Use of Photosensitizers in Semisolid Formulations for Microbial

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Use of photosensitizers in semisolid formulations for microbial photodynamic inactivation Jose Antonio González-Delgado, Patrick Kennedy, Marta Ferreira, Joao P. C. Tome, and Bruno Sarmento J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01129 • Publication Date (Web): 16 Nov 2015 Downloaded from http://pubs.acs.org on November 17, 2015

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Journal of Medicinal Chemistry

Use of photosensitizers in semisolid formulations for microbial photodynamic inactivation José A. González-Delgado†‡, Patrick Kennedy∇∥⊥, Marta Ferreira †, João P. C. Tomé ‡§, Bruno Sarmento*†∥⊥# †

Inovapotek, Pharmaceutical Research & Development, Edifício Inovar e Crescer, Salas 23 e 39, Rua Alfredo Allen, 455/461, 4200-135 Porto, Portugal ‡

∇

QOPNA and Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

ICBAS - Instituto Ciências Biomédicas Abel Salazar, 4150-180 University of Porto, Portugal

∥

I3S - Instituto de Investigação e Inovação em Saúde, University of Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal

⊥ INEB

- Instituto de Engenharia Biomédica, University of Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal

§

Department of Organic and Macromolecular Chemistry, Ghent University, B-9000 Gent, Belgium

# CESPU

- Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde

and Instituto Universitário de Ciências da Saúde, 4585-116 Gandra PRD, Portugal

ACS Paragon Plus Environment

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ABSTRACT

Semisolid formulations, such as gels, creams and ointments, have recently contributed to the progression of photodynamic therapy (PDT) and microbial photodynamic inactivation (PDI) in clinical applications. The most important challenges facing this field are the physicochemical properties of photosensitizers (PSs), optimal drug release profiles and the photosensitivity of surrounding tissues. By further integrating nanotechnology with semisolid formulations, very promising pharmaceuticals have been generated against several dermatological diseases (PDT) and (antibiotic-resistant) pathogenic microorganisms (PDI). This perspective focuses on the different PSs and their associated semisolid formulations currently found in both the market and clinical trials that are used in PDT/PDI. Special emphasis is placed on the advantages that the semisolid formulations bring to drug delivery in PDI. Lastly, some potential considerations for improvement in this field are also discussed.

1. INTRODUCTION Antimicrobial resistance, a consequence of the excessive use of antibiotics, has become a global threat to human health.1 To address this serious issue, alternative microbial control techniques are currently being explored. Photodynamic inactivation (PDI) is a promising, clinically-proven treatment against a broad spectrum of (antibiotic-resistant) microorganisms.2-5 PDI relies on three essential components: 1) a non-toxic photosensitizer (PS), 2) light at a wavelength specific to the PS, and 3) cellular oxygen. Briefly, the irradiated PS can produce radical species in contact with a substrate or generate highly reactive oxygen species (ROS) when interacts with the molecular oxygen (O2). These species react immediately with the surrounding biomolecules to


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produce irreversible damage to the cells/microorganisms (Figure 1).6 These microorganisms can be bacteria, fungi, parasites, yeasts or viruses.7-9 However, depending on their structure or physicochemical properties, PSs can be more active against certain types or strains of microorganisms. For example, neutral and anionic PSs are much more efficient against grampositive bacteria,10 whereas cationic PSs are effective against both types of bacteria.11-13 Administration of these PSs within a suitable pharmaceutical formulation is essential for their clinical efficacy. Topical drugs come in liquid, semisolid and solid formulations, and they can be classified as creams, gels, pastes and ointments according to the pharmacopoeial nomenclature or as emulsions, liposomes, suspensions and transdermal patches according to the structural matrix.14 Some of these formulations have been used in both PDI and PDT due to favourable properties such as penetration and deep contact with damaged tissues and a suitable structure that allows for complete removal once the treatment is finished. Thus, topical treatment such as PDI is a less invasive and more attractive alternative for clinical eradication of microorganisms. In this perspective, we provide an overview of the characteristics of PSs incorporated into semisolid formulations, such as creams, gels or ointments, used in PDI for topical applications, with an emphasis on the design and optimization of these platforms for the clinical applications.

2.

PDI MECHANISM OF ACTION

There are two mechanisms that act during the photodynamic processes (Figure 1). When a PS absorbs a photon, it becomes an excited PS (1PS*). This 1PS* can return to the initial state (1PS) by fluorescing, or it can convert to a triplet excited state (3PS*) by intersystem crossing. From


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this triplet state, the PS can phosphoresce until it returns to the ground state, or it can generate ROS through two pathways (Type I or II). The type I pathway is based on electron or hydrogen atom transfer between 3PS* and a substrate, generating radical species such as the superoxide anion, hydroxyl radical or hydrogen peroxide.15 The type II pathway is the most important, because the 3PS* transfers energy to triplet molecular oxygen (3O2) producing a highly reactive singlet oxygen (1O2) destroying the cell wall/membrane. During mammalian cell targeting, cell death occurs depending on the type, dose and location of the PS inside the cell and the amount of singlet oxygen produced.16-17, 18

Figure 1. Diagram of the physical and chemical processes involved in PDI.

3.

SEMISOLID FORMULATIONS APPLIED TO PDI

Topical semisolid formulations are currently used in PDI due to their multiple advantages over other modes of administration. These systems provide direct contact of the PS with the infected area to enhance drug localization, controlled release and selectivity; however, antimicrobial PDI


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does have its share of challenges. Namely, most PSs have poor solubility in water or biofluids, a special light is needed to excite the drug, and the microbial cell wall often has low permeability. The cell wall of gram-positive bacteria (e.g. Staphylococcus, Propionibacterium and Clostridium) is composed of teichoic acid and a relatively permeable layer of peptidoglycan; thus, allowing for easier penetration of the PS. However, gram-negative bacteria (e.g. Escherichia, Pseudomonas, Acinetobacter and Helicobacter) have a more rigid wall composed of an additional outer layer of lipopolysaccharide and protein causing poor PS penetration. For fungi and yeast, PS penetration is intermediate. Of note, cationic PSs bind more rapidly to microorganisms allowing a temporal selectivity and decreasing the damage to surrounding tissues.19 There are a wide number of compounds that provide photodynamic activity, such as porphyrinoids

and

related

compounds

(porphyrins,

chlorins,

bacteriochlorins,

isobacteriochlorins, porphycenes or phthalocyanines) and non-porphyrinoids (anthraquinones, phenothiazines, xanthenes, acridines, curcuminoids or coumarins, among others). The most important properties in a PS are: 1) proper localization, 2) absorption at specific wavelengths, 3) little or no dark toxicity, 4) safety and 5) efficacy.18 The use of PSs in semisolid formulations in the clinic is still in its infancy (Table 1); therefore, only well-known PSs have been tested in these platforms. For this reason, the newer classes of photoactive compounds might be studied in combination with nanotechnologies20, 21 and a suitable optimization into semisolid formulations for further clinical trials, especially against gram-negative bacteria, which are the ones most that are the most difficult to treat with PDI.22


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Table 1. Examples of photosensitizers incorporated in semisolid formulations for use in antimicrobial photodynamic inactivation. Semisolid

Composition

Photosensitizer

Microorganisms

Ref

Cream

Metvix

(3)

Malassezia folliculitis

23

Gel

1 (5%)

(1)

Acne vulgaris

24

Gel

Photogem

(4)

Candida spp. and

25

Formulation

(50 and 100 mg/L) Gel

Photogem (5 mg/L)

Staphylococcus spp. (4)

Staphylococcus aureus

26

(7)

E. coli

27

(7)

S. epidermidis and Proteus

28, 29

Chitosan (0.25 g) Ascorbic acid aqueous solution (0.16 M) Gel

THES-PEG 600 TMOS-PEG 600

Gel

HEMA-MAA

mirabilis Gel

Radachlorin (0.1%)

(12)

Streptococcus mutans

30

Gel

Verteporfin

(15)

Mycobacterium bovis

31

(17)

Candida albicans,

32

Collagen Non-aqueous gel

NMP Labrafil M1944 CS

Aspergillus niger,

Labrafil M2130 CS

Pseudomonas aeruginosa,

Labrasol

S. aureus and E. coli

Transcutol PEG 400 Cremophor RH40


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HPC-M

Vulnofast

(23)

MRSA

33, 34

(25 or 7)

MRSA

35

(25 or 7)

MRSA and B. cepacia

36

23 (1 mM) Gel

PVA (8.0% w/w) Borax (2.0% w/w) 25 or 7 (up to 1 mg/L)

Gel

PMVE/MA (10, 15 or 20% w/w)

Gel

26 (0.2%)

(26)

Trichophyton rubrum

37

Cream

Unguentum M

(27)

Leishmania major

38

27 (0.026%)

3.1 NATURAL PHOTOSENSITIZERS. 5-AMINOLEVULINIC ACID (5-ALA) AND DERIVATIVES 5-Aminolevulinic acid39 (1, 5-ALA, Figure 2) is a natural amino acid precursor of the heme group. Eukaryotic cells metabolize 1 into the excellent PS protoporphyrin IX40 (2, PpIX, Figure 2). Compound 2 is activated from UV to visible wavelengths with the Soret band at 405 nm and a Q band at 630 nm in DMF. 2 was the first topical PDT agent to receive regulatory approval by the U.S. Food and Drug Administration (FDA, 1999) for the dermatological treatment of actinic keratosis and has also been used to treat carcinomas (e.g. Bowen’s and Paget’s disease) as well


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as psoriasis.41 Although 1 has been widely used off-label to treat acne (Propionibacterium acnes),42 the mechanism of action is not completely understood, and neither 1 nor 2 has been approved as yet for this disease by the FDA.

Figure 2. Chemical structures of the natural photosensitizer 1 and its derivatives (2 and 3). 1 is a hydrophilic zwitterion that penetrates poorly through biological barriers, such as the cell membrane or the stratum corneum of the skin.43, 44 Liposomal encapsulation, ALA prodrug esters and other approaches have been explored to increase cell permeability.45-48 As 1 is unstable in aqueous formulations with pH > 6, it is usually produced immediately before use when applied in creams.49 Compared to 1, methyl levulinate (3, methyl-ALA or MAL, Figure 2) is more lipophilic, accumulates 2 more selectively in the infected skin cells and absorbs more deeply and rapidly into lesions; thus shortening the incubation time.50, 51 3 is usually activated with red light at 630 nm. However, as red light can cause erythema and hyperpigmentation, other lasers and light sources have been investigated for photorejuvenation and to treat inflammatory processes promoted by acne vulgaris.52


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Iontophoresis, widely used in neuropharmacology and therapeutic or diagnostic applications, is a technique in which ions are transported by applied electric field acting like a non-invasive needle to bypass the skin barrier. Combining iontophoresis with PDI, 1 delivered from Carbopol® gel through the stratum corneum demonstrated a better uptake, penetration and higher accumulation of 2 than other formulations, such as a solution or sponge.53 Drug stability over time is a central aim for a clinical formulation, and many oil-in-water (O/W) emulsions are used extensively to help increase this stability. For example, a monoolein/water (70:30, w/w) gel with a low concentration of 1 (0.2% w/w) demonstrated stable physicochemical properties (e.g. fluorescence) after 30 days.54 Monoolein, or glyceryl monooleate, a well-known amphiphilic lipid which forms a cubic liquid-crystalline phase when in an aqueous medium, possesses the ability to incorporate hydrophilic or hydrophobic drugs. Such properties make these platforms efficient drug delivery systems for different cutaneous diseases and PDI. Other O/W microemulsions have been developed in order to improve the stability, in vitro skin permeability and skin delivery of 1 or 3.55 The concentration of 1 in water-in-oil (W/O) emulsions is usually 20% but varies from 2% to 40% depending on the application time. For example, 10-20% 1 (Levulan® Kerastick®), 78 mg/g 1 (Ameluz®), and 16% 3 (Metvix®), are all different formulations to treat actinic keratosis. The first patch formulation for PDT (Alacare®), also developed for this disease, contains 2 mg/cm2 of 1 and is activated with red light over the wound.56, 57 The above semisolid formulations have been sold in many countries for the treatment of nonmelanoma skin cancers as well, but none of them, as of yet, are licensed for antimicrobial applications58 with the exception of Levulan, which has been prescribed for the treatment of acne. A cream composed by 8% 3 (Visonac®) is also being tested for severe acne vulgaris in


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phase III in Europe and Russia.59 Ma and colleagues (2013) performed a clinical study with 5% w/w 1 in a gel that showed excellent efficacy against acne in Chinese patients using an LED light of 633 nm at dose levels of 96-120 J/cm2.24 This approach is very interesting, because generally when 1 is used in this disease, optimal results are obtained only with high PS concentrations, long incubation times and high doses of light resulting in severe side-effects. In PDI applications, 3 and derivatives have been also studied against viruses, parasites, yeasts and fungi but these studies are not very extensive.39 In a preliminary in vivo study, Lee and colleagues (2010) tested a cream composed by 3 in diverse patients with recalcitrant Malassezia folliculitis caused by Malassezia furfur, a fungus that also aids in the formation of dandruff. During each session, the cream was applied on the lesions of six Korean patients for three hours and followed by irradiation with 37 J/cm2 of red light (630 nm) during 7.5 minutes. After three sessions per week for two weeks, inflammations decreased in the majority of the patients without severe side effects.23 Yet, the number of patients and long-term evaluations are essential to achieve a statistically successful clinical outcome.

3.2 PORPHYRINOIDS Porphyrins are tetrapyrrolic macrocycles interconnected by four methine carbons. According to Hückel’s rule (4n+2), the porphyrin skeleton has 18-π electrons and is considered highly aromatic. This aromatic structure contributes for many of their useful biomedical applications. Due to the acidic and basic properties of the inner pyrrolic nitrogens, porphyrins can be found as amphoteric compounds. Marketed products such as Photofrin® (Axcan Pharma, Montreal, Canada) utilize these advantages in PDT for the treatment of malignant tumours.60


10

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Because of their structural characteristics, many porphyrins have also been used in PDI61-64 by penetrating into bacteria and interacting with the cytoplasmic membrane65 and/or DNA66-71 to produce different types of photodamage. However, when porphyrins are used in aqueous systems, they tend to aggregate and interact with other biomolecules. This process can be induced by changing the pH or ionic concentration or by addition of a surfactant. In physical terms, non-covalent interactions such as van der Waals, hydrogen bonding, electrostatic interactions or metal-ligand coordination are present during these spontaneous processes.72 Aggregation is minimized or non-existent at low porphyrin concentrations. For this reason, a balance between porphyrin dose and concentration is required for an optimal clinical formulation.


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Figure 3. Chemical structures of porphyrinoid PSs used in semisolid formulations for PDI. 3.2.1 HEMATOPORPHYRIN (Hp) AND DERIVATIVE (HpD) Hematoporphyrin derivative73 (4, HpD, structure not disclosed) is a mixture of several derivatives (monomer, dimer and oligomers) and was the first porphyrin-type PS approved in Europe, USA, Japan and Canada (1993) in the treatment of several kinds of cancers (e.g. cervical, endobronchial or esophageal). Although hematoporphyrin (5, Hp, Figure 3) is still successful, its drawbacks (e.g. long persistence in skin, no optimal wavelength, selectivity or photosensitizing efficiency) leave room for improvement through novel innovations. Compound 5 was recently used in PDI against gram-positive (Staphylococcus epidermidis, Streptococcus pyogenes, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA))74 and gram-negative bacteria (Pseudomonas aeruginosa and Acinetobacter baumannii). Tsai et al. (2011) combined chitosan, a polymeric biomaterial with excellent biocompatibility, non-toxicity and adsorption, with micellar 5 at low concentrations (0.1 µM) for 30 minutes and 25 J/cm2 of irradiation at 635 ± 5 nm for complete microbial eradication. Incorporation of poly-L-lysine, a cationic polymer with well-known antimicrobial properties, showed similar results but with a relevant dose-dependent dark toxicity (>0.0025% w/v).75 Porfimer sodium (6, Figure 3)

58

or Photofrin is a partial purification of 4 and is administered

by intravenous injections.76 It has a weak molar extinction coefficient (ε = 1170 M-1 cm-1 in ethanol); however, it has a high singlet oxygen quantum yield (Φ∆ = 0.89 in phosphate buffer with 1% Triton X-100) and safety. Compound 6 has weak absorbance in the red region of the spectrum (≥600 nm), where the penetration of light in tissues is optimal; thus, its use is limited to superficial diseases. Unfortunately, skin photosensitivity can endure one month or more due to retention in cutaneous tissues necessitating avoidance of direct sunlight.77, 78


12

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Photosan-3® and Photogem® are other commercial formulations of 479 that have been approved for clinical use in Russia and Brazil,80 but only Photosan-3 has European Union (EU) approval.81 Fontana et al. (2008) combined chitosan and Photogem (CG-P) to eradicate S. aureus using the disk diffusion method.82 Importantly, the dark cytotoxicity decreased when CG-P was used, demonstrating improved control and precision during drug administration.26 Ribeiro and colleagues (2012) used Photogem suspensions and gels to disinfect dentures colonized with Candida spp., Staphylococcus mutans spp. and streptococci.25

3.2.2 5,10,15,20-TETRAKIS(N-METHYLPYRIDINIUM-4-YL)PORPHYRIN (TMPyP) 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin83 (7, TMPyP, Figure 3) is an interesting water-soluble PS with unique photophysical and photochemical properties that strongly interacts with quadruplex DNA and is able to inactivate gram-negative bacteria.84 Recent studies showed that the compound 7 interacts with different external bacterial structures85 leading to inactivation of bacterial and yeast biofilms.86 The electrostatic forces generated by the cationic nature of 7 and the negatively-charged outer surface of bacteria leads to the accumulation of the PS in the cell wall. With this in mind, Rychtarikova et al. (2012) prepared microporous silica gels by the sol-gel process using tetrakis(2-hydroxyethoxy)silane (THES) or tetramethoxysilane (TMOS) in combination with 7. THES-7 composites, along with polyethylene glycol (PEG) 600 and water, were stable for at least 3 months and exhibited higher photobactericidal activity against E. coli than the TMOS derivative.27 Intraocular lens surgery is prone to bacterial attack, and PDI can help prevent these kinds of infections as well. To address this, Parsons and colleagues (2009) impregnated the compound 7 in anionic copolymers of 2-(hydroxyethyl)methacrylate (HEMA) and methacrylic acid (MAA)


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for ocular biomaterials. This system reduced Staphylococcus epidermidis by 99% under intense light and 91% in dark conditions.28, 29

3.2.3 5,10,15,20-TETRAKIS(3-HYDROXYPHENYL)PORPHYRIN (mTHPP) Carboxymethyl starch (CMS) and dextran sulphate (DS), delivery vehicles widely used in the pharmaceutical, medical and cosmetic industries, were used as hydrogels to encapsulate 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (8, mTHPP, Figure 3).87 In this study, the size of the CMS-DS hydrogels ranged from 40 to 100 nm with a particle size of 3 nm. Drug entrapment was 92% and drug release was complete in 10 hours. The encapsulation of the PS into the hydrogel increased the lifetime of the triplet state, which is essential in the production of ROS to consequently decrease the necessary dose. Importantly, the transparency of the final product provides more antimicrobial effectiveness when light is applied. Although no biological studies were performed, this study paves the way for future applications.

3.2.4 OTHER PORPHYRINS 5,10,15,20-tetrakis(4-sulfophenyl)porphyrin88 (9, TSPP, Figure 3) is a water-soluble dye with an absorption band around 640 nm and has been widely used in clinical assays but not in antimicrobial applications. The diffusion of the compound 9 in different media while encapsulated in poly(vinyl alcohol) (PVA), a non-toxic and biocompatible polymer, was evaluated by the repeated freeze/thaw technique. Pore size distribution by scanning electron microscopy (SEM) was 80-950 nm suitable for the encapsulation of 9.89 Mass transfer and agitation experiments identified phosphate-buffered saline (PBS) and hydrogels agitated at 100 rpm and 37°C to be the optimal parameters.90 These kind of physicochemical studies are


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essential before evaluating the antimicrobial activity of PSs within semisolid formulations, because the drug release rate must be optimal for excellent efficacy of the final product.91 Love and co-workers (2007) developed and patented the novel and interesting XF porphyrins (Figure 3).92 Their dicationic skeleton provides excellent mechanisms of action against the bacterial membrane, even without irradiation.93 This may handicap them in clinical trials; however, a suitable chemical modification and/or delivery vehicle may quench their powerful activity. Few antimicrobial studies have been performed, but the promising results justify their clinical investigations. Among them, 5,15-bis-[4-(3-trimethylammonio-propyloxy)-phenyl]-porphyrin dichloride (10, XF-73, Figure 3) has demonstrated excellent bactericidal activity. In 65 gram-positive bacterial species, there was a minimum inhibitory concentration (MIC) of 0.25-4 mg/L, and a little higher in 36 gram-negative bacterial species, with a MIC of 1-64 mg/L. These differences were attributed to resistance and low permeability; thus, there was less accumulation of the PS in the cytoplasmic membrane barrier of gram-negative microorganisms. S. aureus biofilms tested with the

compound

10

and

(5,15-bis-[3-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin)

dichoride (11, XF-70, Figure 3) demonstrated a low biofilm MIC (1 mg/L) and minimum biofilm eradication concentrations (2 mg/L).94 When both PSs were compared to some antibiotics (e.g. chlorhexidine and cetyltrimethylammonium bromide) in stationary phase cultures, a higher reduction (≥5 log10) in viability during 2 hours was obtained as compared to the other antibiotics (3 log10). Consequently, these studies have promoted the development of a novel gel formulation composed by 10 for the nasal decolonisation of S. aureus, currently in clinical phase by Destiny Pharma Ltd (Brighton, UK).


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Using blue light (418 nm), the compound 10 performed better than 7, in planktonic and biofilm Candida albicans cells, the most common fungus to infect humans.95 0.5 µM 10, 4x lower than 7, was enough to eradicate 6 log10 of planktonic cells with 12 J/cm2 of irradiation for 15 minutes. For the biofilms, 1 µM 10 was effective in comparison with 50 µM 7 to eliminate 5 log10 cells with 4 hours of irradiation at a light dose of 48 J/cm2. This low concentration of 10 in biofilms cells may justify further studies in clinical trials. In a liquid formulation (ethanol/water, 1:9), the compound 10 was also incorporated to eradicate MRSA using an ex vivo porcine skin model.96 10 was localized only in the stratum corneum and had a higher efficacy (3.6 log10) at 10 µM. This topical formulation may be developed in similar semisolid platforms in order to improve the localization and selectivity of this promising drug.

3.3 CHLORINS Chlorins, or β-dihydroporphyrins, have the same backbone structure as porphyrins (tetrapyrrolic macrocycles linked by four methine carbons) but with a reduced ring (18-π electrons) giving rise to an increased absorption (εmax ~ 40,000 M−1 cm−1) in the red region of the spectrum (λmax = 650-750 nm). They are efficient generators of 1O2 and change colour from purple to green. Like many PSs, chlorins are highly hydrophobic making their administration, delivery and uptake in the human skin difficult. As porphyrins, the ionizable groups of chlorins exist in different ionic forms depending on the pH, important for the selective tissue uptake and retention at physiological pH.97


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Figure 4. Chemical structures of chlorins used in pharmaceutical formulations for PDI.

3.3.1

CHLORIN E6 (Ce6)

Chlorin e698 (12, Ce6, Figure 4) is a potent PS with excellent physicochemical properties. For instance, it has a high singlet oxygen quantum yield (Φ∆ = 0.65 at pH 7-8) and an absorption maximum at 654 nm with a molar absorptivity of about 40,000 M-1 cm-1. A very important advantage of this PS in PDI is its easy penetration into the cell membrane, leading to optimal accumulation inside cells.99 However, 12 does have some drawbacks: high instability, long-term skin photosensitization and high dose needs.100,

101

In this sense, the incorporation of 12 into

suitable delivery vehicles may minimise these issues. For example, the compound 12 encapsulated in a superparamagnetic chitosan (CS) – dextran sulphate (DS) hydrogel significantly enhanced the lifetime of the PS triplet state.102 Similar to


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the above-mentioned system of the compound 8, the average drug entrapment was 92% with an encapsulation efficiency of 90%, and the CS-DS hydrogel size ranged from 40 to 100 nm. The drug was released in about 9 hours, allowing for a suitable deposition in the desired sites to be determined in future biological studies. Due to its insolubility in water, 12 is usually formulated in liposomes. Yang et al. (2013) reported the efficacy of the liposomal-12 in vitro against susceptible and drug-resistant clinical isolates of C. albicans and infected burn wounds.103 This interesting strategy may be studied in semisolid formulations such as creams or ointments. Photolon®, a complex of 12 and PVP (polyvinylpyrrolidone) at a ratio of 1:1 (w/w), is marketed for intravenous treatment of several (non-)cancerous human diseases. Compared to the properties and PDT activity of free 12, the 12-PVP formulation more selectively accumulated and retained in the tumour tissue and had a faster clearance rate from skin and normal tissues.104 In order to enhance drug penetration and minimise phototoxicity, Photolon ointments using PEG-400, PEG-1500 or DMSO were developed.105 A formulation with PEG-400 and 25 µM Photolon had no absorption spectrum changes in relation to a Photolon solution. Yet, the addition of PEG-1500 caused red-shifted absorption and fluorescence bands. An increase in the lifetime of the excited singlet and triplet states and in the fluorescence quantum yield was also observed, without changes in the lifetime of the singlet oxygen. Further studies on biological tissues and basal-cell carcinoma of skin determined this topical formulation to be efficient and safe for the treatment of superficial diseases.106 More recently, Sobaniec et al. (2013) developed a gel of Photolon containing 20% 12 and 10% DMSO to treat oral lichen planus, the most common carcinogenic disease of oral mucosa.107 The gel was directly applied onto the wound in human patients one hour before illumination with a


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light dose of 90 J/cm2 in 2-week intervals at 660 nm. This treatment regimen reduced the lesions by 55%; nevertheless, further efficacy and durability studies are needed. An aqueous formulation composed of 12 (90-95%), sodium chlorin p6 (5-7%) and purpurine 5 is marketed under the trade-name Radachlorin® in Russia since 2009 (also known in the EU as Bremachlorin®). This pharmaceutical is the first water soluble chlorin with a short clearance time and has a high maximum absorption wavelength at 403 nm and a Q band at 646 nm in water, demonstrating high efficacy in the inactivation of microorganisms, such as S. aureus and E. coli,108 and disinfection.30 Other water-soluble derivatives of 12 containing alkyl ester groups or mono-L-aspartyl and called Talaporfin sodium (13, NPe6, Lasephyrin®, Figure 4) were more efficient at lower concentrations and, thus, may be excellent for topical use.109 It is noteworthy that 13 was approved in 2004 in Japan, and is currently in phase III clinical trials in the USA.

3.3.2

5,10,15,20-TETRAKIS(4-CARBOXYLATEPHENYL)CHLORIN (TPCC4)

Hyaluronic acid (HA) is an anionic polysaccharide with excellent properties such as biocompatibility, viscoelasticity and water absorption and retention, among others. When HA is combined with chitosan, the electrostatic interactions generate a stability that makes this formulation a promising drug delivery vehicle for transdermal layers. Hyaluronate-chitosan systems have not yet been used in PDI; however, some PSs such as 5,10,15,20-Tetrakis(4carboxylatephenyl)chlorin (14, TPCC4, Figure 4), 9 and 12 were encapsulated into this platform in PDT studies.110 Highly positive nanoparticles are cytotoxic and/or non-selectively taken up by cells. For this reason, anionic 14 was nanoencapsulated into chitosan hydrogels by electrostatic forces and surface decorated with hyaluronate to become negatively-charged. Initially,


19


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cytotoxicity in the dark was evaluated for each PS-nanogel with adherent murine RAW 264.7 and non-adherent human THP-1 macrophages. 12-nanogels were the only ones showing dark toxicity for concentrations higher than 20%; therefore, all PS-nanogels were tested at a maximum concentration of 17% in further in vitro PDT studies. Similar uptake of the three nanogels was found, although LD50 values in function of light doses were different for each PSnanogel (< 0.5 J/cm2 for 9, 2 J/cm2 for 14 and 12 J/cm2 for 12 at 652 nm).

3.3.3

BENZOPORPHYRIN DERIVATIVE MONOACID A (BPD-MA)

Benzoporphyrin derivative monoacid A111 (15, BPD-MA, Figure 4) is the first chlorin-type PS with marketing clearance. It has remarkable photophysical properties,112 such as λabs ≈ 688 nm, ε ≈ 33,000 M-1 cm-1 in methanol and ɸ∆ = 0.78 in phosphate buffer with 1% Triton X-100, however due to its hydrophilic properties and tendency to aggregate in aqueous solution, its bioavailability is limited. In terms of formulations, Verteporfin is the first liposomal photosensitizer in clinical practice, commercially called Visudyne®, and is composed of a 1:1 mixture of two regioisomers of 15 with 70x more phototoxicity than 4 in some cell lines.113 Verteporfin has short-term skin photosensitivity and has been used for diverse medical applications, including destruction of viruses114 and ophthalmological diseases.115-117 In PDI, a subcutaneously-inserted collagen gel with Verteporfin has been applied in the inactivation of Mycobacterium bovis bacillus Calmette-Guérin (BCG), the microorganism which causes tuberculosis in cattle and has the ability to infect humans. Combining 5 µM 15 and a diode laser system (690 nm) with a fluence light of 60 to 100 J/cm2 yielded a 26% survival of cells in vitro. Furthermore, this system was also developed in in vivo studies using infected mice


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and obtaining a noteworthy reduction in viable cells. This procedure could be a promising treatment in localized mycobacterial infections.31

3.3.4

5,10,15,20-TETRAKIS(3-HYDROXYPHENYL)CHLORIN (mTHPC)

5,10,15,20-tetrakis(3-hydroxyphenyl)chlorin118 (16, mTHPC or Temoporfin, Figure 4) is one of the most potent and effective PSs. It is not approved by the FDA, but has received clinical clearance in the EU for treating head, neck and other cancers.79, 119 Photoactivation is produced at 652 nm (ε = 30,000 M-1 cm-1) by red light and is commercially called Foscan® in solution form. Liposomal formulations of 16 in aqueous environments display similar absorption spectra as free 16 in ethanol120,

121

Some formulations, such as Fospeg® (PEGylated liposomes) and Foslip®

(dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol

liposomal

water-soluble

formulation), have been optimized to improve the pharmacokinetics, systemic side effects and specific drug delivery of 16.122-126 Johansson et al. (2006) developed a thermo-reversible gel called Fosgel® comprised of Foslip and Lutrol® F-127 20% (w/v).127 This gel is liquid at 4°C but solidifies at body temperature and was tested in a murine skin tumour model. Important selectivity between the lesion and healthy tissues was shown after 4 hours with a low concentration of 16 (6 ng/mg of tissue). The challenge with 16 is its high potency and prolonged skin sensitivity.111, 128, 129 To solve this issue, a gel formulation of liposomal 16 for topical applications in humans was used to treat nonpigmented skin malignancies.130 Gao and colleagues (2007) tested an ultrafine polyacrylamidebased hydrogel containing 16 in living cells.131 Efficiency was similar to free 16, but its ultrasmall size (~2 nm), low accumulation and easy removal from the body could make 16 a very interesting PS to use in PDT and topical PDI.


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Additionally, Bombelli et al. (2008) used Foscan as an antibacterial agent against S. aureus (MRSA and wild type).132 Several cationic liposomal formulations composed of Foscan, DMPC and some cationic surfactants derived from L-prolinol in different concentrations were evaluated for the inactivation of MRSA; however, only one of the tested formulations had comparable antimicrobial values to free PS with a 77% of entrapment efficacy. This study demonstrated how the stereochemistry of the surfactants affected the aggregation behaviour of the formulation and how a zeta potential (charge) of about 40 mV is mandatory for disrupting the cell membrane. Most recently, Yang et al. (2011) also used liposomal 16 but modified with an antimicrobial peptide (WLBU2), since its cationic character aids in the penetration of bacterial membranes.133 In vitro experiments showed this system to be effective for 16 delivery into microbial cells. Using a concentration of 1 µM after 90 minutes, there was a 3.3 log10 reduction of Pseudomonas aeruginosa and MRSA was completely eradicated.

3.4 PHTHALOCYANINES Phthalocyanines (Pcs) are porphyrin analogues composed of four isoindoles interconnected through nitrogen atoms. Pcs, and their metalloderivatives (MPcs), are also highly aromatic with 18 π-electrons (Figure 5) and used in the preparation of dyes and pigments. They have recently been applied to the synthesis of new molecular materials with electronic and optoelectronic properties. Yet, Pcs could be widely applied in topical gels or creams with promising results,134 as they show excellent physicochemical properties.135-137 For example, they have strong absorption in the red region (600-850 nm), greater tissue penetration138 and high singlet oxygen yields.139 Due to all these advantages, some formulations have been marketed, but only with essential chemical modification or addition of substituents surrounding the Pcs core.78


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Regardless, more efforts are necessary to solve the poor water solubility and strong tendency to form aggregates; for instance, a formulation into polymeric gels or nanoemulsions.140

Figure 5. Chemical structures of phthalocyanines used in pharmaceutical formulations for PDI.

3.4.1

ZINC PHTHALOCYANINE (ZnPc) AND DERIVATIVES

MPcs are often used as they are more stable against oxidation by singlet oxygen compared to pure Pcs. For instance, a gel incorporating zinc (II) phthalocyanine141 (17, ZnPc, Figure 5) has been developed for PDT of various skin diseases such as psoriasis or melanomas. This PS is very lipophilic, and only a few solvents are able to solubilize it. N-methyl-2-pyrrolidone (NMP) is the best solvent and was formulated in a non-aqueous topical gel composed of HPC-M (Hydroxymethyl cellulose type M) and PEG 400. This antibacterial gel formulation decreased the viable counts of C. albicans, Aspergillus niger, P. aeruginosa, S. aureus and E. coli strains, although these properties were also found with free NMP.32 Using oleic acid in propyleneglycol formulations to enhance penetration of 17, da Silva et al. (2011) demonstrated a higher uptake in


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moderate concentrations (5 or 10% w/w). However, when ≥20% (w/w) concentration was used, topical delivery was increased, but not transdermal.142 Recently, Wang et al. (2013) developed a novel, hybrid hydrogel (HHG) containing 17, poly(ethylene glycol) double acrylates (PEGDA), PEG 400, and phosphotungstic acid (PTA) for photodynamic applications. Here, compound 17 had a dual function. On the one hand, it acted as photoinitiator of hydrogel formation and, on the other hand, it was the PS for producing singlet oxygen. The PTA also played an important role as the co-initiator in the photopolymerization and as the catalyst for singlet oxygen generation. Important advantages of this system are the very short time to form the HHG shell around the cells after irradiation and highly ROS generation decreasing the diffusion of the PS and the damage to normal cells. Moreover, exciting the HHG shells with near-infrared light reduces the damage to the host and penetrates deeper than UV light.143 Rodriguez et al. (2010) combined several 17 derivatives (Figure 5) with different formulations of gels for topical uses. The polymeric gel platforms studied were: 1) a hydrophilic gel composed of 25% Cremophor® RH 40, 2) a gel with moderate lipophilicity composed of 20% Lutrol F127 and 30% polypropylene glycol, and 3) a lipophilic gel composed of 15% Lutrol F127, 10% Cremophor RH 40 and 15% polypropylene glycol. In the Lutrol-containing gels, fluorescence and

singlet

oxygen

quantum

yields

butylphthalocyaninato

zinc(II)

(18,

(ɸF

and

ɸ∆)

Figure

5)

were and

increased

using

tetra-t-

tetrakis(1,1-dimethyl-2-

phthalimido)ethylphthalocyaninato zinc(II) (19, Figure 5) in comparison with solutions of 17. However, 2,3,9,10,16,17,23,24-octakis(decyloxy)phthalocyaninato zinc(II) (20, Figure 5) and 2,3,9,10,16,17,23,24-octakis[(N,N-dimethylamino)ethylsulfanyl]phthalocyaninato zinc(II) (21, Figure 5) led to the formation of aggregates. The authors demonstrated how the photophysical


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parameters in vitro may predict the in vivo skin behaviour of semisolid formulations. Furthermore, the permeation studies showed that the location of Pcs in the epidermis occurs without risk of photosensitivity to surrounding tissues, showing the potential of the 17 derivatives in the treatment of psoriasis or dermatitis, for instance.144, 145 Most recently, Fraix and co-workers (2014) developed a novel multi-photoresponsive hydrogel platform based on the hydrosoluble polymers, poly-β-cyclodextrin and alkyl side chain-modified dextran, ZnPc tetrasulphonate (22, Figure 5) and a tailored NO (nitric oxide) photodonor. This hydrogel has: 1) stability in the dark, 2) the ability to release 1O2 and/or NO species (anticancer and antibacterial) under suitable wavelengths, 3) no competitive photoprocesses and 4) dualcolour red and green fluorescence. The remarkable stability of this hydrogel in a buffer solution at pH 7.4 at room temperature demonstrates its potential biomedical applications; however, the photobiological activity will be reported in further studies.146 [(1(4),8(11),15(18),22(25)-Tetrakis[(N,N,N-trimethylammonium-3-yloxy) phenyl)]phthalocyaninato]zinc(II) (23, RLP068/Cl, Figure 5) is a tetracationic 17 derivative with optimal physicochemical properties useful in PDI because of its penetration into the bacterial cell wall and membrane.147 1 mM RLP068/I (analogue of iodine of 23), easily formulated into a gel by Fabris et al. (2005), quickly penetrated into the epidermal layers of mice, but not into the dermis. Irradiation with red light (600–700 nm) produced a severe cutaneous damage without photosensitivity in distal skin areas.148 Consequently, this promising drug was marketed by Molteni Therapeutics S.R.L. (Italy) under trademark Vulnofast® as a gel formulation intended for topical administration in surgical wound infection by MRSA.33, 34

3.4.2

SILICON PHTHALOCYANINE DICHLORIDE (SiPcCl2)


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Similar to all Pcs, silicon phthalocyanines149 (SiPcs) also have high hydrophobicity. Liu and co-workers (2014) developed microgel particles encapsulating silicon phthalocyanine dichloride (24, SiPcCl2, Figure 5) and used lipid decoration to improve water solubility and minimize aggregation. This strategy is very interesting due to the combination of gel properties (or lipogel) with those of micro-/nanoparticles. Encapsulating the compound 24 into poly(Nisopropylacrylamide) (pNIPAM) using the lipids 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4yl) (NBD-PE) led to high release of the 24 (~80%) from the microspheres and an optimal encapsulation of HeLa cells, which were destroyed upon light irradiation. The authors proposed the pNIPAM/lipid system as a suitable carrier for other drugs in diverse applications.150

3.5 PHENOTHIAZINES Phenothiazines family are planar tricyclic compounds where the middle ring has heteroatoms such as sulphur and nitrogen, which are responsible for the potent biological activity.151 The backbone can be modified with substituent groups, which vary their photochemical and photophysical properties. They are amphiphilic compounds with cationic character in physiological pH, exhibiting a strong affinity to the cell membranes due to its lipophilicity. Also, they are widely used as antipsychotic and neuroleptic drugs.79

3.5.1

METHYLENE BLUE O (MBO)

Methylene blue O152 (25, MBO, Figure 6), belonging to the phenothiazine family has a strong absorption band in the 550-700 nm region with a maximum molar absorptivity of 71,500 M-1 cm-


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1

at 653 nm in methanol. It has a low fluorescence quantum yield (ɸF = 0.04) in water, but a high

singlet oxygen quantum yield (ɸ∆ ≈ 0.5) with low dark toxicity. Unfortunately, its medical applications are limited due to the enzymatic isomerization to leuko-25, thus losing its photoactivity.153, 154

Figure 6. a) Isomerization of 25 and b) chemical structures of phenothiazines used in pharmaceutical formulations for PDI.

PVA combined with boric acid and borax is widely used in many pharmaceutical applications, including PDI, as a promising novel drug delivery system.35 In fact, Donelly et al. (2007) reported an optimised gel formulation (8.0% w/w PVA, 2.0% w/w borax and up to 1 mg/mL 25 or 7) against MRSA. To simulate the conditions of an exuding wound, newborn calf serum was used without significant effects on the phototoxicity or hydrogel properties. However, when a combination of a calf serum and 25-based hydrogel were used, the photodynamic effect was considerably reduced. Eradication of C. albicans155 or P. aeruginosa required ~100-fold lower 25 concentrations than toluidine blue O (26, TBO, see section 3.5.2).156


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In a separate study, these same authors used iontophoresis in order to increase the efficacy of 25 or 7 against both gram-positive and gram-negative bacteria. The chosen polymeric platform contained crosslinked PEG with different concentrations of poly(methyl vinyl ether-co-maleic acid) (PMVE/MA - 10, 15 and 20% w/w). Hydrogels containing 10% PMVE/MA loaded with 7 or 25 offered the best results. Under an electric field at 37°C, PS release was ~40%. Incubation of 50 µg/mL 25 or 5 µg/mL 7 for 15 minutes followed by irradiation with a Paterson lamp (635 nm, 100 J/cm2) killed 100% of planktonic MRSA. Under the same conditions, 250 µg/mL 25 or 7 killed 100% and 99.9% of Burkholderia cepacia, respectively. It should be noted that dark cytotoxicity was observed in all conditions.36 Marketed formulations containing 25 to eradicate P. aeruginosa have been studied in vitro.157 Periowave® (Ondine Biopharma Corporation, Vancouver, Canada), composed of 0.01% w/w 25 in PBS and some preservatives, is approved in Canada and the EU for the photodesinfection of oral and nasal cavities. In an example study, the exposure of planktonic P. aeruginosa at >15.5 J/cm2 (670 nm) resulted in 100% eradication, whereas on biofilms grown for 48 hours, the viability was reduced by >99.999%. Recently, a clinically-tested gel composed of carboxymethylcellulose (CMC), 0.2% propylparaben, 0.1% methylparaben and 0.2% 25 obtained good cosmetic results and patient satisfaction for nodular basal cell carcinomas. Furthermore, the long-term effect was relatively safe.158 Additionally, 25 encapsulated in many systems and loaded into polyacrylamide-based hydrogels was tested in suspended cultures of S. aureus, P. aeruginosa, E. coli or Acinetobacter spp.159 Encapsulation decreased the dark toxicity of 25. Although all microorganisms were killed, the efficiency was much higher in gram-positive strains (S. aureus) than for gram-


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negative strains. However, additional optimization should maximise the efficiency of the nanoparticles.

3.5.2

TOLUIDINE BLUE O (TBO)

Compound 26 is chemically similar to 25 and partially soluble in pure water or alcohols with a maximum molar absorptivity of 51,441 M-1 cm-1 at 630 nm in aqueous solution.160 The physicochemical properties allows 26 to permeate the cell membrane and perform photobactericidal activity.161 A 26-based hydrogel incorporating the highly flexible liposomal nanoencapsulation system has been developed for topical application in locating tumours. The hydrogel composed of phosphatidylcholine, cholesterol, sorbitan monostearate (Span 60), sodium deoxycholate (SDC), CMC and 26 demonstrated an excellent entrapment efficiency (>82%), particle size (1.4 µm) and drug release (52%) within 2 hours. When a diode laser was applied at 650 nm and 90 J/cm2 for one hour in a mouse tumour treated with this hydrogel, an increase in the survival time and a decrease in the tumour size were observed. Furthermore, the higher tumour cell necrosis was attributed to the deep penetration of 26 in the skin.162 Very recently, a gel formulation using 0.2% 26 has been used against Trichophyton rubrum in a murine PDI model. A light dose of 42 J/cm2 using 630-nm LEDs for 10 minutes over a period of 7 days reduced fungal burden by 87%.37

3.5.3

3,7-BIS(DI-N-BUTYLAMINO)PHENOTHIAZIN-5-IUM

BROMIDE

(PPA904)


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3,7-bis(di-n-butylamino)phenothiazin-5-ium bromide (27, PPA904, Figure 6) was used at 0.026% in an Unguentum® M cream.163 A topical application time of 90 minutes for three consecutive sessions was found to be the optimal administration conditions. This novel formulation had excellent results in the treatment of cutaneous leishmaniasis in mice.38

4

CONCLUSIONS

Current semisolid formulations based on PSs have been obtaining very good results in PDT and PDI; however, their development and clinical practice is not yet widespread. Optimization of their physicochemical properties, creation of novel PSs and an improved understanding of the mechanistic processes involved in microbial inactivation are crucial to the development of this field. Some of the problems encountered during the development of these pharmaceuticals include: 1) low PS solubility in water, 2) aggregation in aqueous systems, 3) dark toxicity or 4) damage produced in surrounding tissues. Fortunately, nanotechnology platforms such as liposomes and polymeric micelles are helping to minimise some of these incompatibilities. In conclusion, the excellent inherent properties of semisolid formulations in drug delivery convert this combination into a powerful tool in the treatment of skin diseases or against microorganisms such as fungi and (multi)resistant bacteria. The study of topical administration of PSs for PDI is still in its infancy, providing excellent research and commercial opportunities for scientists (chemists, pharmacists, biochemists or engineers) in this field.

AUTHOR INFORMATION Corresponding Author


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*B.S.: phone +351 226074900; fax: +351 226094567; e-mail, [email protected] Notes The authors declare no competing financial interest Biographies José A. González-Delgado was born in Huelva (Spain) in 1986, and he graduated in Chemistry in 2009 from the University of Huelva. He received his Ph.D. degree in 2014 under the supervision of Prof. A. F. Barrero, Dr. M. M Herrador del Pino and Dr. J. F. Arteaga at the University of Granada. He is currently a Marie Curie post-doctoral fellow at Inovapotek, Pharmaceutical Research & Development, Lda (Portugal) under the supervision of Prof. Dr. Bruno Sarmento and MSc. Marta Ferreira working on the nanoencapsulation of photosensitizers and their formulation in semisolid systems (gels and creams) for photodynamic inactivation of microorganisms. Patrick J. Kennedy is a multi-disciplinary scientist in biotechnology and its applications in human health with an emphasis on drug discovery and delivery. He received a BSc in Ecology and Conservation from the University of Florida (USA) and an MSc in Marine Biology from the University of North Carolina (USA). He has worked in diverse academic (e.g. Scripps Research Institute) and industrial (e.g. Genomics Institute of the Novartis Research Foundation, Ablynx) settings in the USA and Europe. His efforts have generated several publications, marketed biotechnology reagents, and potential diagnostic and therapeutic compounds. Patrick is currently pursuing his doctorate, under the supervision of Prof. Bruno Sarmento, in targeted nanomedicine for cancer detection/treatment at the Instituto de Inovação e Investigação de Saúde (i3S).


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Marta Ferreira is graduated in Pharmaceutical Sciences and has two MSc in Pharmaceutical Sciences and Pharmaceutical Technology from the University of Porto (Portugal). Beginning her research career in pharmaceutical development as a graduate student, Marta Ferreira won the “Formulário Galénico Português” Award. Later, she specialized in the safety and efficacy evaluation of cosmetic products. In the follow up of the development and coordination of several R&D projects for the pharmaceutical and cosmetic industries, she started Inovapotek, Pharmaceutical Research and Development, in 2008. She is a member of the International Society for Biophysics and Imaging of the Skin (ISBS), the Portuguese Society of Cosmetic Sciences and the International Federation of Societies of Cosmetic Chemists (IFSCC). João P. C. Tomé graduated in chemistry from the University of Aveiro (UA, Portugal), where he also received his Ph.D. in 2001 working on porphyrin chemistry under the supervision of Profs. Graça Neves and José Cavaleiro. Following postdoctoral appointments at UA and CUNY, he returned to UA in 2008 as an Auxiliary Researcher. In 2013, he became a Senior Researcher at UA and Assistant Professor at Ghent University (Belgium). He has also been a Visiting Associate Researcher at Fluminense Federal University (Brazil) under a project funded by the Brazilian Government (CNPq). His current research interests include the synthesis of porphyrins and phthalocyanines with appropriate molecular properties mainly for: i) photomedicine; ii) photoinduced energy- and electronic-transfer materials; iii) optical (chemo)sensors; iv) catalysis and photocatalysis. Bruno Sarmento has a PhD in Pharmaceutical Technology from the University of Porto, is Assistant Professor of Pharmaceutical and Biopharmaceutical Technology at IUCS, and is an Affiliated Researcher and Team Leader at 13S and Institute of Biomedical Engineering (INEB), Porto, Portugal. He is also a co-founder of Inovapotek, Pharmaceutical Research and


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Development, that was started in 2008. His research is focused on the development of functionalized nanomedicines and their application in the pharmaceutical and biomedical fields. His team also develops biorelevant and optimized in vitro cell and tissue engineering models as tools for diagnosis and to evaluate the transport of drugs and nanoparticles through biological barriers.

ACKNOWLEDGMENT Thanks are due to the Universities of Porto and Aveiro, Fundação para a Ciência e a Tecnologia (FCT, Portugal), the European Union (EU), QREN, FEDER, COMPETE, for funding the QOPNA (project PEst-C/ QUI/UI0062/2013; FCOMP-01-0124-FEDER-037296), the INEB (PEst-C/SAU/LA0002/2013 and North Portugal Regional Operational Programme (ON.2 – O Novo Norte) research units. The research leading to these results has received funding from the European Union Seventh Framework Programme [FP7-PEOPLE-2012-ITN] under grant agreement n° [316975]. José A. González-Delgado thanks the EU for his Post-doctoral grant within the “SO2S” ITN.

ABBREVIATIONS USED PDI, photodynamic inactivation; DNA, deoxyribonucleic acid; PS, photosensitizer; ROS, reactive oxygen species; PDT, photodynamic therapy; UV, ultraviolet; FDA, Food and Drug Administration; DMF, dimethylformamide; DMSO, dimethylsulfoxide; W/O, water-in-oil; O/W, oil-in-water; w/w, weight/weight; w/v, weight/volume; LED, light-emitting diode; MRSA, methicillin-resistant Staphylococcus aureus; THES, tetrakis(2-hydroxyethoxy)silane; TMOS,


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tetramethoxysilane; CG-P, Chitosan gel-Photogem; HPC-M, hydroxymethyl cellulose type M; Pcs, phthalocyanines; MPcs, metallophthalocyanines, NMP, N-methyl-2-pyrrolidone; PEG, polyethylene glycol; HEMA, 2-(hydroxyethyl)methacrylate; MAA, methacrylic acid; CMS, carboxymethyl starch; DS, dextran sulphate; PVA, poly-vinylalcohol; SEM, scanning electron microscope; PBS, phosphate-buffered saline; MIC, minimum inhibitory concentration; PVP, Polyvinylpyrrolidone; LD50, lethal dose 50%; DMPC, dimyristoylphosphatidylcholine; EPG, egg phosphatidylglycerol; BCG, bacillus Calmette-Guérin; HHG, hybrid hydrogel; PEGDA, poly(ethylene glycol) double acrylates; PTA, phosphotungstic acid; NO, nitric oxide; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine;

NBD-PE;

1,2-dioleoyl-sn-glycero-3-

phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl); PMVE/MA, poly(methyl vinyl ether-co-maleic acid); CMC, Carboxymethylcellulose; pNIPAM, poly(N-isopropylacrylamide); SDC, sodium deoxycholate.

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EP/1578750-A2, EP/1578750-B1, ES/2301864-T3,

JP/2006515296-A, JP/4786182-B2,

JP/2011116766-A, NZ/540918-A, PT/1578750-E,

EP/1897883-A2, EP/1897883-A3, US/2004143001-A1, US/7244841-B2, WO/2004056828-A2, WO/2004056828-A3 and US/8084602-B2, 2011; Destiny Pharma Ltd.


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TABLE OF CONTENTS GRAPHIC


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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