Accepted Manuscript Iron oxide nanoparticles functionalized with novel hydrophobic and hydrophilic porphyrins as potential agents for photodynamic therapy Oriol Penon, María J. Marín, David B. Amabilino, David A. Russell, Lluïsa Pérez-García PII: DOI: Reference:
S0021-9797(15)30230-7 http://dx.doi.org/10.1016/j.jcis.2015.09.060 YJCIS 20770
To appear in:
Journal of Colloid and Interface Science
Received Date: Revised Date: Accepted Date:
27 July 2015 23 September 2015 24 September 2015
Please cite this article as: O. Penon, M.J. Marín, D.B. Amabilino, D.A. Russell, L. Pérez-García, Iron oxide nanoparticles functionalized with novel hydrophobic and hydrophilic porphyrins as potential agents for photodynamic therapy, Journal of Colloid and Interface Science (2015), doi: http://dx.doi.org/10.1016/j.jcis. 2015.09.060
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Iron oxide nanoparticles functionalized with novel hydrophobic and hydrophilic porphyrins as potential agents for photodynamic therapy
Oriol Penon,a,b María J. Marín, c David B. Amabilino,d,1 David A. Russell,c Lluïsa PérezGarcíaa,b,*
Departament de Farmacologia i Química Terapèutica, Avda. Joan XXIII s/n, 08028 Barcelona, Spain
Institut de Nanociència i Nanotecnologia UB (IN2UB), Universitat de Barcelona, 08028 Barcelona,
School of Chemistry, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.
Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus Universitari de Bellaterra,
08193 Cerdanyola del Vallès, Spain
* Corresponding author at: Departament de Farmacologia i Química Terapèutica, Universitat de Barcelona, Avda. Joan XXIII s/n, 08028 Barcelona, Spain. Fax: +34 934024539. E-mail address: [email protected]
(L. Pérez-García). 1
Present address: School of Chemistry, University of Nottingham, University Park, NG7 2RD, United
Abstract The preparation of novel porphyrin derivatives and their immobilization onto iron oxide nanoparticles to build up suitable nanotools for potential use in photodynamic therapy (PDT) has been explored. To achieve this purpose, a zinc porphyrin derivative, ZnPR-COOH, has been synthesized, characterized at the molecular level and immobilized onto previously synthesized iron oxide nanoparticles covered with oleylamine. The novel nanosystem (ZnPR-IONP) has been thoroughly characterized by a variety of techniques such as UV-Vis absorption spectroscopy, fluorescence spectroscopy, X-ray photoloectron spectroscopy (XPS) and transmission electron microscopy (TEM). In order to probe the capability of the photosensitizer for PDT, the singlet oxygen production of both ZnPR-IONP and the free ligand ZnPR-COOH have been quantified by measuring the decay in absorption of the anthracene derivative 9,10-anthracenedipropionic acid (ADPA), showing an important increase on singlet oxygen production when the porphyrin is incorporated onto the IONP (ZnPR-IONP). On the other hand, the porphyrin derivative PR-TRIS3OH, incorporating several polar groups (TRIS), was synthesized and immobilized with the intention of obtaining water soluble nanosystems (PR-TRIS-IONP). When the singlet oxygen production ability was evaluated, the values obtained were similar to ZnPR-COOH/ZnPR-IONP, again much higher in the case of the nanoparticles PRTRIS-IONP, with more than a twofold increase. The efficient singlet oxygen production of PRTRIS-IONP together with their water solubility, points to the great promise that these new nanotools represent for PDT.
Keywords: Photodynamic therapy (PDT), Water soluble photosensitizers, Zn-porphyrins, Iron oxide nanoparticles (IONPs), Singlet oxygen production
1. Introduction The use of nanomaterials for biomedical applications has increased substantially over the past decade [1-5]. One of the main advantages of working with nanoparticles is the manipulation of their properties by controlling the material, size and shape of the nanocore, and the chemical functionalization with specific ligands, which provides multifunctionality to the nanosystem . Quantum dots , gold , silver  and especially iron oxide [10-15] nanoparticles have been widely used used for biological and medical applications  due to their easy of fabrication and functionalization, and biocompatibility. In particular, iron oxide nanoparticles (IONPs) have attracted great interest due to their magnetic properties  and have been used in the area of medicine for application including cell labelling , gene delivery , as contrast agents for magnetic resonance imaging (MRI) [20, 21], in hyper/photothermal therapy [22, 23], and as drug delivery systems . IONPs have also been used as drug delivery systems in photodynamic therapy (PDT) [25, 26], a therapeutic modality for the treatment of cancer where cancer cells are killed by reactive oxygen species formed by a photoactive drug (photosensitizer) following irradiation with light of a specific wavelength . Examples of photosensitizers include porphyrin- and chlorin-based molecules such as Photofrin® and Foscan®, which have shown excellent clinical results for the treatment of specific cancers . However, the hydrophobic nature of some photosensitizers result in drawbacks such as aggregation in aqueous media yielding to low efficacy, reduced delivery in blood circulation, and insufficient tumor selectivity . The use of nanoparticles as vehicles for the delivery of photosensitizer drugs to overcome important drawbacks of the molecular photosensitizer is a growing field of research . For example, gold nanoparticles functionalized with a hydrophobic zinc-phthalocyanine derivative and a polyethylene glycol (PEG) derivative covalently bound to jacalin have been shown to selectively target and kill HT-29 colon adenocarcinoma cells [30, 31]. IONPs 3
present significant advantages as drug delivery systems for PDT since they are excellent candidates for multifunctional medicine . IONPs can be used, for example, as imaging agents (for diagnosis) in combination with PDT and hyperthermia therapy (for treatment) and the same nanoplatform can provide targeted drug delivery [33-35]. To apply IONPs in the biomedical field, the particles have to be stable in the physiological media which can be achieved by functionalization of the nanoparticle’s surface  with hydrophilic ligands such as PEG , dextran , phospholipids , chitosan [40, 41] and catechol derivatives  including dopamine . These ligands have been used in combination with hydrophobic photosensitizers to yield water stable functionalized IONPs that can be used for PDT of cancer. For example, Sun et al. have coated IONPs with a layer of the polysaccharide chitosan where a porphyrin derivative drug was encapsulated yielding water soluble IONPs . Another polysaccharide, dextran, has also been used by Mbakidi et al. to coat the surface of IONPs yielding water soluble nanoparticles with chlorin p6 bound to the dextran . However, the functionalization of IONPs with PEG derivative ligands to render water solubility to the nanoparticles is possibly one of the preferred methodologies when designing water stable IONPs for PDT. Following this strategy, several groups have been able to prepare water stable IONPs containing photosensitizer drugs such as chlorin e6 [46-48], hematoporphyrin monomethyl ether , dendrimer porphyrin  and fullerenes . PEG based liposomes were synthesized by Di Corato et al. containing IONPs within the liposome core and Foscan ® in the lipid bilayer and were used for a combined therapy of cancer including magnetic hyperthermia and PDT both in vitro and in vivo . Following a different strategy, some authors have synthesized water soluble photosensitizer derivatives that have been used to directly functionalize the surface of IONPs. Lee and co-workers conjugated pheophorbide A to heparin which provided a water soluble photosensitizer drug that was directly attached to the surface of IONPs making them water soluble [53, 54]. Gu et
al. synthesized a dopamine-porphyrin derivative ligand that was used to functionalize IONPs which were stable on a methanol/water mixture and were uptaken by HeLa cells . Although a large number of iron oxide based nanosystems useful for PDT have been reported over the past ten years, the synthesis of novel water soluble porphyrin based ligands could provide with functionalized IONPs for innovative use in multimodal nanomedical applications. In this context, the work here presented describes the synthesis and characterization of IONPs coated with novel porphyrin based photosensitizer drugs, and the study of their ability to produce singlet oxygen following irradiation. Importantly, one of the porphyrins reported in this work is a water soluble molecule that yields water soluble functionalized IONPs that exhibit great potential for biological applications. First, a zinc porphyrin derivative composed of three phenyl groups and one phenoxy, incorporating a carboxylic acid chain in the meso position, was synthesized (ZnPR-COOH) and used to functionalize the surface of IONPs yielding non-water soluble nanoparticles (ZnPR-IONPs). In order to increase the water solubility of the nanosystem, a hydrophilic porphyrin derivative was synthesized containing three tris(hydroxymethyl)aminomethane (TRIS) groups (PRTRIS3OH). The water soluble ligand was immobilized on the surface of IONPs yielding water stable nanoparticles (PR-TRIS-IONP). The photosensitizer drugs and the respective nanoparticle conjugates have been fully characterized and their ability to produce singlet oxygen has been demonstrated, confirming their potential use for PDT of cancer.
2. Experimental 2.1. Materials and methods 1
H-NMR spectra were recorded using a Varian-300 (300 MHz) and a Varian Mercury 400
MHz spectrometer. NMR spectra were determined in CDCl3, DMSO-d6 or CD3OD, and chemical shifts are expressed in parts per million (δ) relative to the central peak of the 5
solvent. Thin layer chromatography (TLC) was performed on Merck coated F254 silica gel plates. Column chromatography was performed on silica gel 60 (Merck 9385, 230-400 mesh). XPS experiments were performed in a PHI 5500 Multitechnique System (from Physical Electronics) with a monochromatic X-ray source (Aluminium Kalfa line of 1486.6 eV energy and 350 W). Samples were prepared by drop casting of THF solution of ZnPRIONPs onto a silicon substrate. UV-Vis absorption spectra were obtained using a UV-1800 Shimadzu UV spectrophotometer using quartz cuvettes with a 1 cm path length. The UV-Vis absorption spectra recorded to measure the production of singlet oxygen were obtained using a Hitachi U-3000 spectrophotometer. Fluorescence excitation and emission spectra were recorded using a Hitachi F-4500 fluorescence spectrometer. The spectra were recorded using quartz cuvettes with a 1 cm path length. MALDI-ToF-MS analyses were performed using a Voyager-DE-RP (Appplied Biosystems, Framingham, USA) mass spectrometer, and high resolution mass spectra (HRMS) were obtained by electrospray ionization (ESI) using a LC/MSD-ToF mass spectrometer (Agilent Technologies, 2006) from Centres Cientifics i Tècnològics de la Universitat de Barcelona (CCiT-UB). Scanning tunneling microscopy (STM) studies were performed using a 5100 SPM system from Agilent Technologies. Zeta potential measurements were obtained using a Malvern Zetasizer Nano-ZS. The nanoparticles were centrifuged using a Beckman Coulter Allegra TM X-22R centrifuge. Commercially available reagents: pyrrole, 4-hydroxybenzaldehyde, benzaldehyde, sodium hydrogen carbonate (NaHCO3), magnesium sulfate (MgSO4), potassium carbonate (K2CO3), ethyl-7-bromoheptanoate, zinc (II) acetate (Zn(AcO)2), potassium hydroxide (KOH), oleylamine, phenyl ether, hexadecanediol, iron (II) acetate, 4-benzyloxybenzaldehyde, tris(hydroxymethyl)aminomethane (TRIS) and palladium on carbon (Pd-C) were obtained from Aldrich. 9,10-anthracenedipropionic (ADPA) was obtained from Molecular Probes. Solvents: propionic acid, ethyl acetate (AcOEt), acetonitrile (ACN), dichloromethane (DCM),
chloroform (CHCl3), methanol (MeOH), N-dimethylformamide (DMF), hexane (Hex), ethanol, tetrahydrofurane (THF) and dimethyl sulfoxide (DMSO) were of analytical grade.
2.2. Synthesis of ZnPR-COOH 2.2.1. Synthesis of 5-[4-hydroxyphenyl]-10,15,20-triphenylporphyrin (PR-OH)  Dry pyrrole (4.57 mL, 65.6 mmol) was added to a solution of refluxing propionic acid (100 mL). Subsequently, 4-hydroxybenzaldehyde (2 g, 16.4 mmol) and benzaldehyde (5 mL, 49.1 mmol) were added to the refluxing solution and the reaction mixture was stirred for 2 h. Then, propionic acid was evaporated under reduced pressure and the crude mixture was dissolved in AcOEt and washed with water (3 x 100 mL) and with a solution of 5% NaHCO3 (2 x 100 mL). The organic phase was dried over MgSO4 and filtered. The solvent was evaporated under reduced pressure. Acetonitrile was added (70 mL) and a purple solid was obtained by filtration. The mixture of porphyrins was separated by silica gel column chromatography using DCM as the eluent. A second silica gel column chromatography with CHCl3 as the eluent was needed to obtain PR-OH as a purple solid (560 mg, 5.4%). 1H-NMR (300 MHz, CDCl3) δ 8.87 (d, J = 4.8 Hz, 2H), 8.83 (s, 8H), 8.21 (dd, J = 7.3, 1.9 Hz, 6H), 8.05 (d, J = 8.1 Hz, 2H), 7.78 – 7.70 (m, 6H), 7.15 (d, J = 8.0 Hz, 2H), -2.75 (s, 2H). MALDI-ToF-MS: 631.2 m/z [M+1]. mp > 300 ºC. 2.2.2. Synthesis of 5-[p-(ethyl 7-heptanoate)-phenyl]-10,15,20-triphenylporphyrin (PRCOOEt) A suspension of K2CO3 (607 mg, 4.4 mmol) in refluxing DMF (150 mL) was stirred for 30 min under argon atmosphere. Next, porphyrin PR-OH (350 mg, 0.55 mmol) and ethyl-7bromoheptanoate (861 µL, 4.4 mmol) were added to the K2CO3 solution. After 24 h, K2CO3 (167.5 mg, 1.1 mmol) and ethyl-7-bromoheptanoate (215 µL, 1.1 mmol) were added and the reaction was kept at room temperature and controlled by TLC until all of the starting material
had been consumed after 48 h. The reaction was cooled down, filtered and the solvent was evaporated under reduced pressure. The crude mixture was dissolved in DCM and washed with water (3 x 100 mL). Then, the organic phase was dried over MgSO4, and the solvent was evaporated under vacuum. A purple oil was obtained that was purified by column chromatography using Hex/AcOEt (9:1) as the eluent. PR-COOEt was obtained as a purple solid (402 mg, 68%). 1H-NMR (300 MHz, CDCl3 ) δ 8.88 (d, J = 4.8 Hz, 2H), 8.83 (s, 6H), 8.21 (dd, J = 7.5, 1.9 Hz, 6H), 8.11 (d, J = 8.6 Hz, 2H), 7.75 (d, J = 6.4 Hz, 9H), 7.28 (d, J = 8.5 Hz, 2H), 4.24 (t, J = 6.4 Hz, 2H), 4.21 – 4.11 (m, 2H), 2.39 (t, J = 7.5 Hz, 2H), 2.30 (t, J = 7.5 Hz, 1H), 1.99 (m, 2H), 1.77 (m, 2H), 1.66 (m, 2H), 1.31-1.23 (m, 5H), -2.76 (s, 2H). MALDI-ToF-MS 786.3 m/z [M+1]. UV-Vis (CH2 Cl2) λmax 412.5, 517, 556.5, 597.5, 649.5. rf = 0.47 (Hex/AcOEt, 85:15). mp = 185 °C. 2.2.3.
COOEt) PR-COOEt (400 mg, 0.5 mmol) was dissolved in a refluxing solution of DMF (150 mL) under argon atmosphere. Zinc (II) acetate (1.646 g, 7.5 mmol) was added, and the purple solution became green after 5 min. The reaction was monitored using UV-Vis absorption spectroscopy. After 24 h the reaction was cooled down and the DMF was evaporated under vacuum. The crude product was dissolved in DCM and washed with water (3 x 100 mL). The organic phase was dried over MgSO4 and the solvent was evaporated under vacuum. The final product was dried in a vacuum oven at 80 ºC during 4 h, obtaining ZnPR-COOEt as a purple solid (353 mg, 83%). 1H-NMR (300 MHz, CDCl3) δ 8.99 (d, J = 4.7 Hz, 2H), 8.94 (t, J = 2.3 Hz, 6H), 8.22 (dd, J = 7.4, 1.8 Hz, 6H), 8.11 (d, J = 8.6 Hz, 2H), 7.79 – 7.71 (m, 9H), 7.27 (d, J = 8.6 Hz, 2H), 4.25 (t, J = 6.4 Hz, 2H), 4.18 (t, J = 6.4 Hz, 2H), 2.38 (t, J = 7.5 Hz, 2H), 1.99 (m, 2H), 1.76 (m, 2H), 1.66 (m, 4H), 1.52 (m, 5H). MALDI-ToF-MS: 848.2 m/z
[M+1]. UV-Vis (CH2Cl2) λmax 424, 553, 591 nm. rf = 0.93 (DCM/MeOH, 95:5). mp = 105 ºC. 2.2.4. Zinc 5-[p-(7-heptanoic)-phenyl]-10,15,20-triphenylporphyrin acid (ZnPR-COOH) The protected carboxylated porphyrin ZnPR-COOEt (350 mg, 0.42 mmol) was dissolved in a mixture of solvents containing methanol (100 mL) and DCM (50 mL) and the stirred reaction was refluxed at 60 °C. KOH (2.6 g, 46 mmol) dissolved in water (10 mL) was added to the solution and the reaction was refluxed at 60 °C overnight. The solution was evaporated under vacuum and the crude product was dissolved in DCM, filtered and washed with water (3 x 100 mL). The organic phase was dried yielding ZnPR-COOH as a purple solid (303 mg, 80%). 1H-NMR (400 MHz, CDCl3) δ 8.98 (d, J = 4.7 Hz, 2H), 8.94 – 8.92 (m, 6H), 8.23 – 8.19 (m, 6H), 8.10 (d, J = 8.6 Hz, 2H), 7.78 – 7.70 (m, 9H), 7.25 – 7.22 (m, 2H), 4.22 (t, J = 6.4 Hz, 2H), 3.33 – 3.29 (m, 2H), 2.71 (t, J = 0.7 Hz, 2H), 2.43 (t, J = 7.4 Hz, 2H), 2.19 (t, J = 8.1 Hz, 2H), 1.97 (m, 2H), 1.77 (m, 2H). MALDI–ToF-MS 820.2 m/z [M]; HRMS-ES: calc. for C51H40N4O3Zn 820.2392, found 821.2508 [M+1]. UV-Vis (CH2Cl2) λmax 424, 553, 591 nm. rf = 0.35 (DCM/MeOH, 95:5). mp = 138 °C.
2.3. Synthesis of PR-TRIS3OH 2.3.1. Ethyl 4-formylphenoxyacetate (CHO-Ac) Ethyl 4-formylphenoxyacetate (CHO-Ac) was prepared following a protocol described in the literature . CHO-Ac was obtained as a yellow solid (10.120 g, 48%). 2.3.2. 5-[benxyloxy]-10,15,20-[ethyl 4-phenoxyacetate] porphyrin (PR-AC3Bnz) 4-benzyloxybenzaldehyde (3.4 g, 16 mmol), ethyl 4-formylphenoxyacetate (10 g, 48 mmol) and dry pyrrole (4.48 mL, 64 mmol) were added to a refluxing solution (140 ºC) of propionic acid (200 mL) under argon conditions. The solution became dark and after 1.5 h the reaction was cooled down and evaporated under reduced pressure. The crude product was
dissolved in DCM and washed with H2O (3 x 200 mL) and with a 5% NaHCO3 solution (2 x 200 mL). The organic phase was evaporated and the resulting solid was dried overnight in a vacuum oven at 80 ºC. The mixture of porphyrins was separated by silica gel column chromatography using DCM as the eluent followed by a second silica gel column chromatography with CHCl3 as the eluent. After washing with acetonitrile, PR-AC3Bnz was obtained as a purple solid (390 mg, 2.3%). 1H-NMR (400 MHz, CDCl3) δ 8.88 – 8.83 (m, 8H), 8.13 – 8.09 (m, 8H), 7.62 (d, J = 7.2 Hz, 2H), 7.49 (t, J = 7.7 Hz, 2H), 7.42 (t, J = 7.4 Hz, 1H), 7.34 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 8.6 Hz, 6H), 5.33 (s, 2H), 4.91 (s, 6H), 4.41 (q, J = 7.1 Hz, 6H), 1.41 (t, J = 7.1 Hz, 9H), -2.77 (s, 2H). HRMS-ES: calc. for C63H54N4O10 1026.3840, found 1027.3903 [M+1]. rf = 0.775 (DCM/MeOH 99:1). mp = 190 ºC. 2.3.3. 5-[benxyloxy]-10,15,20-[Tris(hydroxymethyl) 4-phenoxyacetamide] porphyrin (PRTRIS3Bnz) Compound PR-AC3Bnz (200 mg, 0.195 mmol) was dissolved in 40 mL of anhydrous DMSO at room temperature under argon conditions. Tris(hydroxymethyl)aminomethane (236 mg, 1.95 mmol) and potassium carbonate (269 mg, 1.95 mmol) were added to the PRAC3Bnz solution. After 48 h, tris(hydroxymethyl)aminomethane (118 mg, 0.975 mmol) and potassium carbonate (134.5 mg, 0.975 mmol) were added and the reaction was finished after 8 h. The resulting solution was filtered, and the DMSO was removed by evaporation under reduced pressure using a dry ice condenser rotary evaporator. The crude product was dissolved in AcOEt/MeOH (9:1) and washed with water (3 x 50 mL). The organic phase was dried over MgSO4, and the solvent was evaporated under vacuum. The sample was purified by silica gel chromatography using DCM/MeOH (95:5) as the eluent to obtain PR-TRIS3Bnz as a purple solid (220 mg, 90%). 1H-NMR (400 MHz, DMSO) δ 8.83 (s, 8H), 8.12 (dd, J = 8.7, 2.7 Hz, 8H), 7.64 (d, J = 7.0 Hz, 2H), 7.50 (t, J = 7.4 Hz, 2H), 7.45 – 7.39 (m, 11H), 5.38
(s, 2H), 4.91 (s, 9H), 4.79 (s, 6H), 4.31 (s, 3H), 3.68 (s, 18H), 3.20 (s, 6H), -2.92 (s, 2H). MALDI–ToF-MS: 1252.5 m/z [M+1]. rf = 0.41 (DCM/MeOH 9:1). mp > 300 ºC. 2.3.4.
porphyrin (PR-TRIS3OH) PR-TRIS3Bnz (130 mg, 0.11 mmol) and 25% of palladium on carbon (32.5 mg, 0.30 mmol) were added to a stirred mixture of DCM/MeOH (1:1) (40 mL) at 60 ºC. Air was removed from the reaction mixture using a vacuum pump. Subsequently, a hydrogen balloon was incorporated into the stirred reaction mixture. After 24 h, the reaction was filtered and the solvent was evaporated under vacuum to obtain the final product PR-TRIS3OH as a green solid (98 mg, 82%). 1H-NMR (400 MHz, MeOD) δ 8.69 (m, 8H), 7.84 (dd, J = 17.7, 7.8 Hz, 8H), 7.14 (d, J = 7.3 Hz, 8H), 4.67 (s, 6H), 3.87 (s, 18H), 3.66 – 3.54 (m, 3H). UVVis (MeOH) 419, 523, 560, 602 and 651 nm. MALDI-ToF-MS: 1165.4 m/z [M], HRMS-ES: calc. for C62H63N7O16 1161.4331, found 1162.4404 [M+1]. rf = 0.33 (DCM/MeOH 8:2). mp > 300 ºC.
2.4. Synthesis of ol-IONPs Magnetite nanoparticles covered with oleylamine (ol-IONP) were prepared as described in the literature .
2.5. Synthesis of ZnPR-IONPs ZnPR-COOH (2 mg, 2.4·10-3 mmol) and ol-IONPs (2 mg) were mixed with THF (3 mL) and stirred overnight at room temperature. The resultant precipitate was washed with THF (3 x 1.5 mL) using centrifugation (14,000 rpm, 5 min) to finally obtain 8 mg of a dark brown powder (ZnPR-IONPs).
2.6. Synthesis of PR-TRIS-IONPs ol-IONPs (2 mg) were dissolved in THF (1.5 mL) and the solution was stirred under an argon atmposphere. Next, PR-TRIS3OH (5 mg, 4.3·10-3 mmol) was dissolved in DMSO (1.5 mL) and added to the ol-IONPs solution. The mixture was stirred at 50 °C during 24 h. The resultant mixture was centrifuged (13,000 rpm, 15 min) and the pellet corresponding to unreacted products was eliminated. The supernatant was evaporated under vacuum and the formed solid was dissolved in water. The solution was washed with water (3 x 1.5 mL) via centrifugation (13,000 rpm, 30 min at 4°C), and the final product PR-TRIS-IONPs (precipitate) was redissolved in phosphate buffer solution pH 7.4 (PBS) at 4 °C.
2.7. Detection of singlet oxygen production Singlet oxygen induces the photobleaching of ADPA forming an endoperoxide which can be detected by UV-Vis . 50 µL of ADPA in methanol (1.1 mM) was added to 550 µL of a solution of ZnPR-IONPs in THF. The sample was placed in a quartz cuvette and stirred thoroughly. A light source with a wavelength between 400 and 500 nm was used to irradiate the solution under stirring during 4 hours. A UV-Vis absorption spectrum of the studied sample was recorded every hour in the range of 300-800 nm and the singlet oxygen production was determined by the decrease of the absorption intensity due to the ADPA. The same protocol was used to measure the singlet oxygen production of free ZnPR-COOH (30 µM) in THF, PR-TRIS3OH (40 µM) in H2O/DMSO (95:5) and PR-TRIS-IONPs in H2O/DMSO (95:5).
3. Results and discussion 3.1. Synthesis of the photosensitizers 3.1.1. Synthesis of ZnPR-COOH The hydrophobic ZnPR-COOH was obtained following four steps as described in Scheme
triphenylporphyrin (PR-OH)  was synthesized using the Adler-Rothemund technique reacting benzaldehyde, 4-hydroxybenzaldehyde and pyrrole in propionic acid under reflux . After purification by column chromatography, the porphyrin PR-OH was obtained in a 5.4% yield. In a second step, ethyl 7-bromoheptanoate was linked to the porphyrin PR-OH via the substitution reaction between the phenol residue of PR-OH and the bromine atom, obtaining the compound PR-COOEt. Subsequently, zinc (II) was integrated into the core of the porphyrin to avoid the incorporation of iron cations within the macrocyclic ring during the synthesis of the IONPs. An excess of zinc acetate was added to a solution of PR-COOEt in refluxing DMF. After 16 h the progress of the reaction was monitored by UV-Visible absorption spectroscopy, since the four Q bands from the free base porphyrin are replaced by two of the Zn system, indicating the formation of ZnPR-COOEt. In addition, the 1H-NMR spectrum of ZnPR-COOEt confirms the presence of the zinc atom since the signal corresponding to the two protons of the core of the porphyrin (-2.76 ppm) disappeared (Fig. S1 for PR-COOEt and Fig. S2 for ZnPR-COOEt). As a final step, a saponification of the acid group was achieved in basic conditions resulting in the formation of the target porphyrin ZnPR-COOH. The chemical characterization of the described porphyrins PR-COOEt, ZnPR-COOEt and ZnPR-COOH is given in the Supplementary Material (Fig. S1 to S6). The porphyrin derivative ligand ZnPR-COOH was not soluble in an aqueous medium.
Scheme 1. Stepwise synthesis of porphyrin ZnPR-COOH.
3.1.2. Synthesis of the water soluble porphyrin PR-TRIS3OH To increase the solubility of the functionalized IONPs in aqueous media, a water soluble photosensitizer PR-TRIS3OH consisting of a porphyrin modified with three TRIS groups was synthesized. The hydrophilicity of this porphyrin derivative was achieved by the incorporation of the TRIS groups . PR-TRIS3OH was synthesized following three steps as indicated in Scheme 2. In the first step, a dissymmetrical porphyrin incorporating a phenoxyacetate aldehyde derivative (CHO-Ac) and 4-benzyloxybenzaldehyde was synthesized. CHO-Ac, synthesized following a reported protocol , allows the incorporation of the TRIS groups while 4-benzyloxybenzaldehyde becomes a phenol group in the final synthetic step. The synthesis of the desired porphyrin (PR-Ac3Bnz) was achieved using the synthesized CHO-Ac, 4-benzyloxybenzaldehyde and pyrrole in boiling propionic acid . Three equivalents of CHO-Ac and one equivalent of 4-benzyloxybenzaldehyde were used to favor the formation of the porphyrin PR-Ac3Bnz over the other possible combinations. PR-Ac3Bnz was isolated following two silica gel column chromatographies. TRIS was then bound to the porphyrin by amide bond formation in dimethyl sulfoxide (DMSO) using potassium carbonate as a base, as reported previously for the formation of other amide bonds . Under these conditions, the amine in TRIS attacks the electrophilic carbonyl in the ester groups of PR-Ac3Bnz forming an amide bond and yielding the PRTRIS3Bnz. The synthesized PR-TRIS3Bnz was soluble in polar solvents such as, methanol, DMSO or water. The deprotection of the benzyl group in PR-TRIS3Bnz was achieved via hydrogenation with palladium on carbon , resulting in the ligand PR-TRIS3OH that was isolated as a green solid with a 82% yield. The solubility of PR-TRIS3OH was tested in water, methanol and DMSO demonstrating the high polarity of the synthesized photosensitizer. The chemical characterization of the described porphyrins PR-Ac3Bnz, PRTRIS3Bnz and PR-TRIS3OH is given in the Supplementary Material (Fig. S7 to S13).
Scheme 2. Stepwise synthesis of the water soluble porphyrin PR-TRIS3OH.
3.2. Preparation of the functionalized iron oxide nanoparticles (IONPs) The novel zinc porphyrin (ZnPR-COOH) was used to functionalize the surface of IONPs taking advantage of the capacity of the carboxylic terminal group to be attached to the colloid's surface . In a first step, iron oxide nanoparticles stabilized with oleylamine (olIONPs, Scheme 3) were prepared following the methodology reported by Sun and Zeng . To functionalize the particles with ZnPR-COOH, a solution of the synthesized porphyrin was mixed with a solution of the ol-IONPs in tetrahydrofuran (THF) and the reaction was stirred overnight. A ligand exchange occurs so that the oleylamine ligands are 16
replaced by the ZnPR-COOH ligands yielding ZnPR-IONPs. The synthesized ZnPRIONPs (Scheme 3) were washed three times to remove any unbound ligand.
Scheme 3. Ideal schematic representation of the functionalization of IONPs with: left) ZnPR-COOH yielding ZnPR-IONPs and right) PR-TRIS3OH yielding PR-TRIS-IONPs.
IONPs were also functionalized with the synthesized hydrophilic PR-TRIS3OH ligand (Scheme 3). The immobilization of PR-TRIS3OH onto the surface of the IONPs was based on the exchange of the oleylamines that cover the IONPs surface with the synthesized porphyrin derivative. PR-TRIS3OH binds to the surface of IONPs via the terminal alcohol group of the TRIS . An example of IONPs functionalized with a porphyrin derivative using diol groups has been reported previously by Gu et al.  A solution of the porphyrin derivative PR-TRIS3OH in DMSO was added to a solution of ol-IONPs in THF and the mixture was left reacting at 50 °C. The functionalization of the IONPs with PR-TRIS3OH 17
yielding PR-TRIS-IONPs (Scheme 3) was successfully achieved after 24 h. THF and DMSO were used as solvents because the ol-IONPs were only soluble in THF, and adding the PR-TRIS3OH in DMSO provided a monophasic solution. Any unbound ligand was removed following several washes and via centrifugation. The synthesized PR-TRIS-IONPs were water soluble achieving one of the main objectives of this present work.
3.3. Characterization of the free ligands and of the corresponding functionalized nanoparticles 3.3.1. UV-Visible absorption spectroscopy UV-Vis absorption spectroscopy was used to characterize the synthesized porphyrins (ZnPR-COOH and PR-TRIS3OH) and the corresponding functionalized IONPs, ZnPRIONPs and PR-TRIS-IONPs (Fig. 1). The UV-Vis extinction spectrum of ol-IONPs was also measured and exhibited a broad absorption band with a shoulder at ca. 310 nm (Fig. S14), as observed previously by other authors . The UV-Vis extinction spectra of the free ligand ZnPR-COOH and of the functionalized ZnPR-IONPs were recorded in THF (Fig. 1a). The UV-Vis absorption spectrum of ZnPR-COOH exhibited the Soret band at 424 nm and the two Q bands at 553 and 591 nm as reported in the literature for other porphyrin derivatives . The UV-Vis extinction spectrum of ZnPR-IONPs exhibited the characteristic porphyrin absorption bands (422, 551, 590 nm) albeit slightly shifted from the free ligand, which confirmed the coating of the nanoparticles with the ZnPR-COOH. The blue-shift may indicate the presence of weakly coupled H-type aggregates at the surface of the particle. The UV-Vis extinction spectrum of ZnPR-IONPs was broader than that of the free porphyrin due to the influence of the IONPs. The UV-Vis absorption spectrum of PRTRIS3OH (Fig. 1b) was characteristic of metal free porphyrins, with the Soret band (419 nm) and the four Q bands (523, 560, 602 and 651 nm) . The UV-Vis extinction spectrum
of the PR-TRIS-IONPs (Fig. 1b) exhibited a red-shifted Soret band (427 nm). The presence of the four Q bands in the UV-Vis extinction spectrum of PR-TRIS-IONPs confirmed the attachment of the porphyrin PR-TRIS3OH onto the surface of the IONPs. Furthermore, the presence of these four bands indicated that the ligand on the nanoparticles was the free base porphyrin, since when a metal such as iron is present inside the porphyrin core only two Q bands are observed. The red-shift in the UV-Vis extinction spectrum from the free ligand to the functionalized nanoparticles indicates the formation of a weakly coupled J-aggregate.
1.0 0.00 500
Fig. 1. UV-Visible extinction spectrum of: a) ZnPR-COOH (0.024 mg/mL, black) and ZnPRIONPs (0.1 mg/mL, red) recorded in THF; and b) PR-TRIS3OH (0.046 mg/mL, black) and PRTRIS-IONPs (0.2 mg/mL, red) recorded in H2O:DMSO. Insets: magnification of the corresponding Q band region.
3.3.2. Fluorescence spectroscopy Fluorescence spectroscopy can be used to identify the presence of the two synthesized porphyrins ZnPR-COOH and PR-TRIS3OH on the magnetic IONPs. The fluorescence emission spectra were recorded for the free ZnPR-COOH compound and the coated ZnPRIONPs in THF (Fig. 2a). The fluorescence emission spectra of both samples exhibit two bands at ca. 602 and 646 nm (excitation at 555 nm) as expected for Zn-porphyrin derivatives
. Fluorescence spectroscopy was also used to identify the presence of the porphyrin PRTRIS3OH on the PR-TRIS-IONPs. The fluorescence emission spectra of the free compound PR-TRIS3OH and of the coated PR-TRIS-IONPs in an aqueous solution exhibited two bands at 656 and 718 nm (excitation at 591 nm), as expected for a free metal porphyrin derivative (Fig. 2b) . A small shift of the emission bands maxima was observed when comparing the free ligands with their corresponding IONPs. This shift may be due to the formation of weakly coupled aggregates on the surface of the functionalized IONPs. These experiments also demonstrate that the fluorescence emission of the photosensitizer is still observable when the porphyrin is linked to the IONPs. The fluorescence excitation spectra of all of the samples are given in the Supplementary Material (Fig. S15) and show similar spectra to the UV-Visible absorption spectra previously described.
b) Fluorescence Emission (a.u.)
Fluorescence Emission (a.u.)
400 350 300 250 200 150 100 50 0 650
Fig. 2. Fluorescence emission spectrum of a) ZnPR-COOH (0.024 mg/mL, black) and ZnPR-IONPs (0.4 mg/mL, red) recorded in THF (λexc = 555 nm); and b) PR-TRIS3OH (0.046 mg/mL, black) and PR-TRIS-IONPs (0.2 mg/mL, red) recorded in H2O:DMSO (λexc = 591 nm).
3.3.3. Transmission Electron Microscopy The size and the morphology of the ol-IONPs were characterized by TEM. Fig. 3a and b show TEM images of ol-IONPs, highlighting their uniform size distribution and relatively 20
low polydispersity. The corresponding size distribution histogram is given in Fig. 3c. The diameter of ol-IONPs was ca. 7 nm, which is in agreement with a previous study . The morphology of the ol-IONPs was diverse, since spherical, hexagonal and cubic shapes can be identified in a sample of ol-IONPs. The nanoparticles are well organized, showing a mosaiclike distribution. Furthermore, no aggregation was observed indicating a good coating of the metal oxide with the oleylamine capping ligand.
40 35 30 25 20 15 10 5 0 4
Diameter size (nm) Fig. 3. a) and b) TEM images of ol-IONPs; and c) size distribution histogram of ol-IONPs (n = ca. 130). Scale bar in a) = 100 nm and in b) = 20 nm.
3.3.4. Scanning tunneling microscopy A sample of ZnPR-IONPs was analyzed using STM and topographic images of the nanoparticles were obtained (Fig. S16). Although the resolution of the images is not high, the 21
morphology and the size of the nanoparticles can be observed. Especially in the topographic phase, spherical nanoparticles can be observed with diameters between ca. 5 and 8 nm, in accordance with the size obtained by TEM.
3.3.5. X-ray photoelectron spectroscopy XPS was used to determine the valence state of different characteristic atoms (Zn and Fe) of the functionalized ZnPR-IONPs. The XPS spectrum of the ZnPR-IONPs (Fig. S17) exhibited a peak at ca. 1020 eV due to the Zn2p3/2, indicating the presence of the ZnPRCOOH . The peaks corresponding to other atoms present on the system such as N, Fe, O or C were also identified. In addition, Fig. S17 shows two characteristic peaks of the formation of the iron oxide nanoparticle, resulting from Fe2p3/2 (706 eV) and Fe2p1/2 (720 eV) electron binding energies. The binding energies of these characteristic peaks are similar to the values reported in the literature for iron oxide nanoparticles . XPS measurements were also attempted with the PR-TRIS-IONPs. However, no conclusive results were obtained since the porphyrin derivative PR-TRIS3OH does not include any other atom other than the C, O and N.
3.3.6. Zeta potential Zeta potential measurements were performed to study the differences in the surface charge between the IONPs functionalized with oleylamine and the IONPs functionalized with the porphyrin derivative ligand PR-TRIS3OH. Untreated ol-IONPs presented a weakly negative zeta potential (-3.22 mV), due to the presence of the oleylamine that covers the iron oxide nanoparticle surface. The influence of the alkyl chains and of the amino groups of the oleylamine should give a charge density close to zero. When the porphyrin PR-TRIS3OH was attached to the surface of the nanoparticles yielding PR-TRIS-IONPs, the zeta potential
of the functionalized nanoparticles was measured to be -22 mV. The negative zeta potential of PR-TRIS-IONPs can be explained by the influence of the hydroxyl groups of TRIS which, at pH 7, should have a negative charge density.
3.3.7. Singlet oxygen production One of the aims of the research here described was to synthesize novel nanosystems that could be applied for PDT of cancer. To achieve this aim, the porphyrin functionalized IONPs should be able to produce significant levels of singlet oxygen when exposed to a light of a specific wavelength. The production of singlet oxygen by the synthesized porphyrin derivative ligands and the respective functionalized iron oxide nanoparticles was examined using the molecular probe 9,10-anthracenedipropionic acid (ADPA). In the presence of single oxygen, the anthracene in the ADPA is converted to an endoperoxide . This change in the structure of the ADPA can be easily followed using UV-Vis absorption spectroscopy since, due to photobleaching, a decrease in the intensity of the characteristic absorption bands of the anthracene moiety between 300 – 400 nm is observed following production of singlet oxygen (Fig. S18). This experiment consisted of the irradiation of the photosensitizer (free or bound to the surface of the IONPs) in the presence of ADPA with a blue light source for 4 hours with continuous stirring. The UV-Vis absorption spectrum of the solution was recorded every hour. Two control experiments were also performed to investigate: 1) the degradation of ADPA without the photosensitizer following irradiation and 2) the behavior of ol-IONPs upon irradiation. A blue light source with a wavelength between 400 and 500 nm was used to excite the Soret band region (near 425 nm) of the porphyrins. To compare all of the studied samples, the percentage of APDA photobleaching for each of the samples was calculated based on the ADPA decay at 378 nm (Fig. 4a). Irradiation of the samples containing either the free porphyrin derivative ligands ZnPR-COOH (Fig. 4a – green) and PR-TRIS3OH
(Fig. 4a – cyan) or the functionalized nanoparticles ZnPR-IONPs (Fig. 4a – blue) and PRTRIS-IONPs (Fig. 4a – pink) show a photobleaching of the ADPA caused by the singlet oxygen produced by the samples. However, when a control sample containing ol-IONPs (Fig. 4a – red) was irradiated in the presence of ADPA, negligible photobleaching of the singlet oxygen probe was observed. These results confirm the need of the photosensitizer porphyrin derivatives to be able to produce singlet oxygen upon irradiation. A final control experiment was performed irradiating a solution of ADPA (without porphyrin) under the same conditions (Fig. 4a – black). As for the ol-IONPs, the production of singlet oxygen by the ADPA probe was negligible. To compare the ability of the free ligands ZnPR-COOH, PR-TRIS3OH and of the corresponding functionalized IONPs to produce singlet oxygen, the maximum rate of ADPA photobleaching for each sample was calculated and compared. The maximum rate of ADPA photobleaching was calculated taking into account the concentration of photosensitizer present in each of the samples using (Equation 1).
Maximum rate of ADPA photobleac hing
(% Abs378 at t 0 min) (% Abs378 at t 60 min) 60 min porphyrin (mM )
Equation 1. Equation used to calculate the maximum rate of ADPA photobleaching.
% ADPA Decay
90 80 70 60 50 40 0
% ADPA Photobleaching Decay
H OO C PR
NP IO IS
Fig. 4. a) Percentage decay of ADPA photobleaching measured at 378 nm upon irradiation (400 – 500 nm) of: ADPA (black line), ol-IONPs (red line), ZnPR-COOH (green line), ZnPR-IONPs (blue line), PR-TRIS3OH (cyan line) and PR-TRIS-IONPs (pink line); and b) % rate of ADPA photobleaching of the synthesized porphyrin derivative ligands and of the corresponding capped iron oxide nanoparticles calculated using the concentration of photosensitizer present.
To calculate the concentration of ZnPR-COOH anchored to the surface of the ZnPRIONPs, the contribution of the ligands to the extinction intensity of the sample should be calculated and the extinction coefficient of the free ligand ZnPR-COOH has to be known. The first parameter was calculated by recording the UV-Vis extinction spectrum of the nanoparticles solution (Fig. S19a) and then determining the contribution of the ligands to the total extinction intensity. The extinction intensity at 554 nm due to the ZnPR-COOH ligands was calculated subtracting the extinction intensity at 580 nm, due to the IONPs, from the total extinction intensity at 554 nm due to both the IONPs and the ZnPR-COOH ligand as indicated in Fig. S19a. The extinction coefficient of the free ligand ZnPR-COOH at 554 nm was determined from the calibration curve, shown in Fig. S20, obtained by measuring the UV-Vis absorption spectrum of samples of ZnPR-COOH of known concentration (from 0 to 0.05 mM). The extinction coefficient of ZnPR-COOH at 554 nm was estimated to be ε554nm = 5.4808 x 103 M-1 cm-1. Using the Beer Lambert Law and the calculated parameters, the concentration of ZnPR-COOH on the surface of ZnPR-IONPs was estimated to be 15.3 µM (Fig. S21). This concentration yielded a maximum rate of ADPA photobleaching following irradiation for the ZnPR-IONPs of 20.5% Abs/(min·mM) (Fig. 4b). The maximum rate of ADPA photobleaching following irradiation of a solution 0.03 mM of the free ligand ZnPRCOOH was 8.5% Abs/(min·mM). These results show that the maximum rate of ADPA photobleaching following irradiation is higher when the porphyrin derivative ligand is attached to the surface of the iron oxide nanoparticles (Fig. 4b). As explained for ZnPR-IONPs, the contribution of PR-TRIS3OH ligands to the UV-Vis extinction intensity of PR-TRIS-IONPs at 418 nm was calculated subtracting the extinction intensity at 458 nm, due to the IONPs, from the total extinction intensity at 418 nm due to both the IONPs and the PR-TRIS3OH ligands (Fig. S19b). The extinction coefficient of the free ligand at 418 nm was calculated using the same calibration curve used for the thiolated
porphyrin derivative recently reported ε418nm = 3.4 x 104 M-1 cm-1 , since the free metal porphyrins present a similar extinction coefficient . Using the Beer Lambert Law and the reported parameters, the concentration of PR-TRIS3OH on the surface of PR-TRIS-IONPs was estimated to be 16.5 µM (Fig. S21). The corresponding maximum rate of ADPA photobleaching following irradiation for the PR-TRIS-IONPs was calculated to be 19.4% Abs/(min·mM). The solution of free ligand PR-TRIS3OH used to measure the singlet oxygen production had a concentration of 0.04 mM which corresponds to a maximum rate of ADPA photobleaching of 7.5% Abs/(min·mM). As for the ZnPR-IONPs, the production of singlet oxygen by the porphyrin derivative ligand PR-TRIS3OH is lower for the free ligand than that for the ligand anchored to the surface of the nanoparticles (Fig. 4b). All of the calculated maxima rate of ADPA photobleaching upon irradiation are compared in Fig. 4b. The highest value of maximum rate of ADPA photobleaching, directly related to the singlet oxygen production, was obtained for the ZnPR-IONPs (20.5%). PR-TRISIONPs present a slightly lower maximum rate of ADPA photobleaching (19.4%) implying a lower singlet oxygen production than ZnPR-IONPs, although these latter measurements were performed in a water/DMSO solvent mix. However, the values are similar and PRTRIS-IONPs have the advantage of being stable in aqueous solutions while the experiments with ZnPR-IONPs had to be performed in THF, which is not compatible with the biological environment. The two free porphyrin derivative ligands had also a very similar maximum rate of ADPA photobleaching, ZnPR-COOH (8.5%) and PR-TRIS3OH (7.5%), and therefore, exhibit a similar ability to produce singlet oxygen. The data presented in Fig. 4b also show that the singlet oxygen production of the free ligands is lower than that of the corresponding functionalized iron oxide nanoparticles. Thus, it has been demonstrated that the anchoring of the ligands onto the surface of IONPs notably increases the capacity of the photosensitizer to generate singlet oxygen, making these nanosystems promising for PDT.
Conclusions Two porphyrin derivatives, a zinc porphyrin derivative containing a carboxylic alkyl chain (ZnPR-COOH) and a water soluble porphyrin containing three polar groups (TRIS) (PRTRIS3OH), were synthesized and used to functionalize the surface of iron oxide nanoparticles yielding two novel nanosystems, ZnPR-IONPs and PR-TRIS-IONPs, respectively. The free ligands and the corresponding functionalized IONPs were characterized using a variety of spectroscopic techniques including UV-Vis absorption spectroscopy and fluorescence spectroscopy confirming the presence of the corresponding porphyrin derivatives on the surface of the nanoparticles. ZnPR-IONPs were not soluble in water making them non-ideal candidates for biological applications. However, the immobilization of PR-TRIS3OH to IONPs produced water soluble and thus biocompatible PR-TRIS-IONPs. Singlet oxygen production experiments were performed on the synthesized ligands and on the corresponding nanoparticle conjugates measuring the maximum ratio of ADPA photobleaching following irradiation with blue light. The free ligands ZnPR-COOH and PR-TRIS3OH produced similar levels of singlet oxygen which were considerably lower than those produced by the corresponding functionalized nanoparticles. The levels of singlet oxygen produced by ZnPR-IONPs and PR-TRIS-IONPs were also similar indicating that both systems were able to produce reactive oxygen species. These experiments have confirmed the capability of the synthesized novel porphyrin derivative ligands to produce singlet oxygen, showing an important increase, in terms of singlet oxygen production, when the porphyrins were bound to the IONPs. Thus, the designed porphyrin functionalized IONPs are potential good candidates for use in PDT, especially PRTRIS-IONPs with regard to their solubility in aqueous media.
Acknowledgements This work was supported by MINECO, Spain (projects CTQ2010-16339, TEC201129140-C03-02 and TEC2014-51940-C2-2). O.P. acknowledges the MICINN for a predoctoral grant. M.J.M. was funded by the School of Chemistry, University of East Anglia.
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Iron oxide nanoparticles functionalized with novel hydrophobic and hydrophilic porphyrins as potential agents for photodynamic therapy Oriol Penon,a,b María J. Marín,c David B. Amabilino,d,1 David A. Russell, c Lluïsa PérezGarcíaa,b,*