Topical photodynamic therapy (PDT) has been applied to treat premalignant and malignant lesions such as actinic keratosis and non-melanoma skin cancer. A limiting factor of the technique is cream permeation and studies using chemical and physical approaches to overcome it have increased over the years. This chapter is going to explore the main techniques described in the literature used to improve the cream permeation or the photosensitizer (PS) distribution concerning homogeneity. Outcomes-based on animal studies and clinical trials comparing different delivery techniques are going to be presented, highlighting the aspects of invasiveness, costs, harmfulness, and effectiveness of those methods.
- topical photodynamic therapy
- drug delivery
- skin cancer
- protoporphyrin IX
Yet topical photodynamic therapy (PDT) principle is simple and the technique is full of potential to provide an effective treatment for very common malignant and pre-malignant diseases, achieving satisfactory outcomes for a varied number of lesion types with a single approach is not feasible.
Light delivery is successful depending essentially on the ability to provide the best combination of the most suitable wavelength for the drug of choice and the optical properties of the tissue, which determine if light reaches the extension of tissue necessary and if undesired side effects will show up.
Conversely, characteristics on the photosensitizer (PS) accumulation in target tissues are less dependent on direct control of the therapist than light delivery. Further, PS accumulation also defines the light dose (fluency). Therefore, PS accumulation must be properly dealt with in order to provide enhancement of the efficacy of PDT, and presenting the most relevant aspects reported in the literature concerning the strategies to do so is the scope of this chapter.
Topical PDT has been widely applied as a treatment option for pre-malignant lesions such as actinic keratosis (AK) and also for non-melanoma skin cancer (NMSC) lesions  such as basal cell carcinoma (BCC) and Bowen disease (BD). The treatment involves the topical application of a PS precursor, followed by the waiting for the drug light interval (DLI) to promote the PS accumulation into the lesion, after which the localized irradiation takes place. Depending on the protocol, this sequence can be repeated with intervals of one week [2, 3, 4, 5] or on the same day .
The molecules more commonly used worldwide are the aminolevulinic acid (ALA) and its derivative, methyl aminolevulinate (MAL). Both of them can promote protoporphyrin IX (PpIX) endogenous accumulation in cells. The DLI for these drugs can vary from 1 to 4 hours , depending on the protocol treatment chosen for the lesion type.
Being clinical PDT still a very empirical field, and since the protocols choices are decisive in determining the therapy outcome, several strategies, both physical and chemical, have been developed aiming to increase the availability of PS
2. Drug delivery strategies
Considering the light attenuation by tissue due to the presence of chromophores and to scattering, the wavelength range preferable for PDT is about 650–850 nm, which is known as the ‘optical window’ for PDT . Once the light penetration can be handled to reach the whole lesion, the remaining concern is to optimize the drug permeation to improve treatment rate success, especially obtaining the improvement of the response of thicker lesions, which shall increase the possibility to make PDT the treatment of choice for those lesions.
Human skin is a complex organ structured to protect the body. The skin layers organization and constitution, as represented in Figure 1 (
Although PpIX precursors have a low molecular weight, the PpIX accumulation after as much as three hours of incubation using exclusively topical application of the cream achieves depths up to 1–2 mm. However, the tissue heterogeneities imply in a non-uniform distribution of the produced PpIX, and portions of the lesion do not produce sufficient PS, impairing the treatment effectiveness [10, 11].
Several techniques have been developed to overcome skin barriers and provide penetration enhancement. There are physical and chemical pretreatment strategies to improve drug delivery that can facilitate precursor permeation through skin and into the lesions, allowing PpIX to be produced in deeper layers and also to be distributed more homogeneously.
3. Physical methods
This section presents physical methods based either on creating holes into the skin or on removing the SC to improve drug delivery in topical PDT.
3.1 Tape stripping
This technique consists in applying a tape onto the skin lesions and then to pull it off. By repeating this procedure a number times, which can be repeated using the same piece of tape, part of the SC is removed, reducing it uniformly by about 30%. This procedure is safe, since it is minimally invasive, and involves a low cost to be implemented; as a further advantage, it does not interfere with the intercellular lipids [12, 13, 14].
Tape stripping is one of the methods indicated to be applied before PDT; it can be used on patient’s lips and also for the preparation of large areas. The recommendation is to choose tapes with strong adhesive properties. According to Christensen
The curettage can be performed using a sharp ring curette or a scalpel blade. The purpose is to remove keratotic tissue by surface scraping the lesion. The scrape might be performed in a checkered pattern, performing parallel horizontal followed by perpendicular movements. Due to the possibility of subclinical lesion extensions, it is recommended that the curettage is performed using at least 5-mm margins surrounding the lesion to minimize the possibility of the recurrence. The curettage is also indicated to be applied before PDT [13, 15].
This technique is effective, quite inexpensive, and although it can offer light discomfort during the procedure, it is commonly acceptable both by health professionals and patients . However, it is important to mention that there is a risk of bleeding, and when bleeding takes place, it may displace the cream and lead to a decreased PpIX formation, and thus lower treatment efficacy . Despite this, studies reported that curettage associated with PDT increases the cure rate of treatment compared with no-curettage topical PDT. The combination may be repeated successfully for non-cure or recurrence cases without compromising the cosmetic effect or deteriorating skin structures. The treatment has shown a clearance rate of 91% for superficial BCC (131 lesions treated), 93% for thin nodular BCC (82 lesions), and 86% for thick nodular BCC (86 lesions) [21, 22].
Debulking (or deep curettage) is considered a surgical procedure and it is often used in thicker lesions, reducing as much as possible the total volume. This procedure is usually performed either immediately or a few weeks before PDT [15, 23]. Even though it is considered a more invasive procedure compared with the simple curettage, it can be applied without anesthesia. The cosmetic outcome is considered favorable  and allows a significant reduction of the BCC thickness, which makes nodular lesions more responsive to the treatment [24, 25].
Whereas the topical PDT efficiency is about 72% when a previous debulking lesion is not applied, for debulked lesions this efficiency can increase up to 92% [23, 26]. Therefore, the debulking associated with PDT is a relevant option for multiple, pigmented, and nodular lesions, improving clinical response .
3.4 Ablative fractional laser
This technique consists of using a laser source to create ablated channels on the lesion surface, vaporizing a small portion of the tissue . Usually, lasers with excitation wavelengths in far-infrared spectrum are used, due to the high absorption by the water in the tissue [29, 30]. The main types used are CO2-laser (10,600 nm), Er:YAG-laser (Erbium yttrium aluminum garnet, 2940 nm), and Er:YSSG-laser (Yttrium scandium gallium garnet, 2790 nm) .
The depth of the microchannels created varies according to the laser energy. The energies between 32 mJ and 380 mJ generate microchannels with a depth between 300 μm and 2100 μm . Due to the increased skin penetration, this procedure makes PpIX fluorescence both more intense and homogeneous in deeper skin regions, and this can be controlled by adequately adjusting laser energy density [32, 33].
Studies using CO2 laser associated with PDT for lesions treatments showed an increase of about 20% in the clearance of different protocols comparing the treatment without and with the use of laser, respectively [34, 35, 36]. Although it is a reliable and effective method, it is also a more invasive procedure with a high potential for skin damage with scarring, discoloration, and even infection. Besides that, the costs of the devices are considered high compared with the abovementioned techniques .
Needles and especially microneedles (MNs) have been used as a method to enhance drug permeation through SC . MNs have the advantages of being minimally invasive, its application is simple, well accepted by the patients, and it is cost-effective . MNs have been developed with lengths up to 900 μm to avoid penetrating in vascular and nervous regions. Therefore, MNs can penetrate the SC without stimulating pain receptors, which is another advantage .
The MNs size cannot be seen by patients, thus reducing possible needle phobia. MNs have strong mechanical properties to enable disruption and penetration through the SC, reaching successfully the deeper skin layers without causing any bleeding due to the small length of needles .
There are MNs with different shapes (tetrahedron, pyramidal, conical, beveled tip, or tapered cone) that promote different insertion pathways and permeation into the skin. They also can be divided according to the material, such as solid (metallic) or polymeric (hydrogel or dissolving) types, and can be assembled either as a roller or as low-cost patches. The MNs can also be produced with a drug coating on their surface, to facilitate delivery, or hollow, to promote the delivery through channels . The use of MNs rollers for enhancing penetration of topical cosmeceuticals has been well described and transdermal patches can be used easily by anyone [13, 39].
Some studies described a higher formation of PpIX in deeper skin regions when MNs are used and compared with topical application in animal models [41, 42]. A recent paper by Requena
Solid MNs have been commonly used for skin pretreatment before conventional PDT, promoting a good PDT response for actinic keratosis . However, their use for the treatment of BCC or squamous cell carcinoma SCC has not been investigated yet .
The use of dermographs has also been investigated to improve drug delivery. A dermograph is a device currently used in esthetic procedures, which works similarly to a tattoo machine, with simpler handling . The device is composed of a handpiece that couples to different types of needles which oscillate vertically. Usually, both frequency and depth can be adjusted in those devices, according to the application. The holes performed into the skin allow for greater skin penetration of liquids by capillarity [45, 46].
A more homogeneous PpIX distribution and a greater penetration in the tissue were observed for animal model using dermographs compared to the topical application of the cream only . A pilot clinical trial was also performed in nodular BCC lesions using a dermograph for intradermal delivery in PDT, and no recurrence was observed after 28 months of follow-up .
Temperature is a parameter with a strong influence on PpIX formation. The increase of the skin temperature (especially in areas that naturally present lower temperatures such as the extremities of the body) during the DLI can improve PDT effectiveness. There are four hypotheses regarding the PpIX formation with the increase of local temperature: increase of ALA penetration in the skin; increase of the conversion rate of ALA to PpIX; increased ALA uptake in the cells due to disruption of the cell membrane, and increase of the amount of skin oxygen due to vasodilation [13, 47].
However, the temperature must be well controlled in order to obtain the desired effect and prevent damage to surrounding tissues . Stringasci
Iontophoresis is a method based on the use of a voltage gradient applied to an electrolytic formulation, using both a positive (anode) and negative (cathode) electrodes on skin to improve transdermal drug delivery . The advantages of iontophoresis are that it is a non-invasive technique that preserves the skin intact, providing controlled and targeted delivery of the drug. However, regarding PDT, special attention should be taken to ensure that the photosensitizing drug does not enter the systemic circulation, which can cause unexpected side effects .
This technique has a potential to decrease the DLI since
4. Chemical methods
Currently, only three commercial pro-drugs are licensed for topical PDT use: 5-aminolevulinic acid (ALA, Levulan®), methyl aminolevulinate (MAL, Metvix®), and the nanoemulsion containing ALA (nc-ALA, Ameluz®) . Besides the physical methods to improve the delivery of these drugs, there are chemical approaches that have been explored for drug formulation modifications, and the use of agents that modify the heme cycle or enhance PpIX formation. Thus, the chemical methods to improve PpIX availability mostly treat on the effects over these substances.
To enhance ALA penetration through the SC, other prodrugs with longer, more lipophilic carbon chains have been explored . In this context, MAL – which has a methyl group associated with the ALA molecule – already presented more lipophilicity properties and increased selectivity.
The use of even more carbon chains has been tested, such as ethyl-ALA, propyl-ALA, butyl-ALA. The main concern about their use is related to the numerous chemical steps necessary to perform the esterification processes for those molecules . The pro-drug hexyl-ALA has been tested in pre-clinical studies [18, 56]. A pilot clinical trial reported that these molecules presented a greater penetration with lower concentrations compared with ALA or MAL which could reduce adverse reactions and decreases the PDT costs . Despite that, ALA and its ester MAL are still the most commonly chosen and investigated molecules to be used as PpIX pro-drugs for PDT.
One aspect explored in literature for the enhancement of the PpIX prodrugs in cells is the addition of iron chelators. Iron has a crucial role in cells within the heme synthetic route, bound to PpIX molecules to form hemoglobin, thus hindering this process preserves the number of available PpIX molecules within cells. Different chelators have been investigated so far as and, such as desferrioxamine, thiosemicarbazones, pyridoxalisonicotinoyl hydrazone, and di-2-pyridyl-derived iron chelators, among others [58, 59, 60]. However, yet thiosemicarbazones have been approved for phase II tests , the use of these chelators is still under discussion, due to toxicity versus efficacy issues.
4.2 Differentiation-promoting agents
In addition to the drug choice, different vehicles such as creams, gels, patch systems, lotions, liposomes, nanoparticles, powders, and microemulsions have been developed to overcome the penetration issue .
The commercial creams Levulan® and Metvix® are prepared with 20% w/w and 16% w/w concentration of prodrug, respectively. Both of them have been widely used with acceptable clearance rates and cosmetic outcomes. Aiming to increase the stability of the cream, the molecule of ALA at 7.8% concentration in a nanoemulsion-based gel has been used to produce the Ameluz® formulation. A multicentric study compared this gel formulation with the Metvix® cream in the treatment of AK, demonstrating similar response and tolerance .
A patch is an adhesive system used for topical or systemic drug delivery. For PDT, there is a commercial patch (Alacare®) containing 2 mg of ALA per cm2. A clinical trial using Alacare® for the PDT treatment of actinic cheilitis (AC) demonstrated high clinical efficacy, good tolerability, and favorable cosmetic effects . However, in the case of thick skin lesions without physical pretreatment, this protocol might be less efficient.
Nanotechnology has an important role in the pharmaceutical industry for the development of new formulations. Studies are concentrated on obtaining drugs with more solubility, biodistribution, bioavailability, uptake, and excretion, decreasing drugs’ toxicity,  and there are different vehicles to promote drug delivery by nanostructures.
Nanoparticles (NP) used to be defined as a particle with a size up to 100 nm in any direction . Lucky
Among the several NP possibilities, liposomes are one of the most used for topical PDT applications. Liposomes are vesicles made up of one or more phospholipid bilayers oriented concentrically around an aqueous compartment to act as drug carriers. This nanostructure has been explored for topical PDT to enhance the PS penetration into the skin while decreasing its absorption into systemic circulation. Examples of PSs currently encapsulated in liposomes for topical PDT are ALA, temoporfin (mTHPC, commercially available as Foscan®), and methylene blue. In most studies, liposomal ALA induced a higher PpIX synthesis than free ALA non-encapsulated delivery .
Other structures are more commonly used for systemic applications of PDT, such as nanofibers and nanomicelles [68, 69] but they are off the scope of this chapter, yet their results are also promising for PDT application.
4.4 Thermogenic and vasodilating substances
Thermogenic and vasodilating substances have been reported as promising to favor the permeation of drugs on the skin [70, 71]. In PDT, vasodilation also increases the oxygen supply in the tissue, which can further optimize the effectiveness of the treatment.
A study was carried out investigating the association of the substances: menthol, methyl nicotinate, and ginger associated with ALA and MAL. The association of methyl nicotinate with MAL demonstrated 50% higher PpIX production after three hours of incubation compared with the cream containing only MAL. These preclinical results are promising as a possible strategy for decreasing the DLI and increase the PpIX production in skin lesions .
This chapter presented several approaches reported in the literature for the improvement of topical PDT outcome. Some of the techniques here presented to address the task of increasing the availability of PS are increasingly being incorporated to PDT protocols, whereas others are very incipient. Yet, the challenge of making PS available is still an open field, plenty of room for further investigation.
We acknowledge support from São Paulo Research Foundation (FAPESP) [grant numbers 2013/07276-1 (CEPOF), 2009/54035-4 (EMU), 2014/50857-8 (INCT)].