Silica nanoparticles functionalized with porphyrins

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Journal of Porphyrins and Phthalocyanines

Published at http://www.worldscinet.com/jpp/

J. Porphyrins Phthalocyanines 2011; 15: 517–533 DOI: 10.1142/S1088424611003653

Silica nanoparticles functionalized with porphyrins and analogs for biomedical studies Flávio Figueira, José A.S. Cavaleiro and João P.C. Tomé* Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal J. Porphyrins Phthalocyanines 2011.15:517-533. Downloaded from www.worldscientific.com by UNIVERSITY OF CALIFORNIA SAN DIEGO on 10/01/14. For personal use only.

Dedicated to Professor Karl M. Kadish on the occasion of his 65th birthday Received 11 May 2011 Accepted 15 June 2011 ABSTRACT: This review focus on the preparation of silica nanoparticles functionalized with porphyrins and related compounds. It is aimed to highlight their features as photosensitizers in the area of photodynamic therapy. In this field, photosensitizers have been covalently and non-covalently linked to silica nanoparticles, in order to study their photophysical and biological properties. Another fascinating scenario for photosensitizer-silica nanoparticles hybrids involves the possibility of including metal cores for conditioning the uptake in the target cells, allowing most of the times the combination of therapies and in certain conditions to facilitate the removal and reutilization of the photosensitizer in environmental applications. KEYWORDS: porphyrins, phthalocyanines, photosensitizer, nanoparticles, silica, ORMOSIL, mesoporous, nanomagnet, theranostic.

INTRODUCTION It is well-known that porphyrin (Por) derivatives play important roles in living organisms. The vital properties (respiration, photosynthesis, drug detoxification and others) have promoted an inexhaustible study on that type of pigments. Also, Por and phthalocyanines (Pc), their synthetic analogs, have been intensively studied due to their potential applications in a number of scientific areas, namely in medicine, in catalysis, as biomimetic model systems of the primary processes of natural photosynthesis, and as materials for nanotechnologies, especially associated with nanomaterials [1–3]. Cancer is a major public health problem for humans, being the second leading cause of death, just behind the heart diseases. Despite several progresses in order to reduce mortality rates caused by cancer, globally no significant decrease has been observed, at least comparing with other diseases [4]. This, in part, explains SPP full member in good standing *Correspondence to: João P.C. Tomé, email: [email protected], tel: +351 234-370-342, fax: +351 234-370-084

the extraordinary efforts to develop new diagnostic and treatment technologies. In medicine, Por and 5-aminolevulinic acid (ALA), a precursor of endogenous PS protoporphyrin IX (Pp IX) have been used as photosensitizers (PS) in photodynamic therapy (PDT). PDT is an emerging methodology for the treatment of a large variety of tumors and age-related macular degeneration, and is also promising for the treatment of cardiovascular and infectious diseases [5–9]. PDT combines three components: a PS, visible light and oxygen, able to generate reactive oxygen species that can induce target cells death. Compared to current cancer treatments (surgery, radiation therapy and chemotherapy), PDT offers the advantage of an effective method for the treatment of early diagnosed and localized carcinoma, without damaging the surrounding healthy tissues (both the photosensitizer and light can be pratically directed to the targets to be treated) [5]. Among the responses to photodamage initiated by PDT are the necrosis, apoptosis and autophagy, however it is still unclear which of these responses are the dominant mechanism of cell death. The efficacy of PDT in tumor treatment depends on various factors: the PS molecule,

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its subcellular localization at the time of irradiation, the dose of irradiation and its wavelength, the time between PS administration and irradiation, the sensitivity of tumor cells and tumor oxygenation level [10]. Amongst the various types of PS used in PDT, Por and prodrug ALA are the most extensively studied and are the basis for the approved compounds in clinical use. However, it must be noted that even the most widely used formulation (Photofrin®) is far from being an excellent PS. It consists of mixtures of oligomeric species, with skin phototoxicity and poor light absorbance properties in the red region of the spectrum where light penetrates deepest into tissues [7]. To surpass this drawback, a second generation of PDT agents, based on chlorins [11] and Pc [12] have been prepared. These new PS have a high absorbance in the red region of the electromagnetic spectrum, facilitating both phototherapy and imaging deeper into tissues. However, both Por and Pc macrocycles do not have proper biocompatibility requirements, mainly solubility in physiological fluids. Approaches using different complex delivery formulations, such as incorporation into liposomes [13], biopolymers [14] or cyclodextrins [15] and more recently into dendrimers [16] have been explored. In fact, the different photodynamic activities may be due to their different extent of cellular uptake and aggregation tendency. Knowing that, the development of more effective PDT agents has been accomplished by appending second generation PS with biomolecular recognition motifs, such as carbohydrates [17], amino acids [12], and monoclonal antibodies [18]. The later strategy allows the preparation of the so-called third generation PS, where the antibody moieties target specific cancer cells receptors. However, this third generation PS did not show the desired in vivo efficacy [19]. To overcome it, mainly the PS uptake issues, and to get a higher PDT efficiency, biocompatible nanoparticles (NP) have been studied and as a result are opening new frontiers for PDT [20–22]. In medicine, some NP open the possibility to build smart materials by the combination of their unique properties with biologically active molecules. The special interest on this type of nanomaterials, in many areas of the nanosciences and nanotechnologies, has been described in several comprehensive reviews [23–29]. NP have been used to improve the pharmacokinetics and pharmacodynamics of many drugs. These two key elements, for an efficient treatment, can be fine-tuned by different factors of these nanoentities, such as size, shape, morphology and surface functionalization. Amongst the several NP studied for biomedical applications, such as gold [30] and silica nanoparticles (SNP) [20, 21], the latter ones have been received special attention. In the PDT area, SNP involvement was already reviewed but it focused only on PS entrapped inside silica matrix [20]. Amorphous silica is a stable biocompatible material, which is also chemically and biologically resistant, and thus compatible with most formulations for medical Copyright © 2011 World Scientific Publishing Company

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applications. Several methods are available to prepare different silica-based materials, allowing covalent and non-covalent immobilization of almost every PS, thus preserving the PS photodynamic properties. Furthermore, SNP show other glass properties, such as permeability and optical transparency, which are important for controlled release and photo-activation of the PS [23, 24]. Highly explored during the last decade, silica-coated metal NP combines the unique properties of the metal NP with the ones of the silica. The silica layer around the metal NP forms a shell; most of the times it allows a richer surface chemistry, a higher biocompatibility and transparency. Drugs can be encapsulated in the covered silica or covalently bonded to different grafted active groups on the surface [26, 31, 32]. One of the metal NP properties that can be further explored is the magnetic behavior [27, 32]. Multi-functional silica-coated magnetic NP have been explored for several applications, mainly in medicine, catalysis and environmental studies [33–35]. The silica shell prevents the intrinsic tendency to aggregate and to oxidize, and allows their functionalization. Following these discussions, the incorporation of Por and related PS in and on SNP, using different strategies, is revised.

DIFFERENT SILICA NANOPARTICLES There are many different shape and size SNP, that have been used in combination with several active molecules [24, 26, 29]. One of the first methods describing SNP with a relatively spherical shape and a smooth surface, were presented by Stöber in 1963. Stöber prepared spherical silica particles in the range of 0.05–2 µm through the hydrolysis and condensation of alkyl silicon in alcoholic solutions under alkaline conditions [36], or in another terms a sol-gel reaction under basic conditions. The solgel process is a wet-chemical technique widely used in the fields of materials science and ceramic engineering and was used primarily for the fabrication of materials (typically a metal oxide) starting from a chemical solution which acts as the precursor for an integrated network of either discrete particles or network polymers. Typically, to prepare SNP the silicon precursors suffer hydrolysis and polycondensation reactions to form a colloidal suspension. Consequently, many researchers have developed this area in order to prepare different NP sizes [37–39]. Modified Stöber syntheses have been developed in recent years allowing non-covalent (I) and covalent encapsulation of several fluorescent dyes in (II) and on (III) the SNP (Fig. 1), for a wide range of applications [40]. While physical encapsulation is achieved when dye is present in the reaction mixture of the SNP, the covalent bound can be achieved by dye silicon derivatives, previously prepared. However, dye can be incorporated during and post-synthesis of the SNP. J. Porphyrins Phthalocyanines 2011; 15: 518–533

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SILICA NANOPARTICLES FUNCTIONALIZED WITH PORPHYRINS AND ANALOGS FOR BIOMEDICAL STUDIES

Fig. 1. Different revised SNP with dyes

A second SNP generation, based on organically modified silica (ORMOSIL), also prepared by sol-gel protocols, has been developed for entrapping dyes in the inner porosity of the silica-based matrix [41]. In general, this nanoparticles are synthesized by alkaline hydrolysis and polycondensation of organotrialkoxysilane precursors within the nonpolar core of Tween-80/water microemulsion [41]. The nanoencapsulation of dyes in SNP prepared with this method can be achieved by non-covalent and covalent approaches. Alternatively to sol-gel microencapsulation described above, the direct microemulsion (oil in water) procedure leads to mesoporous SNP (MPSNP). The preparation of this type of silica combines the sol-gel process of silicon oxides with cationic surfactants, which act as template agents to form the ordered structures. These SNP contain hundreds of empty channels, with a large surface area (z 1000 m2.g-1) and high volume, which can be used to non-covalently (IV) and covalently (V) load active drugs (Fig. 1). Typically, they have ordered hexagonal pores with a mean particle diameter of 70–100 nm and uniform pore diameters (z 3 nm) [42, 43]. In the synthetic procedure the surfactant CTAB, at low concentrations, was initially dissolved in a basic aqueous solution and the mixture vigorously stirred at high temperature. After addition of TEOS and further reaction for 2 h, treatment with acid wash or calcination to remove the organic surfactant furnished a silica framework that may have an ordered hexagonal, disordered, or cubic pore Copyright © 2011 World Scientific Publishing Company

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structure, depending upon the specific synthetic conditions. Recently, much effort has been focused on preparing the organic/inorganic hybrids through functionalization of the interior and/or the exterior of the nanoparticles, controlling the particle morphology. The most popular way of covalently functionalize MSNP is by grafting the nanoparticles post-synthesis with organotrialkoxysilanes. The reaction takes place between the silanol groups on the surface of the silica and the organoalkoxysilanes. Unfortunately, it has been found that materials functionalized via this grafting method contain an inhomogeneous surface coverage of organic functional groups. Another common method for synthesizing organically functionalized mesoporous silica materials is the co-condensation method. This functionalization method is a direct synthesis method, in which the organoalkoxysilane is introduced to the basic, aqueous CTAB and TEOS solution during the condensation. With this synthetic approach, it is possible to control the morphology of the particles by the addition of functional co-condensing reagents. Silica-coated metal nanoparticles can be formed by amorphous or MP silica, using the methods described above in the presence of metal nanoparticles. Both strategies allow non-covalent encapsulation (VI) and entrapping (VII) dyes (Fig. 1) around several metal cores, such as iron oxide and NaYF4 nanocrystals [25, 33, 35]. Dyes covalently graphted on the surface of these nanoparticles (VIII) have also been described (Fig. 1). The success of such hybrids prompts the application of these materials in separation, sensor design, catalysis, theranostic, etc.

PORPHYRIN AND PHTHALOCYANINE DERIVATIVES IN SILICA NANOPARTICLES Currently SNP incorporating supramolecularly or covalently bonded PS have been successfully included in PDT with tetrapyrrolic macrocycles as PS for improving the cancer therapy through a combination of enhanced drug delivery and selectivity towards cancer cells. The first attempts to immobilize porphyrins in silica were performed by the Stöber procedure [36], where the PS was non-covalently immobilized inside pure SNP by the sol-gel process [44]. The sol-gel methodology is a compatible framework for the immobilization of different compounds, including porphyrins and metalloporphyrins. This process is an efficient co-condensation one to afford monodisperse SNP and it is of particular interest for designing catalytically active and biologically compatible materials. J. Porphyrins Phthalocyanines 2011; 15: 519–533

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These particles can be prepared with the desired size, shape, and porosity, and are extremely stable. Their ultralow size (less than 50 nm) avoids capture by the reticuloendothelial system, they are not vulnerable to microbial attack, and protect doped molecules (enzymes, drugs, PS) against denaturation induced by extreme pH and temperature values. Also they are nontoxic, chemically inert and optically transparent. Non-covalent encapsulation in silica nanoparticles

HO -

O


One of the main goals in the development of drugs for PDT is the design of molecules/materials, which specifically can target the tumor cells and not the healthy ones, with a rapid cellular uptake. This specificity can be achieved by incorporating certain bio-compatible subunits on the active molecules or tailor-made nanomaterials capable to carry and to delivery efficiently the PS to the target cells. This is shown with the successful loading in SNP of meso-tetrakis(m-hydroxyphenyl) chlorin (mTHPC (1), Fig. 2) in 2003 by Yan and coworkers; it generates singlet oxygen while the photosensitizer effect is maintained [44]. In fact, the authors have shown that the singlet oxygen production by the SNP (4.8 × 108 M-1.s-1) obtained by this method exceeds the one from the free mTHPC (2.7 × 108 M-1.s-1). The entrenching of 1 inside SNP was at the time accomplished by a modified Stöber sol-gel process, in which 3-aminopropyltriethoxysilane (APTES) is introduced during the reaction. In the range of SNP used in PDT, some of the most extensively studied PS are the protoporphyrin IX (2, Pp IX) and its derivatives. Pp IX is a naturally occurring porphyrin constituent of hemeproteins and other biologically relevant molecules. Following the interest in this PS, Thienot et al. [45] obtained a silica hybrid carrier with phosphate bilayer coatings NP IX, where Pp IX is physically entrapped in the SNP (Scheme 1). The synthesis of this nanocarrier was achieved under the micellar system Tween 80 and 1-butanol in distilled

NH

N

N

HN

OH

Sol-gel process

P

OH

HO -

P

OH

O O O O + Na+ Na+ NaO O OSi Si Si O O O O

O

HO 2

= Pp IX (2)

IX

Scheme 1.

water. Further dissolution of 2 in DMF and addition to the previously prepared system along with the addition of VTES afforded the silica-based nanocarriers after 20 h of reaction and purification over dialysis. The SMTP layer was obtained by reacting the silica-based nanocarriers with SMTP in aqueous suspension under mild conditions. Some of the advantages obtained with the method implemented by these authors were the size of the silicabased nanocarrier within the nanometer scale from 10 to 200 nm by adjusting the temperature and the co-surfactant amounts. These nanocarriers have also revealed enhanced stability upon aging in mouse serum media and the ability to produce reactive oxygen species up to 12 h. This last advantage was confirmed by in vitro assays against HCT-116 cells, where the phototoxicity of nanocarrier IX was noticeable when compared with the nanocarrier without the PS 2. Qian et al. [46] and Simon et al. [47] synthesized and studied the properties of Pp IX incorporated in colloidal mesoporous SNP X and in different sized SNP XI (Fig. 3), respectively. In both cases they used a modified sol-gel protocol using a combination of organically modified silica (ORMOSIL) to prepare the SNP. This methodology has opened the door towards the design and synthesis of PS-embedded nanoplatforms, which can be used for the delivery of PS to target cells. H2N

OH

NH2

10 nm

NH2

H 2N

NH2 H2N

HO

N NH

NH2

H2N

HN

H2N H2N

N H

OH

25 nm

NH2 NH2

NH2

X

60 nm

H H H

= Pp IX (2)

1 HO

Fig. 2. meso-tetrakis(m-hydroxyphenyl)chlorin 1 Copyright © 2011 World Scientific Publishing Company

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XI

Fig. 3. Silica nanoformulations with Pp IX (2) J. Porphyrins Phthalocyanines 2011; 15: 520–533

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The incorporation of Pp IX in ORMOSIL NP also contributed to the formulation of colloidal mesoporous SNP X and different sized SNP XI. This alternative method combines APTES and vinyltriethoxysilane (VTMS) as precursors. In vitro PDT studies of nanocarriers X, performed on HeLa cells by a 532 nm light source irradiation at a fluence rate of 2 mW.cm-2, have shown to be effective in cell destruction upon 8 min of irradiation. Simon et al., with the synthesis of three different sized Pp IX encapsulated SNP XI, have shown that the internalization of SNP is dependent on the type of cells and not by the NP size as expected from previous studies [48, 49]. The exact mechanism of internalization remains unknown although the authors suggested a passive internalization with accumulation in the cytoplasm of cells. This type of mechanism is coherent with the absence of effect of NP size on cell uptake [47]. In vivo studies with these NP on nude-tumor-bearing mice have shown to be in agreement with the previous results, for the three cancer models studied, high tumor uptake of Pp IX SNP was observed, but maximal accumulations were reached at different times: 2 h for gliobastoma, 16 h for A549 and 20 h for HTC cells. This shows that the accumulation in tumor was, at least partially, a passive process due to the known tumor vessel characteristics like the enhanced permeability and retention effect. These NP have revealed efficient accumulation within tumors, and these results can be further adjusted in case of NP X where the surface of these NP can be grafted with appropriate functionalized groups and conjugated with certain biomolecules for specific targeting. Other porphyrin derivatives like 2-devinyl-2-(1hexyloxyethyl)pyropheophorbide (3, Fig. 4) have been also entrapped into SNP by ORMOSIL protocol [50]. Nanocarrier loaded with 3 was synthesized in the nonpolar micelle core of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/1-butanol/water micelles. Removal of the O

N

NH

HN

N

3 HO

O

O

N

O O 4

Fig. 4. Pyropheophorbide 3 and anthracene 4 Copyright © 2011 World Scientific Publishing Company

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surfactant and co-surfactant by dialysis has afforded the ultrafine ORMOSIL NP with a mean diameter of 30 nm. These NP have been used in phototoxicity studies against UCI-107 and HeLa cells exhibiting significant death features upon irradiation at 650 nm, as analyzed by the MTT assay. These characteristics and the resistance of the SNP loaded with 3 to aggregate, normally observed for 3 alone in polar solvents, gives the possibility to exploit this nanoformulation for imaging of biological systems. This same PS was latter imbibed in ORMOSIL SNP along with a TPA (two photon absorption) dye, anthracene 4 (Fig. 4), under similar methods to the ones previously described [51]. This combination aimed to explore the energy-transferring combination of an existing photosensitizer with TPA dyes. The photosensitizing unit (3, the energy acceptor) is indirectly excited through fluorescence resonance energy transfer from the TPA absorbing dye unit 4. The size of the SNP doped with PS and TPA dye (1.1 wt.% of 3 and 20 wt.% of 4, Fig. 4) and the TPA dye alone (20 wt.%, 4) were around 25 and 30 nm, respectively. Two-photon laser scanning fluorescence spectroscopy demonstrated that the co-encapsulated NP was actively uptaken by HeLa tumor cells in vitro, having a preferential localization in their cytoplasm. The cytotoxic effect of these NP was also verified under twophoton irradiation at 850 nm, and drastic changes in the cells morphology were observed when compared with the NP loaded with 4 in the absence of 3. These changes are associated with cell necrosis induced by the reactive oxygen species generated by the PS, which is excited as a result of two-photon excitation of 4. This is in complete accordance with the fact that the TPA dye 4 encapsulated with the PS 3 was efficiently converting the energy of the near-IR light to excite the PS 3. This was proved with excitation at 532 nm in D2O where the characteristic 1O2 emission at 1270 nm was observed under photoexcitation of the NP co-encapsulating 3 and 4. Recently the same author making use of a new formulation for photodynamic therapy imbibed the same PS 3 in SNP, which was composed of covalently iodinated ORMOSIL SNP (diameter < 30 nm) [52]. Comparative studies with iodinated silica and non-iodinated silica matrixes NP has demonstrated that the iodinated ones significantly enhances the efficiency of 1O2 generation in about 1.7 times. The in vitro PDT efficiency carried out on RIF-1 tumor cells also have shown that the iodinated O NP were more efficient in PDT when comO pared with the non-iodinated NP. Another second-generation PS incorpoN rated in ORMOSIL SNP for in vitro delivery to cancer cells was chlorin 1 (mTHPC, Fig. 2) [53]. The encapsulation of 1 was accomplished by the same method used J. Porphyrins Phthalocyanines 2011; 15: 521–533

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by Roy et al. [37] to capture PS 3. The NP obtained by entrapping 1 (approximately 33 nm diameter) in a monomeric form, delivered singlet oxygen with high efficiency and a drug loading around 1%. The cellular uptake, localization and phototoxic activity of NP loaded with 1 was determined in human esophageal cancer cells (KISE 510) after its delivery by the NP. The phototoxicity of increasing concentrations of 1, delivered by the nanoparticles after 24 h of incubation followed by irradiation with a light dose of 0.12 J.cm-2, has displayed a significant cell viability reduction with dosages of 0.2 µM. Increasing the concentration of 1 to 1.25 µM led to complete loss of cell viability. Unexpectedly the variations observed in cell viability with nanoparticles containing 1 when compared with 1 alone has shown similar PDT results. Fluorescence resonance energy transfer measurements, using NP with 1 physically entrapped and ultracentrifugation experiments suggested that 1 is leached from NP to the media when serum proteins are present. This can explain why the viability assays with these nanoparticles has given such a similar result as 1 alone. However, the coating of the NP surface with poly(ethylene glycol) largely prevented the leaching of the PS to serum proteins. Phthalocyanines, have also been entrapped in SNP by ORMOSIL protocol. Pc 4 (5) encapsulated into SNP preserves their photodynamic activity and provides a basis for future conjugation with targeted molecules as shown by Zhao and coworkers (Fig. 5) [54]. In fact, encapsulation of 5 in ORMOSIL SNP using AOT surfactant instead of Tween 80 led to NP with 25–30 nm of diameter, with enhanced aqueous solubility, stability and PDT efficiency when compared to the free 5. Cell viability studies against A375 melanoma cells showed that the SNP encapsulating 5 were more phototoxic in the MTS assay than 5 with a IC50 of 5 and 30 µM, respectively. The in vitro imaging studies and subcellular localization after incubation for 2 h and excitation at 633 nm, the A375 and BF16F10 melanoma cells treated with the NP have shown strong fluorescence indicating a substantial uptake. In addition 5 was largely diffused throughout the cytoplasm while the NP have localized themselves preferentially in the

O

lysosomes, suggesting that SNP encapsulation enhances lysosomal accumulation. Covalent encapsulation in silica nanoparticles Since the release of the PS drugs is not a prerequisite for their therapeutic action in PDT, and that a premature PS release can probably give side-effects, to overcome this difficulty Rossi and coworkers [55] have strategically modified Pp IX with an organosilane reagent to get 6 (Fig. 6), assuring its covalent attachment to the inorganic framework. The preparation of the SNP with this synthon 6 was obtained through a modified Stöber sol-gel process [56]. This process has revealed to be an excellent methodology to obtain high loadings of PS in the SNP with an increased potential to perform PDT when compared with the free PS. In fact, while the PS is bonded covalently to the silica matrix, it can be excited by irradiation to generate 1O2 that diffuses to the solution with an efficiency of 1 O2 delivery higher than the 1O2 quantum yield of the free PS [57]. Based in this same model, Ohulchanskyy and coworkers [41] have reported the synthesis of a novel ORMOSIL SNP of ultralow size (z 20 nm), highly monodispersed and stable in aqueous suspension by covalently incorporated iodobenzylpyropheophorbide derivative 8 (Scheme 2). The highly monodispersed aqueous ORMOSIL SNP, with covalently linked iodobenzylpyropheophorbide 7, was synthesized upon co-precipitation of iodobenzyl-pyrosilane 8 with the commonly used ORMOSIL precursor VTES with different molar ratios in respect to 8 (1:0, 1:1, 1:2, 1:4) in the nonpolar core of Tween-80/water microemulsion. This provided four different nanoformulations whereas the size of the nanoformulation increases with the increased amount of VTES, and iodobenzyl-pyrosilane derivative 8 is getting diluted with the increasing NP size. Further in vitro studies with these nanoformulations, have presented that these NP are capable of being actively taken up by colon-26 tumor cells, and the phototoxic effect is not only dependent on the cellular uptake but also is proportional to the average amount of 8 within the NP. In this case the nanoformulation without VTES showed the highest phototoxicity to the tumor cells. Hocine and coworkers [58] in 2010, in a study comparing several silica, silica with radial porosity and

N

Si N

O N

N Si

N N

N N

N NH

HN

N H

N

H N

N OH Pc 4 (5)

Fig. 5. Silicon phthalocyanine Pc 4 (5) Copyright © 2011 World Scientific Publishing Company

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O O Si O

6

O O Si O

O

Fig. 6. Pp IX organosilane synthon 6 J. Porphyrins Phthalocyanines 2011; 15: 522–533

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I

I

O

O

NH

N

N

HN

OEt Si OEt OEt

H2N

EtO

O

EtO

HO

OEt Si

N

N

HN

O N H

O

7

NH

O

8

Scheme 2.


N

O3S

N

SO3

N H

N H N

N

N

N

H N N

H N

9

NH HN

O Si O O

S

10

O3S

O Si O O

NH

523

1115 m2.g-1 and a loading of porphyrin 10 of about 6.70 µmol.g-1. The size of this NP obtained by SEM/TEM microscopy is about 150 nm. Studies performed with these three NP incubating 20 µg.mL-1 of each one on human breast cancer cells (MDA-MB-231) for 24 h at 37 ºC, showed that they have no cytotoxic effects in the dark. The 1O2 quantum yield quantifications showed that the first NP loaded with 10 was the one who generated more 1O2. Nevertheless, upon irradiation of the cells treated with this NP, revealed to be the one with the lowest phototoxicity of this set of NP. Still, SNP with PS 9 from which the 1O2 quantum yield was not possible to be determined by the direct method, has shown to be the most phototoxic of all NP considered in this study and have killed about 52% of the cells upon irradiation. This can be explained by the shape and porosity of these NP, which is already demonstrated to have an important role on cellular uptake and cell function [59–61].

HN O

PORPHYRINS AND PHTHALOCYANINES IN MESOPOROUS SILICA NANOPARTICLES

Fig. 7. Silicon porphyrins 9 and 10

mesoporous SNP for photodynamic therapy, have covalently incorporated the cationic and anionic water-soluble PS 9 and 10 (Fig. 7). In this section only the silica with radial porosity NP and the MPSNP with porphyrins 9 and 10 are discussed. The mesoporous materials with grafted sugar moieties are further discussed in the next section. The preparation of SNP was performed with the addition of Si(OEt)4 as silane agent in basic conditions (tetrapropylammonium hydroxide 1 M in water). The solution was then heated for two days at 80 °C in a polyethylene flask, without stirring. Removal of the template by exchange with a mixture of HCl (12 N) and EtOH and then subsequently centrifuged with H2O and EtOH, afforded the silica NP with porphyrins 9 and 10 with a specific surface area of 100 m2.g-1 and 300 m2.g-1, respectively. The size of these NP obtained by SEM/TEM was around 90 and 80 nm for NP loaded with porphyrins 9 and 10, respectively. The monodispersed Por functionalized porous SNP with radial porosity was prepared using a one-pot method, mixing a water solution of dodecyltrimethylammonium bromide with ethylene glycol. Porphyrin 10 was added to the previous mixture and after complete micellar solubilization, the addition of the silane agent, tetramethoxysilane, was performed. After complete reaction, removal of the surfactant by centrifugation, afforded the radial porosity SNP with a specific surface area of Copyright © 2011 World Scientific Publishing Company

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Covalent encapsulation To overcome premature release of physically entrapped PS, leading to a reduced therapeutic efficiency, and most probably to side-effects, covalent linked PS inside the mesopores have been explored. This is possible when the inner surface, or the pore orifices, have functional groups that can be used to support active molecules. These groups can be grafted directly, during the MPSNP formation, or post-synthetic surface functionalization of the mesopores with a silane linker [20, 58]. Different types of spacers have been used to connect the PS to the NP, however the most common is the propylamino group. In the studies performed by Hocine et al. [58], previously discussed, also the synthesis of MPSNP imbidding porphyrins 11 and 12 (Fig. 8), was achieved by adding the silylated porphyrin to a mixture of CTAB in 0.2 M of NaOH. TEOS was added dropwise and left to react during 6 min at 25 °C then rapidly neutralized to pH 7 by addition of aqueous HCl (0.2 M). After getting a suspension with centrifugation and sonication to re-suspend the NP in ethanol, CTAB was extracted with 50 mL of solution of EtOH/HCl 12 N. After extraction by centrifugation the NP were put in water suspension. The specific surface area obtained for NP loaded with PS 11 was 1192 m2.g-1, with a maximal loading of 11 around 4.43 mol.g-1. MPSNP loading PS 12 was obtained under a similar procedure after reaction of porphyrin 12 with J. Porphyrins Phthalocyanines 2011; 15: 523–533

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N

N

N N H

11

N

O

H N N

N H

(EtO)3Si(H2C)3S N

N

O

N

N H

O

N H N

O

N 12

Fig. 8. Activated cationic porphyrins 11 and 12

APTES in methanol. After completion of this reaction the same procedure followed for acquiring the previous NP led to the synthesis of NP with 12 with a specific surface area of 1031 m2.g-1, and a maximal loading around 0.97 µmol.g-1. The set of 1O2 studies performed on NP loaded with 9, 11 and 12 showed a higher 1O2 production for the later ones. However the viability assays against tumor cells


has shown that these NP exhibited less cytotoxicity than the NP loaded with 9. Cheng et al. [62] in 2008 and Tu et al. [63] in 2009 prepared MPSNP covalently linked to meso-tetra(4-carSi(OEt)3 boxyphenyl)porphyrin palladium(II) and Pp IX, respectively for PDT appliHN cations. This was accomplished by N adding the MPSNP to a solution of S H the previously activated PS 13 or 14. The covalent attachment of the PS 13 was accomplished in methanol in the presence of EDC and APTES (Scheme 3). The resulting mixture was then reacted for 12 h at 60 ºC affording the MPSNP XII with a maximal loading of 2.3 wt.% of 13 with respect to MPSNP after a washing process. The significant photo-induced cytotoxicity at 532 nm observed for MDA-MB-231 breast cancer cells is most likely due to the high concentration of 13 conjugated within the MPSNP. Also the role of MPSNP plays as N

Scheme 3. Copyright © 2011 World Scientific Publishing Company

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but also as a nanoreactor facilitating photo-oxidation reaction as shown by the photo-oxidation monitoring of these nanocomposites. Intriguingly the efficacy of the nanocomposites in photo-oxidation is about 15 times higher than that of the PS in solution. This can be explained by the encapsulation of the 1O2 quencher into the mesoporous channels. Once in this place of the NP, the quencher reaction with 1O2 is much more efficient than the one with free PS in solution. The doping of fluorescent dyes into the nonporous core gives the NP imaging features, which is demonstrated with cell imaging experiments on HO-8910 PM cells. Fluorescent confocal images have shown that these NP are mainly located at the surface of the tumor cells. In spite of the excellent results that MPSNP have shown in the last years allowing surface functionalization and incorporation of highly hydrophobic PS, more detailed studies concerning their biocompatibility need to be performed before going ahead to clinical treatment. In addition to these works, the surfaces of MPSNP can also be modified with different functional groups allowing the use a variety of monoclonal antibodies or other ligands to target them to the desired sites in vivo. Based in this knowledge Brevet and coworkers in two different publications have grafted sugar moieties to MPSNP loaded with PS 10 [58, 66]. The synthesis of SNP XIV and XV (Scheme 4) containing two different maximal loadings of porphyrin 10 (3.5 and 5.8 Mmol.g-1 of material) was achieved by similar methods used for the mesoporous materials loading porphyrins 11 and 12. TEM microscopy revealed monodispersed NP with a diameter of approximately 100 nm in both cases. Variation in the porphyrin concentration during the NP assembly led to mesoporous materials XIV and XV with 3.5 and 5.8 µmol of porphyrin 10 per gram of nanomaterial, respectively. In an attempt to improve the selectivity of the NP towards cancer cells the authors have functionalized the surface of the MPSNP with mannose moieties. This was prepared in a two steps reaction. First the MPSNP surface was functionalized with NH2 reacting the NP XIV3.5 and XV-5.8 with APTES in neutral pH for 20 h under vigorous agitation. Further centrifugation and washing with EtOH furnished both NP XVI-3.5-NH2 and XVII5.8-NH2 with a maximal loading of 1.8 mmol of APTES per gram. Further reaction of the amines on OH HO O O the surface of the NP with the ethyl squaratefunctionalized mannose, linked the mannose OH NH N moieties furnishing XVIII-3.5-0.1 and XIXCOOH 5.8-0.1, where 0.1 represents the equivalents N HN number of mannose residues used in the reaction of XVI-3.5-NH2 and XVII-5.8-NH2 NP NCS 16 per NH2 groups. In order to study the efficiency of these NP 15 (EtO)3Si Si(OEt)3 for PDT, human breast cancer cells (MDAN N O O H H MB231) were incubated for 24 h with 20 µg.mL-1 of XVIII-3.5-0.1 and XIX-5.8-0.1 and Fig. 9. Fluorescein isothiocyanate (15) and silicon hematoporphyrin derivative 16 then submitted to monophotonic irradiation


facilitator for endocytotic cell uptake that dramatically increases the intracellular density of singlet oxygen upon photoirradiation. The direct measurement of the 1O2 by its luminescence at 1270 nm in D2O indicated the ability of this material to form reactive 1O2 species and displace them into the media. Combined with the ability of XII to serve as an in vivo contrast agent for oxygen sensing and imaging, XII holds a pronounced potential as a useful nanoplatform for cancer diagnostic and direct treatment materials (theranostics materials [64]). The process to synthesize MPSNP XIII (Scheme 3) was similar to the one already described, although a previous functionalization of the MPSNP with APTES was needed to acquire in the following step the conjugation with the activated Pp IX 14. This work has presented a simple modification process that allows a high loading of PS into the mesoporous channels. Monitoring the oxygen luminescence by excitation at 514 nm using a continuous wave argon laser has resulted in a characteristic singlet oxygen emission at 1270 nm. A control using the MPSNP alone, without PS, has shown no emission at 1270 nm. The cellular uptake tested in HeLa cells has displayed an uptake dependent of the dosage of XIII and significant intracellular staining due to the presence of XIII was also possible to obtain by merged confocal images of the cells treated with 60 µg.mL-1 for 24 h. The cell viability assays have indicated a threshold of cell tolerance to singlet oxygen generated by these nanoplatforms XIII when the dosage is increased from 19 to 40 µg.mL-1. Zhang and coworkers [65] recently have designed and prepared a multifunctional core-shell NP that contains a non-porous silica core doped with dye 15 (Fig. 9). The additional external mesoporous layer of these NP was obtained with TEOS and PS 16 by a sol-gel condensation reaction. The final NP has a mesoporous silica shell containing hematoporphyrin derivative 16. These nanocomposites are stable and can be stored over a month at room temperature. The elegance of the bi-functionality of this system is related with its capacity to act not only as a carrier for the photoactivable drug which is covalently linked to the mesoporous silica shell


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APTES

Si

NH2 n

XIV-3.5 (3.5 mmol of 10 / g SNP) XV-5.8 (5.8 mmol of 10 / g SNP) EtO

XVI-3.5-NH2 XVII-5.8-NH2

HO O

H N

OH OH OH

O = PS 10

NH2

H N

Si Si J. Porphyrins Phthalocyanines 2011.15:517-533. Downloaded from www.worldscientific.com by UNIVERSITY OF CALIFORNIA SAN DIEGO on 10/01/14. For personal use only.

O

n-m

O

H N

HO O O

O

OH OH OH

m

O

XVIII-3.5-0.1 (reaction with 0.1 equiv. of mannose residue / NH2) XIX-5.8-0.1 (reaction with 0.1 equiv. of mannose residue / NH2)

Scheme 4.

Theranostic materials

Table 1. Amount of mannose on the MPSNP XVIII NP Titration of mannose (mmol.g-1)

XVIII-3.5-0.1

XVIII-3.5-0.25

0.16

0.38

(630–680 nm; 6 mW.cm-2) for 40 min. It was possible to note that NP XVIII-3.5-0.1 was able to cause cytotoxicity without the presence of irradiation killing around 19% of cancer cells. Their irradiation caused 99% cell death and this should be due to an active endocytosis supplied by mannose receptors. Last, in this same work a grafting study using for this XVI-3.5-NH2 was performed to determine the optimum amount of mannose bonded to the surface of the NP towards cancer cells. Simple changes in the procedure include increased amounts of squarate mannose between 0.1 to 2 equivalents which were added to the NP and have furnished NP (XVIII-3.5-0.1 to XVIII-3.5-2, Table 1). PDT experiments with this series of NP after incubation for 24 h (20 µg.mL-1 of NP XVIII-3.5-0.1 to XVIII3.5-2, for 24 h) and then submitted to laser irradiation during 40 min (6 mW.cm-2) have indicated that XVIII3.5-0.1 induced around 99% cell death, while the induced death of XVIII-3.5-0.1 to XVIII-3.5-2 was decreasing along the NP series with higher loadings of mannose moieties. This effect has been explained by the differences in the endocytosis pathways of the NP. The uptake of XVIII-3.5-0.1 is mediated by mannose receptors, which are over-expressed in the cell membrane. This seems to be endocytosis pathway that occurs during 24 h. These 24 h are a pre-requisite since the tests performed with 2, 4 and 6 h of incubation of XVIII-3.5-0.1 did not present the same results. Copyright © 2011 World Scientific Publishing Company

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XVIII-3.5-0.5

Recently, Cheng and coworkers [67] continuing their work with MPSNP have 0.545 incorporated a fluorescent dye (ATTO 647N) directly into the mesoporous silica framework for traceable image, the PS 13 into the MPSNP channels and consequently grafted PEG along with cRGD peptides onto the NP outer surface for targeted delivery to cancer. The introduction of the fluorescent dye onto the MPSNP framework was accomplished by conjugating APTES with the dye. This modified silane agent with the fluorescent dye was then introduced in the NP by sol-gel chemistry. Prior to the removal of the surfactants (CTAB) from the MPSNP the amino groups on the NP surface have been modified by adding APTES. MPSNP XX (Scheme 5) was obtained by the introduction of the previously modified PS 13 with APTES into the MPSNP channels. Finally, the modification of the MPSNP XX surface was possible employing poly(ethylenoglycol) (PEG) containing two functional groups, hydroxysuccinimide ester and maleimide, which reacted with the amine groups on the MPSNP to furnish the PEG-MPSNP XXI. Then at the distal ends of the PEG molecules cyclic RGD peptides were attached to the maleimide, resulting in the theranostic agent MPSNP-PEG-RGD XXII. Characterization of these NP by TEM microscopy and N2 absorption and desorption has concluded that these NP possess a uniform size of 100 nm with a surface area of 682 m2.g-1. The evaluation of the specificity and uptake of the XXII, was made in tumor cells that are deficient in AvB3 integrin (MCF-7 breast cancer) and those over-expressing AvB3 integrin (U87-MG gliobastoma). Confocal fluorescence microscopy performed in these cell lines have J. Porphyrins Phthalocyanines 2011; 15: 526–533

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demonstrated that the pH dependent acidbase equilibrium of chlorin 17 (Fig. 10) in aqueous media is affected in the presence of positively charged SNP. Curiously, in the presence of SNP and at lower pH, the partially or fully protonated species of 17 get bound to the NP instead of forming non-fluorescent aggregates. This has been shown by the fluorescence emission even at pH = 3 that chlorin 17 firmly bounds to the positively charged NP. This spectroscopic signature is also seen at pH between 5 and 8 because a significant amount of fluorescence still persists at these pH values. J. Porphyrins Phthalocyanines 2011.15:517-533. Downloaded from www.worldscientific.com by UNIVERSITY OF CALIFORNIA SAN DIEGO on 10/01/14. For personal use only.

SILICA-COATED METAL NANOPARTICLES FUNCTIONALIZED WITH PHOTOSENSITIZERS

Scheme 5.

shown that XXII has preferentially been uptaken by the integrin expressing cells (U87-MG cells) confirming the receptor mediated endocytosis. The PDT analysis performed with a 532 nm diode laser (250 ± 5 mW.cm-2), delivering the total energy of 1.2 J.cm-2 have exhibited substantial cell death at concentrations of 50, 25 and 10 µg.mL-1 of XXII combined with the U87-MG cells (approximately 10% of cell survival). The same conditions used on MSF-7 breast cancer cells have shown only a modest loss (80% of cell survival) demonstrating that this tri-functionalized MPSNP XXII, possesses a great potential for target delivery of cancer therapeutics.

Another very interesting property of the amorphous silica is the possibility to incorporate metal nanocrystals inside SNP. This procedure has been used to prepare iron oxide [27] and gold nanorods [69] within PS-SNP. For example, the incorporation of a magnetic core allows a controlled delivery or a recovery/reutilization of the PS by a simple applying of an external magnetic field [33]. Simultaneously, MPSNP have also been used to coat different metal cores and entrapped PS for MRI, magnetic hyperthermia and PDT. Silica-coated metal nanoparticles non-covalently functionalized with photosensitizers The findings that SNP could easily be combined with therapeutic drugs for chemical therapy, photosensitizers for PDT and magnetic NP for hyperthermia treatment and magnetic resonance image (MRI) led Liu et al. [70] to prepare a hybrid NP by loading purpurin 18 into SNP within a Fe3O4 core (Fig. 11). The synthesis of NP XXIII was achieved at room temperature adding Fe3O4 NP, the silane coupling agent

PORPHYRINS NON-COVALENTLY IMMOBILIZED ON SILICA NANOPARTICLES As described previously, SNP provide an outstanding platform to covalently and non-covalently immobilize PS inside them. However, these two molecular and supramolecular strategies can also be accomplished on the SNP surface. In 2009, Jain and coworkers [68] investigated the binding of chlorin 17 with amine modified SNP. This study Copyright © 2011 World Scientific Publishing Company

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N

NH

HN

N

CO2H CO2H HO2C

17

Fig. 10. Chlorin p6 (17) J. Porphyrins Phthalocyanines 2011; 15: 527–533

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Fig. 11. Purpurin 18 and magnetic silica coated NP XXIII


APTES and concentrated ammonia to a solution of PS 18, dissolved in deionized water and ethanol. The resulting suspension was transferred to a mixture of cottonseed oil and VTMS. This method afforded Fe3O4 silica coated nanospheres with 20–30 nm of diameter and its characterization was performed by UV-vis spectroscopy. The 1O2 was produced via laser irradiation that was monitored photometrically by following the absorbance decay of RNO at 440 nm. In spite of the magnetic purpurin SNP XXIII being less efficient in generating 1O2 than 18 alone, the simple synthetic method has shown to be promising in the synthesis of monocarriers with potential applications in PDT. In 2009, Chen and coworkers [71] have prepared and studied the anti-tumor effect of loaded magnetic MPSNP in targeting PDT. MPSNP allow a high drug loading, which can be released by controlling size, shape and electrostatic porous modification. To have an ideal therapeutic effect, the drug should be free in the target cells cytoplasm [24]. The preparation of the silica based Fe3O4 nanocarriers was performed with the micellar system AOT/1-butanol/distilled water making use of the sol-gel methodology in the presence of 19 (Fig. 12). The NP obtained (20–30 nm) and the encapsulation of 19 into the mesoporous silica was obtained in 21% efficiency. Fluorescence imaging of the incubated SW480 colon carcinoma cells with the magnetic SNP revealed a subcellular distribution and a significant uptake. The in vitro photodynamic efficacy obtained with the MTT assay showed that the combination of 24 h exposure of tumor cells

O

OH

HN

N O

NH

19

N

N

N

Zn

N

N

N

N

N

OH

N

20

Fig. 12. Porphyrin derivative 19 and zinc phthalocyanine (20) Copyright © 2011 World Scientific Publishing Company

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to 80 µM of the loaded magnetic SNP with 19 and 4.35 J.cm-1 irradiation during 10 min cause approximately a loss of 40% cell viability. The potential of the magnetic core of these particles for MRI and magnetic hyperthermia has not yet been tested. On the other hand, Kim et al. [72] in 2009 incorporated zinc phthalocyanine 20 (Fig. 12) into MPSNP with iron nanomagnets. The nearspherical NP with a mean size around 60–120 nm were prepared through a one pot procedure under basic conditions with a low surfactant concentration (cetyltrimethylammonium bromide) and Fe3O4 NP. Magnetic studies have shown that these NP have a supermagnetic behavior with 1.8 emu.g-1 at 1.0 T, which is enough to submit these NP to MRI and magnetic hyperthermia. Drug delivery experiments with ibuprofen as a guest molecule have shown that this drug can easily penetrate the SNP pores and the low release rate monitored by UV-vis spectroscopy, when compared to the MPSNP loaded with ibuprofen alone. This late release effect can be explained with an interaction between 20 and ibuprofen. Another method to use NIR light absorption into silica based nanomaterials where the light can penetrate deeper into tissues, is the use of NaYF4 (Yb/Er) nanocrystals wrapped with MPSNP loaded with a PS [73]. NaYF4 nanocrystals, upon excitation with a NIR laser, convert the NIR light to visible light, which further activates the PS to release reactive 1O2 to kill cancer cells. Qian and coworkers pursuing this aim have synthesized MPSNP coated NaYF4 with 20 in a multistep process. NaYF4 nanocrystals were coated by an amorphous silica shell (10 nm) and then by a mesoporous silica layer (11 nm). Finally, 20 is loaded into the NaYF4 coated MPSNP by soaking the NaYF4 coated mesoporous silica in a solution containing 20. Cell imaging studies using these NP with MB49 bladder cancer cells incubated for 24 h were carried out using a confocal microscope equipped with a 980 nm NIR laser. The images taken have exhibited an uptake of the NP mainly by the cytoplasm. This work also showed that the absorption peak of 20 (670 nm) overlaps with the red emission peak of the NaYF4 nanocrystals. This means that the light emitted by the NaYF4 nanocrystals activates 20 leading to 1O2 production. This is in agreement with the amount of 1O2 produced by the NP loaded with 20 when exposed to NIR laser. In contrast, the NP without the PS 20 did not show any 1O2 production. In vitro studies of NaYF4 coated SNP with the PS 20 on cancer cells exposed to NIR irradiation for 5 min exhibited cell destruction. Silica-coated metal nanoparticles covalently functionalized with photosensitizers In 2010, Zhao and coworkers [69] have synthesized novel multifunctional NP that combine two J. Porphyrins Phthalocyanines 2011; 15: 528–533

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Silica-coated metal NP covalently functionalized with photosensitizers on the surface

Au NR

= PS 16 XXIV

Fig. 13. Silica-coated gold nanorod XXIV with hematoporphyrin 16


functionalities, a gold nanorod (NR) core and a porphyrin-doped mesoporous silica shell, in one entity (XXIV, Fig. 13). Due to the encapsulation of mesoporous silica, the porphyrin can be well-protected against the external bioenvironment. Metal NP, in particular gold NR, have been known as promising two-photon imaging contrast agents. In addition, gold NR are nontoxic, biocompatible, and chemically inert. All of these features judge gold NR as an ideal candidate for two-photon imaging in vitro and in vivo. The preparation procedures of gold NR XXIV wrapped in silica doped with 16 were first prepared by using a seed-mediated, CTAB-assisted method. The silica coating was then obtained using a sol-gel method in a two steps process. The silica-coated gold NR were prepared via base-catalyzed hydrolysis of TEOS and were then isolated from solution and redispersed in a CTAB solution. These NP subsequently reacted with hematoporphyrin silicon precursor 16, effectively doping the second silica layer. To assess the capability of 1O2 generation of XXIV, the photo-oxidation of ABDA was employed as a probe molecule to monitor the singlet oxygen generation. ABDA reacts directly with the generated singlet oxygen to yield an endoperoxide, which causes a decrease of ABDA absorption centered at 380 nm. The photo-oxidation of ABDA in the presence of XXIV was monitored for 75 min under the irradiation with a diode laser at 633 nm. This methodology evaluated that the photo-oxidation efficiency of gold NP XXIV is slightly higher (^20%) than that of the same amount of free PS molecules. By incubating XXIV with MDA-MB-231 cells for 24 h it is possible to obtain the images of the cells using bright-field imaging and two-photon fluorescence imaging methods, as well as their merged images. The twophoton fluorescence imaging was performed by using a confocal microscope coupled with a femtosecond laser with central wavelength at 770 nm as the excitation source. It can be seen that the composite NP were mainly located at the cytoplasm.


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As stated before, PDT studies demonstrated that it can be also very effective in the photoinactivation of microorganisms. This has open the possibility of using this technique not only for application in the clinic field but also in environmental applications, more specifically for the inactivation of pathogenic microorganisms in water and wastewater. However, the extension of the photodynamic principle to a new environmentally friendly technology can only become economically viable if the PS is immobilized on a solid matrix in order to allow its complete recovery after the photoinactivation process. Recently Cavaleiro and coworkers, following their developments in efficient photosensitizers for photoinactivation of environmental microorganisms [9, 74, 75], have published a study comprehending cationic porphyrin magnetic SNP and antimicrobial activity [33]. The remarkable antimicrobial activity, associated with their easy recovery, just by applying a magnetic field, makes these novel materials interesting for wastewater disinfection. The synthetic route to these novel materials using porphyrins 21–23 (Scheme 6) was carried out in DMSO at 140 °C with the iron SNP obtained previously by a co-precipitation method [76]. The ability for these new hybrids to generate 1O2 was evaluated using DPBF as a 1O2 quencher. DPBF reacts with 1O2 in a [4+2] cycloaddition being oxidized to colorless o-dibenzoylbenzene. The absorption measurement of DPBF decay at its maximum absorbance (415 nm) is a method to qualitatively measure the ability of NP XXV to XXIX to generate 1O2. The results of these assays have shown that they can generate singlet oxygen at 20 µM of immobilized PS with a similar rate of the non-immobilized porphyrins 21–23, at concentrations around 0.5 µM. The anti-microbial efficiency was performed against gram-negative E. coli and gram-positive E. faecalis bacteria, and also against T4-like phage. In these cell lines XXVIII turned out to be the most effective nanomaterial since it was able to cause total photoinactivation of the three tested microorganisms, at a PS concentration of 20 µM, upon irradiation with a white light and a total dose of 21.6, 43.2 and 14.4 J.cm-2, respectively. The use of silica coated magnetic nanoparticles as contrast agents has resulted in the production of highly stable, nontoxic solutions that can be manipulated by an external magnetic field. In a work performed by Nowostawska and coworkers [77], Pp IX was reacted with APTES in the presence of the carbodiimide coupling agent and then reacted with the silica coated nanoparticles. The silica shell in this case provides an effective barrier between the magnetic core and the PS attached to the silica surface (XXX, Fig. 14). The combination of both magnetic and fluorescent properties in one nanocomposite can


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H2N

H2N

Si O O Si O O OO O Silica O Si O O Si O Fe3O4 O OO O O Si O Si OO O O Si

H 2N

H 2N

Si O O Si O O OO O Silica O Si O O Si O Fe3O4 O OO O O Si O Si OO O O Si

H2N

NH2

F

F PS

F F

F

NH2

F PS

NH2

F

NH


F

F F

F

Ar

F Ar

F

NH2

F

N

N

F

PS F

Si O O Si O O OO O Silica O Si O Si O O Fe3O4 O OO O O Si O Si O O O O Si

37-39

21, XXV 22, XXVI, XXVIII 23, XXVII, XXIX N

PS

F

NH2

N

Ar

F

F

CH3I

N HN NH N

F

H N

XXV-XXVII

F

F

Ar F

H2N

H2N

N I-

F PS

F

F

NH F N

F

H N F

F F

PS

.nIN

XXVIII, XXIX

Scheme 6.

imaging. Confocal and composite images of the cells incubated with the NP taken at different periods of time (24 h, 48 h and 5 days) have demonstrated that there was no cellular uptake of the fluorescent nanocomposites but only binding to the outer cell membranes.

Fe3O4

= PS 6

CONCLUDING REMARKS XXX Fig. 14. Silica-coated magnetic nanoparticle XXX with PS 6

provide additional benefits as they can serve as multimodal assays for in vitro and in vivo bioimaging applications such as MRI and fluorescent microscopy. This can be further combined with the ability to be used as agents for anticancer therapy by PDT or hyperthermia. Initial co-incubation experiments performed with THP-1 macrophage cells in the presence of these NP XXX, showed a distinct photobleaching of the PS upon exposure to light under a fluorescent microscope. This bleaching was neutralized by the addition of B-ME to the cell cultures and resulted not only in increased fluorescence intensity levels, but also allowed enhanced Copyright © 2011 World Scientific Publishing Company

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This work tried to emphasize the importance of SNP on the treatment of degenerative diseases and microorganisms control, revising some methodologies that can be assessed by combining Por and related PS properties with SNP, in order to achieve functional materials for different PDT applications. Comparatively to other carriers, such as liposomes, biopolymers and more recently dendrimers or even other “bio”compatible nanoparticles, such as the well-known gold nanoparticles, SNP can incorporate many of the features of the other ones, accumulatively with a very strong mechanical and chemical stability, and versatile covalent and non-covalent immobilization characteristics, allowing many different combinations/formulations. For example, SNP open the possibility to entrapped PS inside silica matrix, contrarily to gold nanoparticles. At the same time silica entrapment J. Porphyrins Phthalocyanines 2011; 15: 530–533

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can protect and provide “bio”compatibility to many other entities, such as metal nanocrystals, that would be impossible in other formulations. In a general matter, two types of sol-gel methods have been discussed for the synthesis of SNP: the Stöber and the microemulsion methods. It is noticeable that the best method for incorporation of high concentrations of PS into SNP is the covalent bonding approach, but it requires previous functionalization of the PS with proper silicon derivatives. In both methodologies, two chemical approaches for the incorporation of PS into silica nanoparticles have been discussed. The first approach consists in using non-covalent process, by entrapping the PS into the silica matrix. On the contrary, the second approach has been described as using covalent bonding of the PS with the silica matrix. Somehow the covalent bonding of the PS to the silica network is usually the preferred method. According to the Stöber method, the PS incorporation yield into the SNP under non-covalent bonding is poor and dependent of the absorption force between the PS itself and the silica precursor. On the other hand, the microemulsion process (Ormosil and MPSNP) avoids this problem, controlling the quantity of incorporated PS into silica NP by using a water soluble PS. Microemulsion methods also allow co-condensation of PS along with an excess amount of other molecules, for instance BDSA. In this case BDSA is a highly twophoton active molecule that allows an excitation in IR region of the spectra in order to treat tissues more deeply. Nevertheless, this methodology also presents disadvantages like the particle size, which may not be uniform, and further different modifications of the particle surface are not easily achieved and sometimes require covalent bounding to achieve proper encapsulation. However, some interesting results have been obtained with the covalent loading of PS into these nanoparticles, particularly when the silica nanoparticles are in the scope of the MPSNP. The last ones, belong to the most emergent NP combined with tetrapyrrolic PS as shown by the recent works developed by Thienot and Cheng. These materials show a high chemical and thermal stability and easy functionalization, making them ideal candidates for biomedical applications with multiple biological active molecules. The most popular way of covalently functionalizing MPSNP with PS has been revealed to be by coupling the PS to the inside groups of the mesopores of these SNP. Additional grafting of biological relevant molecules on the outer surface of the MPSNP has prompted these materials to the theranostic field, where multiple functions (diagnostic, therapy and vector) have shown promising results. Additionally, core shell SNP combining a metallic core coated with a layer of silica, which can be obtained by the different synthetic methodologies, already found applications in a wide variety of fields. Recent developments in this area led researchers to combine different materials with SNP superparamagnetic iron oxide and Copyright © 2011 World Scientific Publishing Company

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gold nanorods, which can be used for MRI and two-photon imaging, respectively, while the outside silica shell, due to its biocompatibility, acts as PS carrier. Also the use of Por-based magnetic SNP hybrids used against bacteria and virus for environmental applications, mainly water disinfection, found to be highly effective PDT agents. In conclusion, these studies reported versatile techniques to get many PS-based SNP in fashion arrangements, which in combination with their photophysical and biological properties provide guidelines to control the PS pharmacokinetics and pharmacodynamics behaviors, highlighting that photoactivable Por-based SNP are still in the very beginning. Therefore, these nanomaterials still need more in vitro and in vivo studies in order to evaluate completely which are the side-effects of SNP in vivo. The combination of several motifs incorporated in organized silica nanomaterials as metallic cores, covalent coupling of the PS, and biological active molecules grafted on SNP surface suggest an outstanding future for these hybrid nanomaterials. Acknowledgements Thanks are due to University of Aveiro, FCT (Portugal) and FEDER for funding the Organic Chemistry Research Unit (QOPNA) and grants PTDC/ QUI/65228/2006 and PTDC/CTM/101538/2008 to J. Tome. F. Figueira also thanks FCT for his PhD fellowship SFRH/BD/46788/2008.

ABBREVIATIONS ABDA AOT APTES CTAB DMSO DPBF EDC IP IPS IR MRI MPSNP MTS MTT NP NR ORMOSIL Pc PDT PdTPP

9,10-anthracenediyl-bis(methylene) dimalonic acid sodium bis(2-ethylhexyl)sulfosuccinate 3-aminopropyltriethoxysilane cetyl trimethylammonium bromide dimethylsulphoxide 1,3-diphenylisobenzofuran N-ethyl-N a-(3-dimethylaminopropyl)carbodiimide hydrochloride iodo benzylpyropheophorbide iodobenzilpyrosilane infra-red magnetic resonance imaging mesoporous silica nanoparticle(s) 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide nanoparticle(s) nanorod organic modified silica phthalocyanine(s) photodynamic therapy palladium tetraphenylporphyrin


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PEG Por Pp IX PS cRGD RNO B-ME SEM STMP SNP TEM TEOS mTHPC


TPA UV-vis VTES

polyethylene glycol porphyrin(s) protoporphyrin IX photosensitizer(s) cyclic Arg-Gly-Asp peptide N,N-dimethyl-4-nitrosoaniline B-mercaptoethanol scanning electron microscopy sodium trimethoxy phosphate silica nanoparticle(s) transmission electron microscopy tetraethoxysilane meso-tetra(m-tetrahydroxyphenyl) chlorin two-photon absorption ultraviolet-visible vinyltriethoxysilane

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