambin, P.; Ward, C.; Meehan, J.; Kunkler, I. H.; Langdon, S. P.; Ree, A. H.; Flatmark, K.; Lyng, H.; Urban, G.; Weltin, A.; Singleton, D. C.; Haider, S.; Buffa, F. M.; Harris, A. L. Targeting Tumour Hypoxia to Prevent Cancer Metastasis . From Biology , Biosensing and Technology to Drug Development : The METOXIA Consortium. J. Enzyme Inhib. Med. Chem. 2014, 6366 (August), 1–33.

(6)

Holgado, M. A.; Martin-Banderas, L.; Alvarez-fuentes, J.; Fernandez-Arevalo, M.; Arias, J. L. Drug Targeting to Cancer by Nanoparticles Surface Functionalized with Special

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Biomolecules Drug Targeting to Cancer by Nanoparticles Surface Functionalized with Special. Curr. Med. Chem. 2012, 19 (19), 3188–3195. (7)

Choi, K. Y.; Liu, G.; Lee, S.; Chen, X. Theranostic Nanoplatforms for Simultaneous Cancer Imaging and Therapy: Current Approaches and Future Perspectives. Nanoscale 2012, 4 (2), 330–342.

(8)

Sharma, S. V; Settleman, J. Oncogene Addiction : Setting the Stage for Molecularly Targeted Cancer Therapy. Genes Dev. 2007, 21, 3214–3231.

(9)

Pastorek, J.; Pastorekova, S. Hypoxia-Induced Carbonic Anhydrase IX as a Target for Cancer Therapy: From Biology to Clinical Use. Semin. Cancer Biol. 2014, 31, 52–64.

(10)

Mahon, B. P.; Pinard, M. A.; McKenna, R. Targeting Carbonic Anhydrase IX Activity and Expression. Molecules 2015, 20 (2), 2323–2348.

(11)

Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C. T. Carbonic Anhydrase: Current State of the Art, Therapeutic Applications and Future Prospects. J. Enzyme Inhib. Med. Chem. 2004, 19 (3), 199–229.

(12)

Sedlakova, O.; Svastova, E.; Takacova, M.; Kopacek, J.; Pastorek, J.; Pastorekova, S. Carbonic Anhydrase IX, a Hypoxia-Induced Catalytic Component of the pH Regulating Machinery in Tumors. Front. Membr. Physiol. Membr. Biophys. 2014, 4, 1–14.

(13)

Svastova, E.; Pastorekova, S. Carbonic Anhydrase IX: A Hypoxia-Controlled “catalyst” of Cell Migration. Cell Adhes. Migr. 2013, 7 (2), 226–231.

(14)

Tan, E. Y.; Yan, M.; Campo, L.; Han, C.; Takano, E.; Turley, H.; Candiloro, I.; Pezzella, F.; Gatter, K. C.; Millar, E. K. A.; Toole, S. A. O.; Mcneil, C. M.; Crea, P.; Segara, D.;

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Page 36 of 45

Sutherland, R. L.; Harris, A. L.; Fox, S. B. The Key Hypoxia Regulated Gene CAIX Is Upregulated in Basal-like Breast Tumours and Is Associated with Resistance to Chemotherapy. Br. J. Cancer 2009, 100, 405–411. (15)

Hussain, S. A.; Ganesan, R.; Reynolds, G.; Gross, L.; Stevens, A.; Pastorek, J.; Murray, P. G.; Perunovic, B.; Anwar, M. S.; Billingham, L.; James, N. D.; Spooner, D.; Poole, C. J.; Rea, D. W.; Palmer, D. H. Hypoxia-Regulated Carbonic Anhydrase IX Expression Is Associated with Poor Survival in Patients with Invasive Breast Cancer. Br. J. Cancer 2007, 96, 104–109.

(16)

Lock, F. E.; McDonald, P. C.; Lou, Y.; Serrano, I.; Chafe, S. C.; Ostlund, C.; Aparicio, S.; Winum, J.-Y.; Supuran, C. T.; Dedhar, S. Targeting Carbonic Anhydrase IX Depletes Breast Cancer Stem Cells within the Hypoxic Niche. Oncogene 2013, 32 (44), 5210–5219.

(17)

Ivanov, S.; Liao, S.; Ivanova, A.; Danilkovitch-miagkova, A.; Tarasova, N.; Weirich, G.; Merrill, M. J.; Proescholdt, M. A.; Oldfield, E. H.; Lee, J.; Zavada, J.; Waheed, A.; Sly, W.; Lerman, M. I.; Stanbridge, E. J. Expression of Hypoxia-Inducible Cell-Surface Transmembrane Carbonic Anhydrases in Human Cancer. Am. J. Pathol. 2001, 158 (3), 905–919.

(18)

Dudutienė, V.; Zubrienė, A.; Smirnov, A.; Timm, D. D.; Smirnovienė, J.; Kazokaitė, J.; Michailovienė, V.; Zakšauskas, A.; Manakova, E.; Gražulis, S.; Matulis, D. Functionalization of Fluorinated Benzenesulfonamides and Their Inhibitory Properties toward Carbonic Anhydrases. ChemMedChem 2015, 10 (4), 662–687.

(19)

Dudutiene, V.; Matuliene, J.; Smirnov, A.; Timm, D. D.; Zubriene, A.; Baranauskiene, L.; Morkunaite, V.; Smirnoviene, J.; Michailoviene, V.; Juozapaitiene, V.; Kasiliauskaite, A.;

ACS Paragon Plus Environment

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Page 37 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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 60

ACS Applied Materials & Interfaces

Jachno, J.; Kazokaite, J.; Linge, D.; Gibiez, P.; Capkauskaite, E.; Zaksauskas, A.; Kazlauskas, E.; Ladbury, J. E.; Matulis, D. Discovery and Characterization of Novel Selective Inhibitors of Carbonic Anhydrase IX. J. Med. Chem. 2014, 57, 9435–9446. (20)

Bimbo, L. M.; Sarparanta, M.; Santos, H. a.; Airaksinen, A. J.; Mäkilä, E.; Laaksonen, T.; Peltonen, L.; Lehto, V. P.; Hirvonen, J.; Salonen, J. Biocompatibility of Thermally Hydrocarbonized Porous Silicon Nanoparticles and Their Biodistribution in Rats. ACS Nano 2010, 4 (6), 3023–3032.

(21)

Par, J.-H.; Gu, L.; Maltzahn, G. von; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. Biodegradable Luminescent Porous Silicon Nanoparticles for in Vivo Applications. Nat. Mater. 2009, 8, 331–336.

(22)

Lu, J.; Liong, M.; Li, Z.; Zink, J. I.; Fuyuhiko, T. Biocompatibility, Biodistribution, and Drug-Delivery Efficiency of Mesoporous Silica Nanoparticles for Cancer Therapy in Animals. Small 2010, 6 (16), 1794–1805.

(23)

He, Q.; Shi, J. Mesoporous Silica Nanoparticle Based Nano Drug Delivery Systems: Synthesis, Controlled Drug Release and Delivery, Pharmacokinetics and Biocompatibility. J. Mater. Chem. 2011, 21 (16), 5845.

(24)

A. Santos, H.; M. Bimbo, L.; Lehto, V.-P.; J. Airaksinen, A.; Salonen, J.; Hirvonen, J. Multifunctional Porous Silicon for Therapeutic Drug Delivery and Imaging. Curr. Drug Discovery. Technol. 2011, 22 (228–249).

(25)

Santos, H. A.; Mäkilä, E.; Airaksinen, A. J.; Bimbo, L. M.; Hirvonen, J. Porous Silicon Nanoparticles

for

Nanomedicine:

Preparation

and

Biomedical

Applications.

ACS Paragon Plus Environment

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ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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 60

Page 38 of 45

Nanomedicine 2014, 9 (4), 535–554. (26)

Xia, B.; Wang, B.; Chen, Z.; Zhang, Q.; Shi, J. Near-Infrared Light-Triggered Intracellular Delivery of Anticancer Drugs Using Porous Silicon Nanoparticles Conjugated with IR820 Dyes. Adv. Mater. Interfaces 2016, 3 (4), 1–11.

(27)

Xia, B.; Wang, B.; Zhang, W.; Shi, J. High Loading of Doxorubicin into StyreneTerminated Porous Silicon Nanoparticles via π-Stacking for Cancer Treatments in Vitro. RSC Adv. 2015, 5 (55), 44660–44665.

(28)

Anderson, R. C.; Muller, R. S.; Tobias, C. W. Chemical Surface Modification of Porous Silicon. J. Electrochem. Soc. 1993, 140 (5), 1396–1396.

(29)

Shahbazi, M. A.; Shrestha, N.; Mäkilä, E.; Araújo, F.; Correia, A.; Ramos, T.; Sarmento, B.; Salonen, J.; Hirvonen, J.; Santos, H. A. A Prospective Cancer Chemo-Immunotherapy Approach Mediated by Synergistic CD326 Targeted Porous Silicon Nanovectors. Nano Res. 2014, 8 (5), 1505–1521.

(30)

Sarparanta, M. P.; Bimbo, L. M.; Mäkilä, E. M.; Salonen, J. J.; Laaksonen, P. H.; Helariutta, K. A. M.; Linder, M. B.; Hirvonen, J. T.; Laaksonen, T. J.; Santos, H. A.; Airaksinen, A. J. The Mucoadhesive and Gastroretentive Properties of HydrophobinCoated Porous Silicon Nanoparticle Oral Drug Delivery Systems. Biomaterials 2012, 33 (11), 3353–3362.

(31)

Rannard, S. P.; Davis, N. J. The Selective Reaction of Primary Amines with Carbonyl Imidazole Containing Compounds : Selective Amide and Carbamate Synthesis. Org. Lett. 2000, 351 (3), 3–6.

ACS Paragon Plus Environment

38

Page 39 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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 60

ACS Applied Materials & Interfaces

(32)

Liu, D.; Mäkilä, E.; Zhang, H.; Herranz, B.; Kaasalainen, M. Nanostructured Porous Silicon-Solid Lipid Nanocomposite: Towards Enhanced Cytocompatibility and Stability, Reduced Cellular Association, and Prolonged Drug Release. Adv. Funct. Mater. 2013, 23, 1893–1902.

(33)

Missirlis, D.; Kawamura, R.; Tirelli, N.; Hubbell, J. A. Doxorubicin Encapsulation and Diffusional Release from Stable, Polymeric, Hydrogel Nanoparticles. Eur. J. Pharm. Sci. 2006, 29 (2), 120–129.

(34)

Csaderova, L.; Debreova, M.; Radvak, P.; Stano, M.; Vrestiakova, M.; Kopacek, J.; Pastorekova, S.; Svastova, E. The Effect of Carbonic Anhydrase IX on Focal Contacts during Cell Spreading and Migration. Front. Membr. Physiol. Membr. Biophys. 2013, 4, 1–12.

(35)

Murphy, D. J. Determination of Accurate KI Values for Tight-Binding Enzyme Inhibitors: An in Silico Study of Experimental Error and Assay Design. Anal. Biochem. 2004, 327 (1), 61–67.

(36)

Williams, J. W.; Morrison, J. F. The Kinetics of Reversible Tight-Binding Inhibition. Methods Enzymol. 1979, 63 (C), 437–467.

(37)

Santos, H. A.; Riikonen, J.; Salonen, J.; Mäkilä, E.; Heikkilä, T.; Laaksonen, T.; Peltonen, L.; Lehto, V.; Hirvonen, J. In Vitro Cytotoxicity of Porous Silicon Microparticles : Effect of the Particle Concentration , Surface Chemistry and Size. Acta Biomater. 2010, 6 (7), 2721–2731.

(38)

Fu, O.; Hou, M.; Yang, S.; Huang, S.; Lee, W. Cobalt Chloride-Induced Hypoxia

ACS Paragon Plus Environment

39

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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 60

Page 40 of 45

Modulates the Invasive Potential and Matrix Metalloproteinases of Primary and Metastatic Breast Cancer Cells. Anticancer Res. 2009, 29, 3131–3138. (39)

El-Abd, E.; Matta, C.; Sheta, M.; El-Kerm, Y.; Afifi, M.; Benhassine, T.; Meftahi, S.; Sakr, S.; Elsherbini, B. Relation between Hypoxic Markers P65, P50, CAIX, and Tumor Stages in Invasive Ductal Carcinoma Subtypes. Adv. Cancer: Res. Treat. 2015, 2015, 1– 19.

(40)

Thiry, A.; Dogne, J. M.; Masereel, B.; Supuran, C. T. Targeting Tumor-Associated Carbonic Anhydrase IX in Cancer Therapy. Trends Pharmacol. Sci. 2006, 27 (11), 566– 573.

(41)

Araújo, F.; Shrestha, N.; Shahbazi, M. A.; Fonte, P.; Mäkilä, E. M.; Salonen, J. J.; Hirvonen, J. T.; Granja, P. L.; Santos, H. A.; Sarmento, B. The Impact of Nanoparticles on the Mucosal Translocation and Transport of GLP-1 across the Intestinal Epithelium. Biomaterials 2014, 35 (33), 9199–9207.

(42)

Almeida, P. V; Shahbazi, M.; Kaasalainen, M.; Salonen, J.; Hirvonen, J. Amine-Modified Hyaluronic Acid-Functionalized Porous Silicon Nanoparticles for Targeting Breast Cancer Tumors. Nanoscale 2014, 6, 10377–10387.

(43)

Haxaire, K.; Maréchal, Y.; Milas, M.; Rinaudo, M. Hydration of Polysaccharide Hyaluronan Observed by IR Spectrometry. I. Preliminary Experiments and Band Assignments. Biopolymers 2003, 72 (1), 10–20.

(44)

Pei, X. W. S.; Li, H. Study on an Antifouling and Blood Compatible Poly ( Ethylene – Vinyl Acetate ) Material with Fluorinated Surface Structure. J. Mater. Sci. 2010, 2788–

ACS Paragon Plus Environment

40

Page 41 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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 60

ACS Applied Materials & Interfaces

2797. (45)

Xu, W.; Thapa, R.; Liu, D.; Nissinen, T.; Granroth, S.; Narvanen, A.; Suvanto, M.; Santos, H. A.; Lehto, V. P. Smart Porous Silicon Nanoparticles with Polymeric Coatings for Sequential Combination Therapy. Mol. Pharm. 2015, 12 (11), 4038–4047.

(46)

Nel, A. E.; Mädler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding Biophysicochemical Interactions at the Nano–bio Interface. Nat. Mater. 2009, 8 (7), 543–557.

(47)

Pelaz, B.; Del Pino, P.; Maffre, P.; Hartmann, R.; Gallego, M.; Rivera-Fernández, S.; De La Fuente, J. M.; Nienhaus, G. U.; Parak, W. J. Surface Functionalization of Nanoparticles with Polyethylene Glycol: Effects on Protein Adsorption and Cellular Uptake. ACS Nano 2015, 9 (7), 6996–7008.

(48)

Huang, X.; Li, L.; Liu, T.; Hao, N.; Liu, H.; Chen, D.; Tang, F. The Shape Effect of Mesoporous Silica Nanoparticles on Biodistribution, Clearance, and Biocompatibility in Vivo. ACS Nano 2011, 5 (7), 5390–5399.

(49)

Herranz-Blanco, B.; Shahbazi, M.; Correia, A. R.; Balasubramanian, V.; Kohout, T.; Hirvonen, J.; Santos, H. A. pH-Switch Nanoprecipitation of Polymeric Nanoparticles for Multimodal Cancer Targeting and Intracellular Triggered Delivery of Doxorubicin. Adv. Healthc. Mater. 2016, 5 (15), 1904–1916.

(50)

Yang, X.; Zhang, X.; Liu, Z.; Ma, Y.; Huang, Y.; Chen, Y. High-Efficiency Loading and Controlled Release of Doxorubicin Hydrochloride on Graphene Oxide. J. Phys. Chem. C 2008, 112, 17554–17558.

ACS Paragon Plus Environment

41

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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 60

(51)

Page 42 of 45

Alabugin, I. V; Manoharan, M.; Peabody, S.; Weinhold, F. Electronic Basis of Improper Hydrogen Bonding : A Subtle Balance of Hyperconjugation and Rehybridization. J. Am. Chem. Soc. 2003, 125 (5), 5973–5987.

(52)

Hobza, P.; Havlas, Z. Blue-Shifting Hydrogen Bonds. Chem. Rev. 2000, 100, 4253–4264.

(53)

Bagalkot, V.; Zhang, L.; Levy-Nissenbaum, E.; Jon, S.; Kantoff, P. W.; Langery, R.; Farokhzad, O. C. Quantum Dot-Aptamer Conjugates for Synchronous Cancer Imaging, Therapy, and Sensing of Drug Delivery Based on Bi-Fluorescence Resonance Energy Transfer. Nano Lett. 2007, 7 (10), 3065–3070.

(54)

Park, D.; Cho, Y.; Goh, S.; Choi, Y. Hyaluronic Acid-Polypyrrole Nanoparticles as pHResponsive Theranostics. Chem. Commun. 2014, 50, 15014–15017.

(55)

Li, Y.; Zhao, Y.; Cheng, H.; Hu, Y.; Shi, G.; Dai, L.; Qu, L. Nitrogen-Doped Graphene Quantum Dots with Oxygen-Rich Functional Groups. J. Am. Chem. Soc. 2012, 134, 15– 18.

(56)

Xiaojing, M.; Xiao, S.; Wang, J.; Wu, Y.; Zhining, X. Synthesis and Spectroscopic Properties of Acridinium-9-Sulfonamides. Chin. J. Anal. Chem. 2009, 37 (7), 970–974.

(57)

Pantoliano, M. W.; Petrella, E. C.; Kwasnosk, J. D.; Lobanow, V. S.; Myslik, J.; Edward, G.; Carver, T.; Asel, E.; Springer, B. A.; Lane, P.; Salemme, F. R. High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery. J. Biomol. Screen. 2001, 6 (6), 429–440.

(58)

Matulis, D.; Kranz, J. K.; Salemme, F. R.; Todd, M. J. Thermodynamic Stability of Carbonic Anhydrase: Measurements of Binding Affinity and Stoichiometry Using

ACS Paragon Plus Environment

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Page 43 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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 60

ACS Applied Materials & Interfaces

Thermofluor. Biochemistry 2005, 44 (13), 5258–5266. (59)

Zubriene, A.; Matuliene, J.; Baranauskiene, L.; Jachno, J.; Torresan, J.; Michailoviene, V.; Cimmperman, P.; Matulis, D. Measurement of Nanomolar Dissociation Constants by Titration Calorimetry and Thermal Shift Assay - Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII. Int. J. Mol. Sci. 2009, 10 (6), 2662–2680.

(60)

Yildirim, A.; Ozgur, E.; Bayindir, M. Impact of Mesoporous Silica Nanoparticle Surface Functionality on Hemolytic Activity, Thrombogenicity and Non-Specific Protein Adsorption. J. Mater. Chem. B 2013, 1 (14), 1909–1920.

(61)

Chiche, J.; Ilc, K.; Laferrière, J.; Trottier, E.; Dayan, F.; Mazure, N. M.; Brahimi-Horn, M. C.; Pouysségur, J. Hypoxia-Inducible Carbonic Anhydrase IX and XII Promote Tumor Cell Growth by Counteracting Acidosis through the Regulation of the Intracellular pH. Cancer Res. 2009, 69 (1), 358–368.

(62)

Harris, A. L. Hypoxia — a Key Regulatory Factor in Tumour Growth. Nat. Rev. Cancer 2002, 2, 38–47.

(63)

Kaluz, S.; Kaluzová, M.; Stanbridge, E. J. The Role of Extracellular Signal-Regulated Protein Kinase in Transcriptional Regulation of the Hypoxia Marker Carbonic Anhydrase IX. J. Cell. Biochem. 2006, 97 (1), 207–216.

(64)

Potter, C.; Harris, A. L. Hypoxia Inducible Carbonic Anhydrase IX, Marker of Tumor Hypoxia, Survival Pathway and Therapy Target. Cell Cycle 2004, 3 (2), 164–167.

(65)

Frederiksen, L. J.; Siemens, D. R.; Heaton, J. P.; Maxwell, L. R.; Adams, M. A.; Graham, C. H. Hypoxia Induced Resistance to Doxorubicin in Prostate Cancer Cells Is Inhibited by

ACS Paragon Plus Environment

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Page 44 of 45

Low Concentrations of Glyceryl Trinitrate. J. Urol. 2003, 170, 1003–1007. (66)

Semenza, G. L. Hypoxia-Inducible Factors : Mediators of Cancer Progression and Targets for Cancer Therapy. Trends Pharmacol. Sci. 2012, 33 (4), 207–214.

(67)

Koch, S.; Mayer, F.; Honecker, F.; Schittenhelm, M.; Bokemeyer, C. Efficacy of Cytotoxic Agents Used in the Treatment of Testicular Germ Cell Tumours under Normoxic and Hypoxic Conditions in Vitro. Br. J. Cancer 2003, 89, 2133–2139.

(68)

Magnusson, E. B.; Halldorsson, S.; Ronan, M. T.; Leosson, K. Real-Time Optical pH Measurement in a Standard Microfluidic Cell Culture System. Biomed. Opt. Express 2013, 4 (9), 77–85.

(69)

Schaufele, F.; Demarco, I.; Day, R. N. FRET Imaging in the Wide-Field Microscope. In Molecular Imaging; Periasamy, A., Day, R., Eds.; Elsevier Inc., 2005; pp 72–94.

(70)

Tkachenko, A. G.; Xie, H.; Coleman, D.; Glomm, W.; Ryan, J.; Anderson, M. F.; Franzen, S.; Feldheim, D. L. Multifunctional Gold Nanoparticle-Peptide Complexes for Nuclear Targeting. J. Am. Chem. Soc. 2003, 125 (16), 4700–4701.

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