Nanotechnology and photodynamic therapy from a clinical perspective

Photodynamic therapy (PDT) is currently applied clinically in many medical centers worldwide. The clinical outcomes are from one side satisfying and from the other side highlighting the areas of further development. Issues like hydrophobicity of the photosensitizer (PS), uncontrolled distribution and limited tissue penetration of the accompanying light sources triggered the interest of many research groups. Nanotechnology outstood among the various suggested enhancement solutions. In this review, the rationale behind using nanotechnology is discussed. Light is shed on the status of nanotechnology from approval for clinical use. Clinical studies of PS‐loaded nanoparticles are summarized and the challenges facing the progress of those systems are enumerated.


| THE NEW PDT AVENUE SHAPED BY NANOTECHNOLOGY
A nanoparticle (NP) is defined by the European union in 2011 as "A natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 to 100 nm." Since only dimension is needed to be in the nanoscale, there are countless number of NPs. They vary widely in composition, shape, physiochemical properties and pharmacokinetic profiles. According to their dimensions, they are classified into four different classes ( Figure 1). The first class of NPs includes all NPs with zero dimension. That implies to quantum dots, solid NPs and hollow NPs. The second class is for the one-dimensional NPs including nanowires, nanofibers, nanorods and nanotubes. The third class is for the two-dimensional NPs such as nanosheets and nanofilms. The last class is for the three-dimensional NPs exemplified by liposomes and dendrimers [1].

| GREEN SYNTHESIS OF NPs AND THE USE OF NATURAL POLYMERS ARE FAVORED
For biomedical applications, a great emphasis is given to biodegradability, biomimicry and safety of NPs. Biogenic and green chemical approaches for the synthesis of NPs is therefore favored over the conventional methods. It was proven that biogenic silver NPs were six times less toxic to normal cells compared to their chemically synthesized counterparts. In vivo studies further confirmed this finding where green silver NPs were four times less toxic to treated mice. It is worthy to mention that they exerted an anticancer activity and a synergistic antibiofilm activity with their conjugated Ciprofloxacin and Gatifloxacin [2]. Moreover, naturally driven polymers are favored over synthetic ones and they exert a therapeutic effect. Chitosan is one of the heavily studied natural polymers for its anti-inflammatory [3], antimicrobial [4], and antioxidant [5] activities. Chitosan-based films, spheres and hydrogels were loaded with various PSs where reports corroborated enhancement in PDT response for both oncologic and nononcologic applications [6]. This versatile delivery system can accommodate both cationic PSs such as methylene blue (MB) and anionic ones such as Rose Bengal. Compared to free PSs, the bactericidal and antibiofilm of both MB and Rose Bengal were significantly enhanced when they were conjugated with polycationic chitosan NPs [7]. Silk fibroin is another example of bio-extracted polymers with many favored characteristics such as facile modification since it is rich of functional groups. It was electrospun in a form of a multi-layered nanofiber along with other polymers such as cellulose acetate and polyethylene oxide. The nanofibers were loaded with MB and used for PDT-mediated antibiofilm and wound healing [8]. Silk fibroin hydrogel managed to secure a sustained release of loaded PS with satisfying skin permeation [9]. Examples are numerous and Table 1 summarizes some of the recently reported studies employing nanotechnology advances for PDT applications.

| HOW NANOTECHNOLOGY CAN ADVANCE PDT
Third generation PSs are highly needed, and numerous studies are investigating methods to overcome the limitations of second generation PSs such as undesired PS accumulation, aggregation, lowered therapeutic responses and limited tissue penetration.

| NPs increase the bioavilability of hydrophobic PSs
Majority of the naturally occurring PSs belong to the tetrapyrrole aromatic structures or phthalocyanines where their highly conjugated chemical structures render them hydrophobic [24]. Consequently, their administration results in very low absorption rates. A Plethora of NPbased formulations were proven to significantly improve the cellular uptake of the loaded PSs and the efficiency of their mediated PDT was consequently increased. In a recent study, molecularly engineered benzo[c]thiophene organic PS was formulated in polyethylene glycol NP. The nanoform of the PS shifted its absorption maximum from 804 to 843 nm and exhibited aqueous stability for 60 days. This enhanced dispersibility allowed for both in vitro and in vivo applications. The preparation of PS in the nanoform did not diminish the ROS production ability upon irradiation. It is worthy to mention that this nanosystem mediated PTT, PDT and photoacoustic imaging with a preferential accumulation in the liver and spleen of the treated mice [25]. Recently, the FDA approved PS meta-tetra(hydroxyphenyl)chlorin commercially known as Foscan ® in some countries and in others as Temoporfin was complexed with different forms of the three-dimensional supramolecular organic frameworks. Self-assembled NPs were formed, and their diameter ranged from 50 to 160 nm. The NPs increased the absorption intensity at 670 nm by 2 to 5 folds, the rate of singlet oxygen production and the quantum yield by 2 to 3 folds compared to free Foscan ® . They were inert to healthy cells and decreased the concentration needed to kill 50% of the tested cancer cells (IC50) by 2 to 3 folds compared to free Foscan ® . Moreover, for 12 days after treatment, the tumor volumes of mice treated with free Foscan and the NPs were 50% and 90%, respectively compared to the control affirming the enhanced PDT effect mediated by the NPs [26]. The devastating nail fungal infection caused mainly by the dermatophyte Trichophyton rubrum may last for years and is characterized by high rate of recurrence. Nails impose a barrier to proper drug accumulation. This obstacle was resolved and nail uptake increased by 2.3 folds with the aid of polymeric NPs loaded with the photosensitizer Chlorin e6 and supplied with nail penetration enhancer [27].

| Nanotechnology allows for targeted accumulation of PSs
Nanotechnology can offer a classic example of passive targeting. Cancerous tissues accumulate the nanoconstruct by means of enhanced permeability and retention F I G U R E 1 Schematic presentation depicting classification of nanoparticles (NPs) into four groups according to the dimensions and listing examples for each group. QDs, quantum dots effect, which is facilitated by the small size of the NPs that can easily diffuse within the defected membranes of the tumor cells. In one of the studies, Chlorin e6 was conjugated with human serum albumin. This conjugate allowed for a better dispersion of Chlorin e6 in phosphate buffer saline in contrast to the insoluble free Chlorin e6. At the in vitro level using Hela cells as a model, the conjugate did not provide a higher cellular uptake or singlet oxygen production or PDT efficiency. However, at the in vivo level, treating the tumor with the conjugate was twice as efficient as using free Chlorin e6 [28]. Another strategy for targeting is to utilize mesenchymal stromal cells for their tendency to migrate and imbed themselves within the tumor stroma. By means of T A B L E 1 Summary of some of the reported studies in 2022 on the use of NP-based PS for PDT enhancement

Strategy
Nanosystem composition Application References

Poly lactic acid NPs loaded with Curcumin and Nisin
Antibiofilm and wound healing [23] Abbreviations: 5-ALA, 5-aminolevuluonic acid; AuNPs, gold NPs; Ce6, chlorin e6; ICG, indocyanine green; MB, methylene blue; MSN, mesoporous silica nanoparticles; PEG, polyethylene glycol. electrostatic interaction, the mesenchymal stromal cells were loaded with tetra-sulfonated aluminum phthalocyanin NPs composed of fluorescent poly-methyl methacrylate. The NPs were internalized in the mesenchymal stromal cells without exerting a cytotoxic or antimigration activity to them. Co-culturing the mesenchymal stromal cells with 2D and 3D osteosarcoma cells followed by irradiation caused a significant decline (below 90%) in the viability of both cell types. The tumoricidal activity was corroborated at the in vivo level and was confirmed by the diminished cell viability of the collected cancerous tissues 35 days after treatment [29]. Platelet-membrane was also employed for targeting purposes of synthesized NPs. For the treatment of breast cancer, liposomes loaded with Chlorin e6 coated with platelet-membrane were significantly superior to the uncoated counterpart in ROS production measured at the in vitro level, accumulation at the tumor site and tumor inhibition rate at the in vivo level [30]. Preferential PS accumulation in cancerous sub-populations that are over-expressing the folate receptor was achieved when titanium dioxide NPs were loaded with Al(III) Phthalocyanine chloride tetrasulfonic acid and decorated with folic acid. The selectively was confirmed by the higher cellular uptake in folic acid over-expressing Hela cells in comparison to the folic acid low-expressing A295 cells. Another proof of selectivity was the Hela low cellular uptake when cells were pretreated with free folic acid in a way that rendered the receptor unavailable for receptor-ligand mediated endocytosis [31]. A sub-cellular level of targeting can be achieved by carefully designed nanoplatforms. Self-assembled micelles of BODIPY-based PS were chemically linked with the mitochondrial targeting tetraphenylporphine. The synthesized NPs caused a red shift in the absorption maximum of the PS which granted a deeper tissue penetration. The PS was released faster in the acidic medium compared to the neutral one, which indicated its suitability for the PS delivery to the acidic cancerous regions. In comparison to the free PS, the NPs accumulated more in the mitochondria, produced more ROS upon irradiation and caused a four time more reduction in the mitochondrial membrane potential. At the in vivo level, the NPs targeted to the mitochondria reduced the tumor weight to 90% of the control while both the free PS or the untargeted NPs declined the tumor weight to only 50% of the control [32]. Lysosomal targeting was also attained using self-assembled NPs loaded with helicene-based PS. Those NPs overcame the troublesome feature of water-insolubility of the free PS and mediated type I and II PDT [33]. Photochemical internalization that involves the irradiation-mediated release of macromolecules such as siRNAs in the cytoplasm is greatly facilitated by NPs. To treat Psoriasis using siRNA in charge of reducing the level of proinflammatory tissue necrosis factor alfa, polymeric lipid NPs were loaded with the PS meso-tetraphenylporphyrin sulfonate. The NPs were also loaded with the desired siRNA. They protected the siRNA and upon irradiation, it was released in the cytoplasm declining the level of tissue necrosis factor alfa and thus improved the skin condition of the treated mice [34].

| Nanotechnology allows for the construction of multimodal systems
One of the greatest advantages of PDT is its suitability for repetitive treatment session without eliciting a significant resistance to therapy compared to other therapeutic modalities such as chemotherapy [35]. Resistance to therapy is a challenging condition and a combinational therapy is one of the successful strategies to overcome it. Chlorin e6 and 5-ALA PDT were reported to be of a lower efficiency to cancer cells over-expressing a transmembrane ATB binding cassette transporter sub-family G2. Combining PDT with an inhibitor to this transporter (KO143) restored PDT efficiency by virtue of increasing the PS cellular uptake [36,37]. PDT can be easily combined with other treatment protocols and NPs can be easily modified to be multifunctional and serve as platforms for multimodal therapies.

| PDT and PTT can be combined through nanosystems
PDT and photothermal therapy (PTT) are commonly combined for an augmented therapeutic outcome. In one of the studies, PDT and PTT were combined for the treatment of breast cancer. Curcumin was the selected PS and magnetic iron oxide NPs coated with silica layer was employed to mediate PTT upon irradiation using 808 nm. The prepared nanocomposite exhibited a colloidal stability at neutral pH for a whole week and Curcumin release was triggered by irradiation. Monotherapies were not efficient in eradicating the tumor mass in 4T1 mouse model. On the other hand, intratumoral injection of the nanocomposite followed by 3 min irradiation using 460 nm diode laser (150 mW/cm 2 ) and then 7 min of 808 nm (0.5 W/cm 2 ) managed to reduce the tumor size by 98% relative to the control group within 2 weeks after treatment. Treated tissues showed an over-expression of the pro-apoptotic markers such as Caspase 3 and Bax indicating that cell death mode involved apoptosis [38]. In another study, PDT was combined with PTT through a smart nanosystem that consisted of mesoporous carbon dopped with manganese dioxide and loaded with Chlorin e6. Irradiating Chlorin e6 with 670 nm resulted in an elevation in ROS level and mediated PDT while irradiating the mesoporous carbon with 808 nm allowed for a hyperthermia and mediated PTT. For targeting, the surface was engrafted with the membrane of the cancer cells 4T1. This nanoplatform was able to deplete the antioxidant enzyme Glutathione oxidase and simultaneously increase the intracellular O 2 level opposing the hypoxiamediated resistance to PDT. Furthermore, this construct was capable of efficiently acting as magnetic resonance imaging contrast agent and allowed for imaging [39]. The generation of oxygen was also reported when zinc peroxide NPs coated lipid nano capsules containing indocyanine green. Irradiating the construct with 808 nm resulted in a significant raise in the temperature (PTT) that was cytotoxic and triggered the release of indocyanine green from the lipid NPs. The subsequent 670 nm irradiation produced ROS that mediated the caspasedependent apoptotic nasopharyngeal cancer cell death both at the in vitro and in vivo levels [40]. Sono-PDT was also combined with PTT. Chitosan NPs were loaded with indocyanine green and gold nanoclusters, and the surface of the NPs was decorated with hyaluronic acid to mediate the delivery to CD44 over-expressing cancer cells. Indocyanine green release was significantly higher in the acidic medium than the neutral one and the rate was the fastest when the NPs were irradiated using ultrasound and near infrared irradiation. The system allowed for a temperature raise up to 65 C and the produced singlet oxygen was three times higher than the control while each monotherapy showed only two folds increase. Treated cancer cells showed a significant decline in the mitochondrial membrane potential. Furthermore, the tumor weight was the lowest when B16F10 bearing mice were treated with the combined therapy through the nanoplatform better than through the physical mixing of gold nanoclusters and indocyanine green [41]. In another recent study, PDT and PTT were combined using iridium dioxide NPs that acted as both the PS and the hyperthermia-mediating agent. The NPs exerted a catalase-like activity as well. They were linked to glucose oxidase that catalyzed the conversion of glucose into hydrogen peroxide. The latter was enzymatically converted into oxygen by the iridium dioxide NPs and thus this nanozyme could drop the hypoxia up to 95% of the control 3D cellular structures and increase the responsiveness of the 4T1 cells to PDT. For targeting purposes, the NPs were linked with hyaluronic acid. The combined therapy aggravated the antitumoral activity of the applied monotherapies and suppressed tumor growth completely for 12 days without exerting any toxicities to the vital organs such as heart, kidney and spleen [42]. Methicillin resistant Staphylococcus aureus and kanamycin-resistant Escherichia coli were completely eradicated using a combined PDT and PTT protocol. Polymerized mesoporous carbon NPs (below 100 nm in diameter) were loaded with oxygen saturated perfluorohexane and indocyanine green. Upon 808 nm irradiation, both hyperthermiamediated and ROS-mediated antibacterial activity was achieved, and membrane disruption was evident. Infected wounds were very responsive to the treatment as they were free from bacterial colonies and had a total wound closure in 12 days. The oxygenation provided by the oxygen saturated perfluorohexane promoted wound healing via stimulating collagen synthesis and neovascularization [43].

| Chemo-PDT combinational therapies mediated by nanosystems
Chemotherapy is one of the standard therapies and is frequently combined with PDT. It was recently reported that to combine PDT and chemotherapy, a self-assembled NP system was synthesized. It consisted of Chlorin e6, the anticancer drug monomethyl auristatin E and the linker between them was a Caspase 3 cleavable peptide. The synthesized NPs were spherical in shape with an average size of 50 ± 20 nm, and maintained their stability in saline for 4 days. Only in the presence of active Caspase 3, which is predominantly found in cancerous microenvironment, the nanosystem was cleaved where the anticancer drug induced an apoptotic cell death and Chlorin e6-mediated PDT resulted in a cytotoxic activity. The viability of the treated mouse squamous cell carcinoma using the nanosystem was three times lower than the respective one when either monotherapy was applied. At the in vivo level, the combined therapy succussed to totally suppress the growth of the tumor mass over 2 weeks after administration while free Chlorin e6-PDT slowed down the tumor growth to be one third of the control group [44]. Clinical ovarian cancer tissues showed an over-expression in CD44 at the mRNA level and samples of patients with poor prognosis showed a significantly lower CD44 protein expression relative to samples of patients with complete remission. Since CD44 expression is associated with stemness, a hyaluronan nanosystem consisting of Pheophorbide A linked through a ROS cleavable linker with the chemotherapeutic SN-38 was synthesized. Upon irradiation, Pheophorbide A triggered the production of ROS that from one side exerted an apoptotic cell death mode and from another side cleaved the linker and released SN-38 in an irradiation dose-dependent manner. This release pattern allowed for paracrine cytotoxic effect where unirradiated cells lost their viability. The cellular uptake was hyaluronic acid-CD44 mediated and a significant accumulation in the tumor tissue compared to other organs was confirmed. The chemo-PDT was twice as effective as PDT alone in suppressing the growth of the tumor for a month [45]. A facile synthesis of the nanosystem was recently reported by the self-assembly of the chemotherapeutic Paclitaxel and porphyrin-based PS by manipulating the selfassembly conditions without the need for external excipients. Those NPs were free from a classical carrier system and Paclitaxel release was tightly controlled [46]. The chemotherapeutic Salinomycin that acts as cancer stem cell inhibitor was co-loaded with indocyanine green and paramagnetic ferric cations along with a fluoreinated magnetic resonance imaging agent in nanoemulsions. This system showed a synergistic chemo-PTT and PDT activity in addition to the bioimaging ability [47].

| NPs CURRENT STATUS FROM APPROVALS
The first U.S. Food and Drug Administration (FDA) approved NP-based drug for therapeutic purposes is Doxil ® which was approved in 1995. Doxil ® is a pegylated liposomal form of doxorubicin [48]. It was in 2000, when the National Nanotechnology Initiative was officially declared in the United States. This governmental research and development initiative assembled a consortium of governmental departments, private sector agencies and commissions to collectively shape the vision of nanotechnology aiming to industrial revolutions with societal impacts (https://www.nano.gov/about-nni). China and Japan preceded since their respective national initiatives were announced in 1990 and 1995, respectively [49]. Dedicating funding, educating future workforce and creating jobs in this field are the top goals of such national initiatives. Those efforts resulted in an increasing number of NP-based preparation that are approved by national health entities for biomedical applications. Currently there are nanoformulations that received both FDA and European Medicines Agency (EMA) approvals for the clinical use in multiple indications including diagnostics and therapeutics (Table 2). It is worthy to mention that there are above 80 formulations that are approved by particular countries [50]. It is anticipated that the number of approved NPs will keep escalating since many clinical trials are registered driven by industrial interest.
With respect to PDT, the liposomal form of Verteporfin commercially known as Visudyne ® manufactured by Novartis AG was approved in 2000 by FDA and EMA for the management of Age-related macular degeneration. This PS is accumulated in low density lipoprotein overexpressing blood vessels at the macula and upon irradiation using a nonthermal 689 nm light source, these blood vessels are occluded which preserves the vision [51].
Visudyne is currently being tested at the clinical level for other indications or combinational treatment protocols. A recruiting phase I/II clinical trial (NCT04590664) is registered by Emory University in collaboration with National Cancer Institute (NCI). The trial aims to study the side effects and best dose of Visudyne for the treatment of high-grade epidermal growth factor receptor -mutated glioblastoma. Verteporfin is administered in this trial as a chemotherapeutic agent without the irradiation step. Another recruiting phase II trial sponsored by Mayo clinic in collaboration with NCI (NCT03033225) studies the possibility of ultrasound endoscope imaging combined with Visudyne-PDT in treating advanced unrespectable pancreatic cancer. A completed phase I/IIa clinical trial registered under the number of (NCT02872064) investigated the use of Visudyne-PDT in the treatment of primary breast cancer. A total of 12 patients were recruited and the study took place in the university college of London. Another completed phase I/II multicenter clinical trial (NCT00007969) was conducted to investigate the possible use of Visudyne-PDT combined with Detox-B adjuvant for the treatment stage III/IV melanoma.

| Acne treatment
In one of the reported clinical trials, MB oil in water nanoemulsion of around 200 nm in diameter was prepared and formulated in the form of a gel for a better applicability on the skin. The gel did not hinder the release of MB since 100% was released within an hour. Ex-vivo assessment proved a reasonable deposition within the rat skin layer where the deposition was as follows: 22.5% ± 1.4 in stratum corneum and 44.3% ± 3.3 in the remaining skin layers. A prospective, split-face clinical trial including 15 patients diagnosed with mildmoderate acne cases was conducted. The facial right side was treated with pulsed dye lasers (Cynosure 585 nm of 10-mm spot size, 10-milliseconds pulse duration and 7.5-8.5 J/cm 2 ). The left facial side was treated with MBnano preparation applied for 10 min and followed with irradiation for 2 min (a 665-nm diode laser at an intensity of 765 mW/cm 2 , 150 mW power and 5 mm spot size). Three sessions were conducted at a rate of 1 session/14days and a follow-up was planned 6 weeks after the last session. Both inflammatory and noninflammatory acne lesions responded very well to the treatments. Mean inflammatory lesion count reduction exceeded 75% at the follow-up interval while the respective one in case of nano-MB-PDT and noninflammatory lesion was 68%.
Overall, there was no significant different between both treatments. Nevertheless, nano-MB-PDT was superior in the reduction of acne severity and tolerability of the treatment where patients did not witness erythema, peeling, swelling, crusting, postinflammatory hyperpigmentation or scarring in contrast to the other modality [52]. This study clearly showed the positive relation between PDT and patient compliance. The treatment protocol was well-suited for an outpatient clinic application where the incubation phase lasted for only 10 min. However, a control group of free MB-PDT was not included and thus the added value of nanoformulation was not addressed at neither the ex-vivo level nor the clinical level.

| Hidradenitis suppurativa treatment
In another study, free MB-PDT was compared to nano-MB-PDT for the treatment of Hidradenitis suppurativa [53]. The nanoformulations prepared were noisomes which are more stable than conventional liposomes [54]. The optimized noisome form showed a loading efficiency of 86%. For an adequate topical application, both the free MB and the nano-MB were formulated in a final gel form. The release kinetics showed that noisomes imposed a delay in MB release from the gel when compared to the free MB. A total of 11 eligible patients were enrolled in a single-blind, randomized, comparative split-body study.
One side was treated with free MB while the other side was treated with the nano-MB-PDT. In both cases, gels were placed for half an hour and then lesions were irradiated using intense pulsed laser fitted with a 630 nm filter (2.5 Â 4.5 cm 2 spot area, total area 11.25 cm 2 , 20 ms pulse duration and 25 J/cm 2 fluence). Sessions were applied once per 2 weeks for a maximum of 6 months. Nano-MB-PDT managed to reduce the lesion size by 77 ± 18% while free MB-PDT resulted in only 44 ± 20% lesion size reduction. Recurrence was reported in some cases 3 to 6 months after free MB-PDT. Although this study demonstrated the superior clinical outcomes nanotechnology can provide to PDT, the design did not provide insights about the possible role played by the laser itself. Clinically, intense pulsed laser was proven to be an effective treatment of hidradenitis suppurativa with minimal recurrence up to 1 year [55].

| Plantar warts treatment
Plantar warts were treated at the clinical level using MB encapsulated within a nanovesicular form known as transferosome [56]. Transfersomes are highly elastic and deformable version of the liposomes that can easily penetrate through tight pores and thus are well suited for topical applications [57]. In this single-single blinded, placebo controlled clinical trial, a total of 38 patients were divided into three groups. The first was treated using the gel that contained free MB. The second group had a total of 14 patients that were treated using the gel containing tranfersomes (≈720 nm) loading with MB with a loading efficiency of ≈60%. The third group was a placebo one treated with a blank gel. Incubation time lasted for 30 min and was followed in all group with irradiation (a diode laser, 670 nm, 81 J/cm 2 , 15 min). Treatment sessions were scheduled as once per week for a maximum of 6 sessions and follow-up lasted for 8 months. Complete lesion healing was achieved in nano-MB-PDT group in 86% of the treated lesions, while it was only 53% in free MB-PDT group. It is worth mentioning that the placebo group showed 10% complete lesion healing. A significantly lower number of treatment sessions was needed in case of transfersomes loaded with MB-PDT group with an average of 2.2 sessions, while free MB-PDT treatment protocol required 4.14 sessions. For the entire follow-up duration, no recurrence was reported in any of the MB treated groups. Combined PTT and PDT was also accomplished at the clinical level. Rose Bengal was conjugated with goldpolypyrrole NPs having a diameter of 50 to 100 nm. The conjugate had a negative zeta potential and was evaluated for the treatment of recalcitrant plantar warts. In this trial, 9 patients with a total of 23 lesions were treated by the application of the prepared conjugated for 10 minutes followed by irradiation using intense pulsed light fitted with a 550 nm filter, with a fluence of 20 J/cm 2 . A total of four sessions were applied. This treatment protocol managed to achieve a total lesion cure in 60% of the lesions. Partial cure was recorded in 27% of the lesions and the rest did not respond [58]. This study did not allow for treatment followup to check for possible recurrence. At the in vivo level, the therapeutic effects of each monotherapy, combined therapy and their respective dark controls were examined. It was concluded that the combined therapy was more efficient and thus it was the only group tested at the clinical level. Although this has spared the number of patients involved in the trail, but the difference in the conditions needed at the in vivo and clinically should be taken into consideration. Although the application was topical and limited to 10 min, the associated nanoformulation allowed for a deep skin penetration.

| Vitiligo treatment
Vitiligo was successfully treated using 8-methoxypsoralen incorporated in transethosome of 200 nm in diameter with a loading efficient of 87% [59]. The latter is a form of polymeric NPs that combine the benefits of ethosomes and transfersome allowing for a deep skin penetration especially for hydrophobic drugs [60]. The ex-vivo evaluation of the permeation showed that the gel containing the nanoform managed to deposit ≈200 μg/cm 2 while the free drug suspension allowed for the permeation of only 20 μg/cm 2 . In this clinical trial, 15 patients diagnosed with acral vitiligo were recruited. One hand was treated using only the narrow band UVB irradiation while the other hand was incubated first for 30 minutes with the nanoform containing 8-methoxypsoralen. Irradiation protocol was 250 mJ/cm 2 with an incremental increase of 10% in the following session according to patient tolerability. Sessions were scheduled two times per week for a total of 12 weeks. Vitiligo extent score for a target area evaluating the repigmentation % showed that the regions treated with nano-8-methoxypsoralen had a significantly better score when compared to the treatment protocol using only the narrow band UVB. Concrete information about the concentration of applied 8-methoxypsoralen was not available. The authors described it as "A very thin film of the prepared optimized formula." The absence of free 8-methoxypsoralen-PDT at the clinical level made it difficult to quantify the added value of the nanoform at the clinical level.
Vitiligo was also treated with bergamot oil-PDT. Bergamot oil was encapsulated in lipid NPs of 210 nm bearing a negative zeta potential and could release 100% of the loaded bergamot oil within 24 hours. In a prospective clinical trial, 23 patients diagnosed with vitiligo were recruited. They were divided into two groups, the first was the light control one in which patients were treated with narrow band UVB with a starting irradiation dose of lower than 0.8 J/cm 2 for adults and 0.6 J/cm 2 for children. The second group was incubated for 30 min with nanobergamot oil followed by irradiation. Sessions were conducted once per week. Results revealed that 40% of the patients diagnosed with grade 2 and grade 3 vitiligo had re-pigmentation. As the severity of the cases increased, the response to nano-bergamot oil-PDT decreased. Only 10% of the patients with grade 4 vitiligo had re-pigmentation [61].

| Palmar hyperhidrosis treatment
Palmar hyperhidrosis is one of the dermatological diseases that were treated using transfersomal hydrogel of eosin yellow. The prepared NPs were spherical in shape with an average diameter of ≈300 nm and the encapsulation efficiency was 33%. A group of six patients diagnosed with bilateral palmar hyperhidrosis were treated with both fractional CO 2 laser and PDT. The procedure was to apply two passes of CO 2 fractional laser to facilitate deep penetration of the afterwards applied transfersomal hydrogel of eosin yellow for 5 min followed by irradiation using intense pulsed light. Treatment sessions were conducted once per week for a total of 6 weeks. Results showed that only four sessions were needed for 33% of the cases to achieve 90% improvement. For three patients, a total of six sessions were required to achieve 75% improvement and only one patient showed 25% improvement at the end of the sessions [62]. The very limited number of patients enrolled in this study made it difficult to investigate possible reasons for varied response. Previous clinical studies showed that free eosin yellow PDT was efficient in treating palmar hyperhidrosis [63]. Thus, this clinical trial design hindered investigating the therapeutic significance of this combined therapy involving NPs of eosin yellow over respective free eosin yellow PDT.

| Tinea capitis treatment
Curcumin is one of the PSs that suffer from poor bioavailability due to its hydrophobic nature [64]. It was formulated in the form of a nanospanlastic that is characterized with high elasticity and deformability making this form suitable for transdermal drug delivery [65]. A randomized controlled comparative clinical study was conducted in which 52 children diagnosed with the fungal infection of tinea capitis were enrolled. A Group of the patients (n = 13) was treated with only the blue light (120 mW/cm [2]), for 16 minutes using light emitting diode system (450 nm, 3 cm spot size) to serve as a light control group. Another group was treated with the standard antifungal drug Griseofulvin. The third group was treated with only curcumin with no irradiation to serve as a dark control group. The last group was treated with nano-curcumin PDT that was spherical in shape with a diameter of 180 nm, loading efficiency of 85% and could release 80% of its loaded curcumin over 24 hours. Sessions were scheduled as one session per 14 days and the maximum number of sessions was 6. Nano-curcumin PDT managed to achieve a complete cure in 48% of the cases. On the other hand, 100% cure was reported in the group treated with the antifungal agent. In the nano-curcumin-PDT group, two patients witnessed mild-moderate pain sensation during the PDT session, while 3 cases of the antifungal treatment group reported GIT side effects in the form of nausea and cholic. None of the control groups showed a significant therapeutic outcome. No recurrence was reported in case of nano-curcumin PDT for the whole 6 month follow-up duration, while one case of relapse was reported in the antifungal group [66]. This clinical trial opens the door for the consideration of PDT as an adjuvant therapy to conventional ones. This combination might decrease the dose required of the conventional therapy lowering their associated side effects.

| Basal cell carcinoma treatment
Indocyanine green was encapsulated in transfersomes of 125 nm, loading efficiency of 53% and release rate of 80% within 2 hours. In this clinical trial, 10 patients diagnosed with basal cell carcinoma were included. They had a single lesion that was treated with the nano-indocyanine green for 30 minutes followed by 160 J/cm 2 irradiation dose using diode laser 820 nm. Sessions were scheduled as once per week for a maximum of six sessions and patients were followed-up for 6 months. Cure rate was 80% with no recurrence over the follow-up period. Mild erythema and edema were reported during the sessions [67].

| CHALLENGES FACING CLINICAL APPROVAL OF NP-BASED PDT
Hundred clinical trials were conducted that were compiled and lead to the approval of Visudyne. To repeat this successful story, many impediments should be resolved. The above-mentioned studies are deficient in: • Sample size: sample size of the reported studies does not allow for proper analysis and grouping. In most of the studies, no group was assigned to the plain nanoform to examine for possible effects and assure their freedom of toxicities. The involvement of multicenter studies is highly recommended. • Long-term follow up studies: most of the studies left questions about possible long-term toxicities or recurrence rates unanswered. • Consistency: although studies conclude that an enhancement in the therapeutic outcome was achieved, the chronological sequence of studies planned as phase I, phase II and phase III was not reported. Neither was the indication nor the composition of the preparation building up. In other words, studies were discrete steps and in such a manner, no accumulation of knowledge is possible. • Industrial interest: the mentioned studies were funded by scholars without financial support from the private sector. Bulky investments and a dedicated R&D sector drive the filing process and boost their progress. Although the used PSs are clinically approved, approving authorities consider a change in the composition as a new drug and require a whole new filing procedure which imposes a financial burden. This aroused the interest towards registering a new indication to the already approved Visudyne. • NP scalability: the reported nanoformulation are advanced modified forms of the conventional liposomes. The fine protocols for the synthesis might impose a hindrance towards large-scale production and associated quality control protocols.
Collective efforts are needed from laser experts, chemists, formulation specialists, physicians and industrial partners to complete the lengthy process of NPbased PS approval for clinical use.

| CONCLUSION
The application of PDT is advantageous over conventional therapeutic modalities in many aspects. From one side, PDT is not associated with major side effects. It is easily combined with many other modalities such as photothermal, radiotherapy, chemotherapy and immunotherapy for a synergistic outcome. Minimal resistance is developed against PDT and multiple session protocols are commonly applied. The use of nanotechnology managed to augment PDT efficiency. NPs resolve the troublesome impediment of hydrophobicity of many PSs. They direct the accumulation of PS towards the needed cellular and sub-cellular targets. NPs can impose a tight control over the release pattern and rate of the loaded PS. Multimodal therapies are greatly facilitated by designed NP systems that can act as theranostics not only therapeutics. The number of preclinical and clinical trials of NP-based PDT is increasing and promising results have concurred the need for further investigations.