Photodynamic Therapy for Bladder Cancers, A Focused Review

Abstract

Bladder cancer is the first cancer for which PDT was clinically approved in 1993. Unfortunately, it was unsuccessful due to side effects like bladder contraction. Here, we summarized the recent progress of PDT for bladder cancers, focusing on photosensitizers and formulations. General strategies to minimize side effects are intravesical administration of photosensitizers, use of targeting strategies for photosensitizers and better control of light. Non-muscle invasive bladder cancers are more suitable for PDT than muscle invasive and metastatic bladder cancers. In 2010, the FDA approved blue light cystoscopy, using PpIX fluorescence, for photodynamic diagnosis of non-muscle invasive bladder cancer. PpIX produced from HAL was also used in PDT but was not successful due to low therapeutic efficacy. To enhance the efficacy of PpIX-PDT, we have been working on combining it with singlet oxygen-activatable prodrugs. The use of these prodrugs increases the therapeutic efficacy of the PpIX-PDT. It also improves tumor selectivity of the prodrugs due to the preferential formation of PpIX in cancer cells resulting in decreased off-target toxicity. Future challenges include improving prodrugs and light delivery across the bladder barrier to deeper tumor tissue and generating an effective therapeutic response in an In vivo setting without causing collateral damage to bladder function.

INTRODUCTION

Bladder cancer is the 4^th most common cancer among men and the 10^th most common in women in the USA (1,2). It was estimated that in 2022 there would be around 81 000 new bladder cancer cases with approximately 17 000 bladder cancer deaths. Bladder cancer is more common among older people, with the average age of patients diagnosed with bladder cancer being 73. The lifetime risk of getting diagnosed with bladder cancer is 1 in 27 men and 1 in 89 women. Bladder cancer is more frequent in Caucasians than in African or Hispanic Americans. Following age as the most critical risk factor for bladder cancer, smoking is also a strong risk factor for bladder cancer. Tobacco smoking has approximately 50% of population attributable risks (PARs) in both men and women (3). Over the years, tobacco smoking has become increasingly associated with bladder cancer in the USA (4).

The term bladder cancer refers to urothelial carcinoma of the umbrella cells in the bladder lumen, consisting of tumors in the upper urinary tract and the proximal urethra as well (5). Approximately 90–95% of urothelial carcinomas are bladder cancer. Bladder cancer can be categorized into non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC) based on the tumor, node, metastasis (TNM) system. NMIBC can be divided into Tis, Ta and T1, where Tis is the tumor in situ comprised of a flat tumor only present in the mucosa. In both Ta and T1, papillary tumors are typically contained in the mucosa. MIBC can be classified into T2a, T2b, T3 and T4. The T2a and T2b tumors invade the muscle layer, whereas the T3 tumors invade beyond the muscle layer, and T4 tumors spread beyond the bladder to nearby organs such as the prostate, vagina, etc (6).

Common approaches to treat bladder cancer, specifically NMIBC, are to remove the tumor with transurethral resection of bladder tumor (TURBT) and then use either intravesical Bacillus Calmette-Guerin (BCG) immunotherapy or chemotherapy (7–10). If the patient is nonresponsive to BCG or not eligible for chemotherapy, then immunotherapy with pembrolizumab is given (11). Although these treatment procedures can help achieve initial tumor control, nonspecific toxicity and side effects along with recurrences remain a major barrier to the treatment of NMIBC (12). For low and intermediate-risk NMIBC, mitomycin C (MMC) is the most frequently used intravesical chemotherapy option to control recurrences and progression (13). MMC can control recurrences up to about 20–30% when used weekly as passive diffusion (14). Recurrences result from incomplete TURBT, which can be due to inadequate diagnosis and reimplantation of tumor cells during the TURBT procedure (15). Recurrence associated with MMC is also related to the pharmacokinetics of MMC in the bladder wall. MMC concentration in the urothelium was 30 folds less than the concentration in the urine (16). Another major concern among bladder cancer patients is the high financial burden (17) due to the high recurrence rate and the required costly ongoing invasive monitoring (18). The cost to manage a bladder cancer patient from diagnosis to death ranges from $89 287 to $202 203, which makes bladder cancer the costliest cancer to manage in the USA (19).

Photodynamic therapy is a unique technique to kill cancer cells with the use of a specific wavelength of light along with the presence of a suitable photosensitizer. Photosensitizers are administered either systemically or in a site-specific manner, intravesically, intratumorally, topically or intraperitoneally. Once illuminated with a specific wavelength laser, a nontoxic photosensitizer can be activated and transfer electrons to molecular oxygen, an electron acceptor that converts into singlet oxygen, ¹O₂ (Fig. 2). Singlet oxygen (SO) can produce a cytotoxic effect by inducing necrosis or apoptosis, thereby causing destruction of the tumor or associated vasculature (20). PDT has been FDA approved for the treatment of esophageal, skin and non-small-cell lung cancer (21) and is currently being evaluated in multiple clinical trials for bladder cancer. The first incident of successful PDT use in bladder cancer dates back to 1975 when Kelly et al. (22) demonstrated cases of tumor destruction using hematoporphyrin derivatives (HpD) and light in a subcutaneous xenograft of human bladder tumor in an immunocompromised mouse model. In 1982, Benson et al. (23) demonstrated the first case of bladder tumor detection using cystoscopic examination after the instillation of HpD. Photofrin-PDT was first approved for the treatment of bladder cancer in 1993 in Canada.

Figure 2.

The basic mechanism of photodynamic therapy in bladder cancer. After either intravenous or intravesical PS administration, PS accumulates in the tumor. After illumination with light of a specific wavelength, PS moves to the 1^st or 2^nd excited singlet state through interconversion. From this state, PS can either return to the ground state releasing fluorescence or move to the excited triplet state via the intersystem crossing. While returning to the ground state from the excited triplet state, PS can release energy as phosphorescence. At the triplet state, PS can also convert molecular oxygen ³O₂ into singlet oxygen (¹O₂) or radicals causes cell death by apoptosis and necrosis.

Others have reviewed the use of photodynamic therapy for cancer treatment in general and bladder cancer treatment in particular (24–34). Here we summarize recent progress in PDT and PDD for bladder cancer since its first use in 1975. This includes a review of the major photosensitizers used in bladder cancer PDT either In vitro or In vivo and the advances in drug delivery using nanotechnology and homogenous light distribution. We also briefly discuss our lab’s strategy to combine photosensitizers with prodrugs to treat NMIBC (Fig. 1).

Figure 1.

Key milestones in the use of PDT and PDD in bladder cancer.

PHOTOSENSITIZERS

Photosensitizer (PS), molecular oxygen and light constitute three major components of PDT. The photosensitizer, after a suitable route of administration, accumulates in different organs of the body. An ideal photosensitizer would selectively accumulate only in the target, tumors in case of cancer treatment. Activation of PS depends on illumination with the appropriate wavelength of light in the excitation range of the PS. Once illuminated, PS can convert molecular oxygen from the ground state (³O₂) to a more reactive singlet state (¹O₂). Singlet oxygen can produce type I (³O₂ to radicals) and type II (³O₂ to ¹O₂) photodamage reactions, both capable of tumor destruction. Fluorescent PS can also be used for cancer diagnosis since the illuminated PS releases energy as fluorescence. Treatment efficacy and diagnosis selectivity and sensitivity depend mainly on the PS. Here we briefly discuss some of the most commonly used PSs used in preclinical and clinical studies of bladder cancer (Figs. 2 and 3).

Figure 3.

Structures of the photosensitizers, hematoporphyrin (35), photofrin (36), hypericin (37), 5-ALA or HAL-based PpIX (36), TLD1433 (38) and chlorin e6 (39).

Hematoporphyrin derivatives (HpD)

To synthesize HpD, Hematoporphyrin was first reacted with a mixture of acetic acid and sulfuric acid to yield Hematoporphyrin mono- and di-acetate and hydrolytic treatment with base yielded HpD (40). Scherer first made hematoporphyrin in 1841 (27), but it was not until 1976 when HpD was first tried to diagnose and treat bladder cancer in patients (41). In 1983, Tsuchiya et al. (42) reported the first total regression of bladder tumors in the clinical setting by photodynamic therapy. With a follow-up of 6–18 months, complete remission was achieved in 6 patients using an argon dye laser to irradiate the bladder. In most studies, HpD was administered IV at a dose of 2.5 mg kg⁻¹, 48–72 h before being illuminated with a 630 nm endoscopically delivered light at a power of 200 mW cm⁻² for 30 min with a total light dose of 100 J cm⁻² or more (43). In an attempt to deliver HpD locally to avoid systemic side effects, a polyurethane film containing HpD (PU-HpD) was developed as a delivery vehicle for the human bladder cancer cell. By controlling HpD concentrations, as well as irradiation times, excessive reactive oxygen species (ROS) were generated, which led to the inhibition of cell growth and death (44). To reduce unwanted toxicity and to improve cisplatin’s selectivity, hematoporphyrin—cisplatin (Hppt) conjugates were developed (45). Hppt was further conjugated to PEG to improve the selectivity and increase water solubility. The PEG-Hppt complex achieved higher DNA platination than cisplatin alone and provided higher efficacy (46). While HpD-PDT achieved a complete response in over 50% of patients with noninvasive transitional cell carcinoma with follow-up for up to two years, HpD caused hematuria, pain and difficulty in urination within a few days after the treatment (43).

Photofrin

Photofrin^® (Porfimer Sodium, QLT Phototherapeutics, Inc., Vancouver, BC, Canada), a second-generation photosensitizer, was purified from the HpD using size exclusion chromatography to remove low-molecular-weight compounds (47). Photofrin was first approved for bladder cancer in Canada in 1993 and then approved for early-stage lung cancer and advanced obstructive esophageal cancer in Japan and by the US FDA in 1994 and 1995, respectively. While FDA approved Photofrin for non-small-cell lung cancer in 1998, it was never approved for bladder cancer in the USA (21). Photofrin can be activated up to 5 mm deep into the tissue when illuminated with 630 nm light, resulting in muscle damage after treatment (48). A study with 36 patients with carcinoma in situ and BCG failure had a 58% response rate to whole-bladder PDT with Photofrin over a three-month period (49). Standard regiments consist of an IV injection of 2 mg kg⁻¹ and the illumination of the bladder after 48 h with 630 nm light with a dose of 120–180 J cm⁻² (49). The main advantage of Photofrin is that it is water-soluble as a sodium salt and is easy to administer as an IV. Disadvantages include Photofrin accumulation in the skin, which can produce skin toxicity for up to 6 weeks post-treatment upon exposure to sunlight or high-intensity light (47). The most serious complication after whole-bladder PDT with Photofrin was permanent bladder contraction, which occurred in ~19% of patients (49).

Hypericin

Hypericin, a phenanthroperylenequinone, selectively accumulates in transitional cell carcinoma upon intravesical administration into the bladder. After intravesical administration, hypericin can be photoactivated at 595 nm, yellow light, and does not damage the detrusor muscle when used with 12–48 J cm⁻² (50). In 2000, Hypericin was first reported for the detection of papillary and transitional cell carcinoma and was especially useful for detecting carcinoma in situ, demonstrating high sensitivity and specificity. A study of 40 patients found that hypericin-induced red fluorescence was present in all papillary tumors, and hypericin could detect carcinoma in situ as well with 93% sensitivity and 98.5% specificity (51). The usual dose for the diagnosis consists of an 8 mM intravesical instillation for 1–2 h followed by a fluorescence cystoscopy (52). The photodynamic effect of Hypericin mediated apoptosis is induced by the photo-oxidative endoplasmic reticulum stress by the activation of PERK (stress sensor) and induction of Noxa, which contributes to the PERK-induced apoptosis (53,54). The hypericin-mediated photodynamic effect is independent of p53, and thus the status of p53 does not influence its efficacy (55). Whole bladder PDT using hypericin was unsuccessful in treating bladder tumors in rats where tumors recurred within three weeks of treatment, possibly due to oxygen depletion during light irradiation (50). Many approaches have been taken to enhance hypericin efficacy, such as fractionated drug dosing, chemical modifiers, combination with hyperthermia, different formulations, etc., but they did not always offer superior efficacy (56). Hyperoxygenation along with oxygenation intervals resulted in complete phototoxicity in spheroids of RT-112 cells (57).

TLD1433

TLD1433 is a Ru(II) polypyridyl complex and was the first ruthenium (II) based photosensitizer to enter clinical trials and successfully completed a phase 1b human clinical trial (ClinicalTrials.gov Identifier: NCT03053635) (38). TLD1433 uses an orange to red (625 nm) light for photoactivation. It took approximately six years from the initial synthesis of TLD1433 to enter into the clinical trial for NMIBC. Although much progress has been gained in understanding the mechanism of action of TLD1433, the subcellular localization and mechanism of phototoxic action are not fully understood yet. TLD1433 is considered to be 200 times more selective toward bladder cancer cells than the normal healthy bladder tissue (58). TLD1433 is administered intravesically into the patient’s bladder using a catheter, followed by an irradiance with the laser. TLD1433 was also tested in cancer cell lines (T24, AY-27) using green light (530 nm) to protect the muscle layer from PDT damage, and successful tumor damage up to 1.5 mm depth was achieved. In a pharmacokinetic study, the plasma concentration of TLD1433 was below 10 ng/kg after a single intravesical instillation of 6 mg mL⁻¹ (59). A phase I clinical trial was conducted to evaluate the safety and tolerability, and primary, secondary and exploratory endpoints were met with no tumor recurrences up to 90 days after treatment. Three patients receiving the optimum therapeutic dose also showed no evidence of disease at 180 days (58). A phase II study is ongoing (ClinicalTrials.gov Identifier: NCT03945162) to evaluate TLD1433 in 125 patients with BCG-unresponsive or BCG-intolerant NMIBC. TLD1433 is also being evaluated in conjunctival melanoma (38) and human lung adenocarcinoma (60).

Aminolevulinic acid (5-ALA)

5-ALA, a common precursor of hemoglobin and chlorophyll, produces protoporphyrin IX (PpIX) in the mitochondria through the biosynthetic pathway (61). Along with other cancers, bladder cancer experiences abnormal activity of ABCG2, a porphyrin transporter that is upregulated in hypoxia, and porphyrin synthetic enzymes (coproporphyrinogen III), which promote PpIX production and inhibit its catabolism (reduced ferrochelatase activity in cancer) (62–66). These contribute to the higher abundance (9–16 times) of PpIX in cancer cells compared with the normal urothelium, thereby greatly improving the cancer selectivity of PpIX (49). PpIX can be photoactivated by visible blue light (375–445 nm) to emit red fluorescence and is used for ALA photodynamic diagnosis (ALA PDD) (62). The phototoxic effect can be achieved when excited by either 450–580 nm (green light) or 630 nm (visible red) light. 5-ALA-PDT was tested in multiple clinical trials, mostly in CIS bladder tumors, which resulted in a 30–50% response rate over 1.5–3 years. Phosphatase-sensitive prodrugs of 5-ALA were developed that can be activated by phosphatases and have reduced acute toxicity than the parent drug, 5-ALA (67). Another group has combined silybin and ALA-PDT to test for synergistic effects with the hope of controlling metastases. While they could not achieve evidence of synergistic or additive effect, the combination was able to impair cell migration as an effect of the ALA-PDT and silybin combination (68). While 5-ALA was great in achieving tumor cell selectivity over non-neoplastic cells, 5-ALA suffered low lipophilicity with poor penetration when applied topically. It was also associated with poor bioavailability and significant toxicity when administered orally or intravenously (69, 70). With the great selectivity achievable with 5-ALA, it is a promising tool to be used in combination therapies with PpIX-PDT.

Hexyl aminolevulinate (HAL)

Hexyl ester of 5-ALA was developed to solve the limited diffusion of the more polar 5-ALA into tissues, i.e. the bladder epithelium (69). HAL was approved by the US FDA in 2010 for the diagnosis of non-muscle invasive bladder cancer (71). HAL-based photodynamic diagnosis (PDD) provides additional curative benefits beyond pure detection. In two studies, positive patient outcomes were found in patients who have undergone HAL-based diagnosis prior to cystectomy compared with white light cystoscopy. In a study of 224 patients, the 3-year recurrence-free survival of the patients who underwent radical cystectomy (RC) after HAL-, 5-ALA- and white light cystoscopy (WLC)-based diagnosis was 77.8%, 53.6% and 52.4%, respectively (72). Another study of 131 patients found that 5-year cancer-free survival for the patients who underwent RC after a HAL-guided TURBT or WLC-guided TURBT was 90.9% vs 55.8%, respectively (73). A recent study found that diagnostic doses of HAL and blue light can be combined with a checkpoint inhibitor to improve therapeutic efficacy. In an orthotopic rat model, 30 days positive antitumor effect was improved from 31% to 38% when PDD was combined with an intravesical anti-PD-L1 checkpoint inhibitor (74). Using HAL reduced the PpIX formation time to just 2 h by using just a fraction of the amount of 5-ALA (8 vs 180 mM) (75, 76). In a clinical trial in 2013, HAL was used with 380–700 nm (white light) for PDT, and almost 53% and 12% of patients were tumor-free after 6 and 21 months, respectively (62). Despite tumor selectivity, low efficacy due to the shallow penetration depth of the excitation light of HAL is still a barrier to this promising drug becoming a mainstream bladder cancer treatment agent.

HAL-based PDT uses green light with a penetration depth of up to about 1 mm. At the time of the treatment if the tumor depth is larger than 1 mm, then it is unlikely that the light will be able to activate the PS (PpIX generated by HAL). Although it is expected that HAL will be able to penetrate the tumor volume and generate PpIX, but treatment efficacy is mostly limited by the penetration depth of the incident light. PpIX can also be activated by 630 nm (red) light, which can penetrate deeper into the tissue (>20 mm), but the excitation efficacy is lower than the green light.

Chlorin e6

Despite the natural abundance, most chlorophyll-based photosensitizers pose poor solubility and form aggregates. While chlorin e6 (Ce6), a chlorophyll derivative, is more soluble with improved photosensitizing properties (39), it is still unstable in an aqueous solution causing fluorescence quantum yield to be reduced (77). Several forms of Ce6 were developed to overcome these issues, which are discussed later in this review in the nanoformulation section. Ce6 PDT was used in the cytotoxicity assay using the human bladder cancer cell lines, T24 and 5637. Cell monolayer and multicellular spheroids were used and found that Ce6 could achieve more than 80% cell kill in both monolayer and multicellular spheroids and it was speculated that Ce6 might exert cytotoxicity by producing ROS and by reducing superoxide dismutase activity (78). Ce6-polyvinylpyrrolidone (Ce6-PVP) was developed to treat human bladder cancer. Patients with BCG nonresponsive recurrent NMIBC were treated with Ce6-PVP at 665 nm and 10–24 J cm⁻² light. Out of five patients who underwent PDT, after a 29-month follow-up, two patients were disease-free, two had tumor recurrence and one had tumor progression to the muscle layer with none experiencing immediate adverse effects (79). Low-dose PDT with Ce6-PVP was also useful at reducing the resistance of bladder cancer cells to other treatments, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (80).

Others

There are a few other less commonly used photosensitizers in bladder cancer PDT. Among those, Chlorin e4 was found to kill about 82% and 85% of cells when tried against T24 and 5637 bladder cancer cells In vitro (81). Another photosensitizer, tetrahydroporphyrin-tetratosylat (THPTS), excites at 760 nm, which can penetrate up to 15 mm, was tried in rat orthotopic muscle invasive bladder cancer and achieved 60% tumor reduction compared with control at two weeks after PDT (82, 83) (Table 1).

Table 1.

Commonly used photosensitizers in bladder cancer with their excitation light wavelength, route of administration and clinical complications.

Photosensitizer (name/class)	Excitation wavelength	Route of administration	Complications
HpD	630 nm	IV	Hematuria, pain and difficulty in urination
Photofrin	630 nm	IV	Muscle damage
Hypericin	595 nm	Intravesical	Low efficacy
5-ALA	531 nm	Intravesical	Poor bioavailability
HAL	531 nm	Intravesical	Low efficacy
Ce6	665 nm	Intravesical	Poor aqueous solubility and low efficacy

PHOTODYNAMIC DIAGNOSIS

In 1975, after the first use of HpD in the fluorescent diagnosis of bladder cancer, several other attempts were made to reach the current stage of PDD. In 1987, Photofrin was first used for bladder tumor diagnosis (84). Synthetic photosensitizers produced low-intensity fluorescence and required prolonged photosensitization time and sophisticated equipment (85). The standard form of bladder cancer diagnosis consists of white light cystoscopy or photodynamic diagnosis (PDD), and it is well-studied that PDD improves sensitivity and decreases recurrences when PDD was used with TURBT (86–94). 5-ALA and hexyl ester of 5-ALA could produce more intense fluorescence, thus requiring simpler equipment than that of Photofrin, and the mechanism of 5-ALA selectivity was discussed earlier. Oral administration of 5-ALA at a dose of 20 mg kg⁻¹ was well-tolerated during the TURBT procedure (95). Another study also found that specificity does not differ with variable oral 5-ALA dosing, but selectivity increases with increased dose, and selectivity was highest among the patients who received ≥20 mg kg 5-ALA (63% vs 96% with white light cystoscopy (WLC) vs fluorescent light cystoscopy (FLC), respectively) (96). Until the development of HAL, bladder cancer diagnosis consisted of cystoscopy combined with biopsy to detect low-grade tumors, which often resulted in false-negative results (97).

Based on a study with 113 patients where 57 patients were given PDD and 56 patients were in the control group with white light cystoscopy (WLC). FLC could detect 26% more tumors than the WLC control group. The FLC group also showed a significant reduction in tumor recurrences, 17%–20% fewer recurrences up to a 5-year follow-up period (98, 99). In another study of 345 patients who received TURBT, the 3-year recurrence rate (3YRR) was 39% for the PDD group compared with the 53% 3YRR in the WLC group. The benefit of PDD was more pronounced in high-risk patients, where the 3YRR was 52% and 80% for the PDD group and WLC group, respectively (100). PDD sensitivity was also about 92% when used for 2^nd-TURBT, muscle invasive bladder cancer and patients with BCG failure (101).

PDD is not only more efficient but also cost effective for both 5-ALA- and HAL-PDD. Burger et al. (102) did an extensive study on the cost-effectiveness of PDD over WLC consisting of 301 patients using 5-ALA PDD. After a median follow-up of 7.1 years, recurrence was 18% vs 42% and 0.8 vs 2 TURBT was performed in the PDD group vs WLC group, respectively. On average, 0.3 vs 1.0 bladder cancer recurrence occurred, costing €420 vs €1750 per patient in the follow-up period for PDD vs WLC groups, respectively. After considering a single expenditure of €135 for PDD setup, WLC was still more expensive by €1195 and cost €168 more per patient per year over the 7.1-year follow-up period. Another study with 115 patients using 5-ALA PDD estimated that with PDD, $425 could be saved per year per patient (103). More recent studies also suggested that PDD with hexyl ester of 5-ALA can also be more cost-effective than WLC. A cost-effectiveness analysis of NMIBC patients in Italy estimated an average savings of €435 per patient with PDD over WLC (104). A study based on US healthcare found that HAL-PDD would cost $25 921 over 5 years vs $30 581 using WLC for the diagnostic cystoscopy (105). There are some recent studies focused on improving fluorescence diagnostic efficiency. One recent study demonstrated that chlorin e6-type photosensitizers can also be a promising tool for diagnosis, followed by TURBT (106). Ce6-PVP was used to diagnose bladder cancer in a chick chorioallantoic membrane (CAM) model (77). Ce6-PVP was found to selectively localize in the implanted human bladder cancer tissue rather than CAM tissue.

One of the barriers to PDD is its potential false-positive result as the tumor detection and analysis are mostly operator-dependent and accuracy improves with experience. It is still controversial whether PDD is more beneficial than WLC, as many believe that with careful evaluation of the bladder using WLC one can detect bladder tumors as efficiently as FLC. Apart from these concerns, FLC requires an upfront cost of sophisticated machinery and personnel training (88). Recently, another cost-effective approach was tried in a rat bladder tumor model where Evans blue 1 and 5 mM were instilled in the rat bladder and found that Evans blue was selectively taken up by the tumor tissue in the malignant bladder compared with the healthy bladder (107) (Table 2).

Table 2.

Pros and cons associated with IV versus intravesical photosensitizer PDT.

IV Photosensitizer PDT	Intravesical photosensitizer PDT
Benefits: The administration of IV PSs is straightforward, and the plasma PK can be monitored over time. An intravenous formulation can be used to solubilize insoluble PS and avoid aqueous insolubility	Benefits: It is possible to avoid systemic toxicity by delivering the drug locally. In contrast to IV administration, plasma PS concentrations can be much lower because only a small fraction enters the systemic circulation. The continuous production of urine can clear out excess drugs quickly from the healthy bladder tissue. Formulations such as hydrogels or polymeric films can be used to achieve sustained drug release
Cons: PS is distributed throughout the body, which makes the patient susceptible to sunlight and photodamage. Achieving therapeutic concentrations and selectivity in bladder tumors can be challenging. Because PS is available in the circulation and in the muscle layer, illumination with red light activates PS in the muscle layer, resulting in significant toxicity	Cons: Selectivity between healthy bladder tissue and the tumor is specific to PS, and not all PS can achieve good selectivity. The pharmacokinetic profile of PS is greatly affected by the continuous formation of urine, which dilutes the instillation solution. There are also factors that influence PK, including permeability barriers, urine washout of absorbed drugs and systemic absorption

COMBINATION OF SO-CLEAVABLE PRODRUGS AND PPIX-PDT

HAL-PDD showed great improvement over the WLC of bladder tumors because more microscopic tumors can be detected with HAL-PDD (108). Incomplete resection or reimplantation of tumor cells after TURBT may still lead to some recurrences. While 5-ALA-PDT can also be used as a treatment for NMIBC, it did not show great promise (109). Intravesical chemotherapy and immunotherapy for NMIBC are inefficient at managing recurrences and produce moderate-to-severe side effects. We have developed a unique combination strategy to solve these problems using PpIX-PDT and SO-activatable prodrugs. The prodrugs can facilitate tumor cell kill by PpIX-PDT while sparing the normal urothelium. Once cancer cells are exposed to HAL, PpIX is preferentially produced in the cancer cell’s mitochondria. If the prodrugs can be targeted to the cancer cell’s mitochondria, they can be activated more efficiently due to their proximity to PpIX.

Based on this hypothesis, our group has developed mitochondria-targeted and SO-activatable prodrugs. Mitochondria localizing small molecule (i.e. delocalized lipophilic cation (DLC)) was conjugated with Combretastatin-A4 (CA4) and Paclitaxel (110, 111). Upon illumination, Rhodamine-conjugated prodrugs could generate 90–100% cell killing with as low as 1.25 μm prodrugs, when incubated with HAL. Another cation (4-carboxy-1-methylpyridinium chloride) or TPP (triphenylphosphonium) linked to paclitaxel has also improved the phototoxicity of PpIX-PDT. However, mitochondria targeting moieties significantly increased the molecular weight of the prodrugs, which in turn negatively affected the drug permeability in the In vivo setting (unpublished data). Hence, we developed non-mitochondria-targeted prodrugs with smaller masking groups (morpholine, piperidine and methyl), which helped to keep the overall molecular weight low (<1000 g mol⁻¹) (112). Parent drugs were chosen with a variety of mechanisms of action (paclitaxel, CA4, SN-38 and Mitomycin C). All of the prodrugs could generate 90–95% cell kill when illuminated with 531 nm light after 2 h of incubation with the prodrugs and HAL.

After PpIX is formed, it can diffuse out of mitochondria, allowing non-mitochondrial localizing drugs to be activated upon illumination. Non-mitochondria targeting prodrugs overcome the limitation of using bulky mitochondria targeting groups. While non-mitochondrial targeted prodrugs could enhance the phototoxicity of PpIX-PDT, those required higher concentrations compared with mitochondria-targeted prodrugs (2.5–5 vs 1.25 μm) to achieve >90% cell killing (112). These mitochondria and non-mitochondria-targeted prodrugs were also tested in a 3D spheroids model and are now being evaluated in an orthotopic bladder tumor model (Fig. 4).

Figure 4.

Schematics of mitochondria-targeted and non-mitochondria-targeted prodrugs. 5-ALA, 5 amino levulinate; HAL, hexyl aminolevulinate; PpIX, protoporphyrin IX; MTPG, mitochondria-targeted protecting group; NMTPG, non-mitochondria-targeted protecting group; CD, chemotherapy drugs; L, light cleavable linker.

NANO FORMULATIONS FOR PHOTODYNAMIC THERAPY OF BLADDER CANCERS

So far more than 100 nanomedicine-based formulations have been approved by the US FDA for various therapeutic applications (113). In this section, we discuss photosensitizer-loaded nanoformulations to improve PDT performance, specifically for treating bladder cancers. Nanotechnology-based formulations offer several advantages such as improved selectivity, efficient targeting, low toxicity and minimal side effects. Nanotechnology-based nanocarriers also improve the permeability, retention and therapeutic effect of anti-cancer agents, antibacterials, proteins, peptides and antibody-conjugated molecules (114). These advantages have been successfully demonstrated using In vitro cell culture and In vivo preclinical animal models. However, the need for clinical investigation and understanding the gaps for successful clinical translation still remains.

A benefit of intravesical drug delivery over parenteral and oral delivery is that it minimizes systemic side effects of narrow therapeutic index drugs and maximizes exposure to non-muscle invasive bladder cancers. However, intravesical delivery has certain limitations such as rapid washout of the drug during voiding, dilution by urine, urothelial permeability barriers and the need for formulations to be instilled in the bladder (115). The following section describes the formulation strategies used for PSs in bladder cancer PDT (Fig. 5).

Figure 5.

Nano formulations used in photodynamic therapy of bladder cancer.

Nanoparticles with passive targeting

Passive targeting facilitates the transport of nanocarriers in the tumor microenvironment, due to the special characteristics of tumors compared with normal healthy tissues. Size, shape, surface charge of nanocarriers and tumor-associated factors (inflammatory responses, hypoxia, lymphatic drainage, leaky vessels) all contribute to the passive targeting of nanocarriers due to their enhanced permeability and retention. A concentration gradient is critical to the passive targeting strategy. In this section, we cover polymeric, inorganic and lipid-based nanocarriers for bladder cancer PDT.

Ce6 solubility was improved by making self-assembled nanoparticles with a nitazoxanide antiparasitic agent (116). This nanoparticle was further coated with fluorinated chitosan as a mucoadhesive biocompatible natural polymer to improve transmucosal efficiency after intravesical instillation. Intravesical administration of these nanoparticles achieved local bioavailability with decreased phototoxicity. The anti-cancer effect of nitazoxanide improved bladder permeability to destroy orthotopic bladder cancers. In another study, self-assembly of Chorin-e6-conjugated catalase and fluorinated polyethyleneimine (F-PEI) nanoformulation improved transmucosal, cross membrane and intratumoral penetration (117). As a result of in situ production of oxygen in tumors from endogenous H₂O₂ by catalase in nanoformulations, PDT reduces tumor hypoxia, thereby enhancing its ability to destroy orthotopic bladder tumors with fewer systemic side effects than hematoporphyrin.

Silica-based inorganic nanocarriers were developed for the delivery of third-generation photosensitizers. These mesoporous silica nanoparticles were grafted with S-glycoside that showed higher cellular uptake and phototoxicity in bladder cancer cell lines, such as HT-1376 and UM-UC-3 compared with free photosensitizer (118). Mesoporous silica nanocarriers loaded with S-galactosylated and S-glycosylated porphyrins were developed to improve the PDT performance of porphyrins PS (119). Galactosylated and glycosylated nanocarriers produced a higher amount of singlet oxygen and showed 3–5 times higher therapeutic effect in bladder cancer cell lines, HT-1376 and UM-UC-3 than free porphyrin PS. In another study, chitosan nanoparticles coated in lipid nanocarriers of porphyrin increased their stability, solubility and physicochemical properties, all of which improved porphyrin delivery to bladder cancer cells (120). In vitro assay confirmed that the porphyrin-loaded chitosan-based hybrid nanocarriers have 3.2-fold higher phototoxicity than free porphyrin in T24 cells (120). Hypericin-loaded nanoformulation of N-methyl pyrrolidone increased its solubility and targeting efficiency to bladder wall urothelium. Hypericin fluorescence was increased significantly in nanoformulation and formulation with 40% N-methyl pyrrolidone showed excellent stability. Polyvinylpyrrolidone-based hypericin formulation was also tested in phase IIA clinical trials for bladder cancer (121).

The PDT effect of polymeric nanoparticles loaded with 5-ALA was studied In vitro for cytotoxicity in T24 bladder cancer cells after irradiation with a 650 nm diode laser. 5-ALA-loaded nanoparticles significantly enhanced the PDT effect on bladder cancer cells (122). Dendrimers conjugated with 5-ALA were studied in rat orthotopic bladder to improve the selectivity of 5-ALA-based fluorescence-guided cystoscopy (123). Rats were administered intravesically with 5-ALA-conjugated dendrimers or HAL. Fluorescence intensities of normal and tumor areas were measured and found to be significantly higher in the rats with 5-ALA-conjugated dendrimers. Prolonged and sustained PpIX synthesis in the deeper tissue and less systemic absorption of PpIX indicate that 5-ALA-conjugated dendrimer is an alternative for HAL. In other work, core-shell nanocarriers, composed of magnetite colloidal supraparticle cores and ALA-Zn (II) coordination polymers as shells, were fabricated for targeting prophotosensitizer delivery to bladder cancer cells (124). These core/shell-structured nanocarriers showed significant cytotoxicity to T24 cancer cells while nontoxic to 293T (normal cells) when irradiated with a 630 nm laser.

In another approach, synthetic polyvinylpyrrolidone copolymer-based micelle was loaded with benzoporphyrin, and its derivatives such as benzoporphyrin with (2,2′-bipyridine) dichloroplatinum (II) (125). These formulations were characterized for their efficiency in generating SO and phototoxicity against HT-1376 bladder cancer cells. IC₅₀ values of the micelles-loaded photosensitizers were found to be around 5.58 mm.

PEGylation is another approach to exploring passive targeting. PEGylation of nanocarriers improved circulation half-life by virtue of the stealth effect, rendering them invisible to phagocytic cells. These nanocarriers prevent early opsonization, phagocytosis and engulfment by reticuloendothelial cells, which is beneficial for sustained release application, and prevent clearance from circulation. PEGylated ruthenium phthalocyanines PS has potential in PDT against human bladder cancer cells (HT-1376). Singlet oxygen generation quantum yield for these compounds was reported in the DMSO (ø_Δ= 0.78) and water (ø_Δ = 0.48). In vitro studies show that PEGylated ruthenium phthalocyanines PS accumulated in HT-1376 cells (0.01–0.8 nmol mg⁻¹ of protein) after incubation with 5 mM solutions showed no dark toxicity and higher phototoxic effect were observed when cells treated with 5 μm PS concentration illuminated with red light with at fluence rate of 20 mW cm⁻² for 40 min (126).

Cyclodextrin and cyclodextrin derivatives are used as a carrier to improve the solubility of poorly soluble drugs in the pharmaceutical formulation. Cyclodextrin derivatives conjugated with phthalocyanines PS was studied for photophysical, photochemical and phototoxicity against UM-UC-3 human bladder cancer cells (127). Overall, results show higher solubility of Pc-α-CD and Pc-β-CD compared with Pc-γ-CD and contribute to the generation of higher singlet oxygen and phototoxic effect against UM-UC-3.

Nanoparticles with active targeting

In another approach, active targeting of nanocarriers was used, by bioconjugation with ligands attached to the surface of nanocarriers for selective uptake, retention in the tumor and minimized off-target side effects to normal tissue. Active targeting of nanocarriers consists of transporting drugs to the target organ by site-specific ligand delivery. In this approach, drug delivery ligands such as folic acid, chitosan, fluorinated chitosan, Bld-1-KLA, PLZ4, hyaluronic acid and C225 antibody showed potential for bladder cancer-targeted therapy.

Conventional PSs have poor aqueous solubility and low selectivity for cancer cells and thus cause collateral damage to the nontumor area of the bladder. To achieve higher selectivity, galactose was used to target the carbohydrate-binding domain overexpressed on bladder tumors (128, 129). Galactose-conjugated nanocarrier of phthalocyanine (PcGa₁₆) was prepared, which accumulated in bladder tumors expressing galactose-binding proteins. In PDT-resistant HT-1376 bladder cancer cells, removing caveolin-1 increased sugar-binding proteins at the membrane of the cells, which improved PcGa₁₆ uptake and PDT efficacy.

Hypoxia and low targeting efficiency are common problems in treating bladder tumors via intravesical instillation. Bladder tumor-targeting peptide ligand (PLZ4) was explored for targeted chemotherapy, photothermal therapy, photodynamic diagnosis and image-guided photodynamic therapy. A nanoscale oxygen generator composed of superparamagnetic iron oxide nanoparticles and organoselenium was decorated with PLZ4 to overcome hypoxia in the bladder and enhance tumor targeting. The oxygen generator showed improved accumulation in bladder tumors and enhanced MRI contrast for bladder cancer diagnosis (130). The nanocarrier consumed more H₂O₂ via Fenton reaction, generated more oxygen to relieve hypoxia in bladder cancers and improved the inhibitory effect of cell proliferation. In another study, PLZ4 decorated porphyrin nanocarriers (PNP) emitted fluorescence, heat and reactive oxygen species upon illumination with near-infrared light (131). Doxorubicin-loaded PNPs slowly released doxorubicin and considerably increased systemic circulation time compared with free doxorubicin. In vivo efficacy study of these nanocarriers in a patient-derived xenograft model showed a significant delay in tumor growth and improved survival.

Phosphonic acid-containing groups are inhibitors of urokinase plasminogen, a key enzyme in metastasis and cell invasion (132). Phosphonic acid functionalized PS showed higher accumulation in bladder cancer epithelial cells UM-UC-3 than ARPE19 cells, higher ROS production and IC₅₀ values of 1.154–1.476 μm for urokinase plasminogen inhibition. Functionalization of phthalocyanines with phosphonic acid was performed and evaluated for inhibition of urokinase plasminogen, matrix metalloproteinase-9 and was evaluated in bladder cancer PDT. Overall, results show phthalocyanines functionalized with phosphonic acid, showing higher phototoxicity unlike control phthalocyanines for bladder cancer.

Hydrogels

Hydrogels are aqueous solutions of a polymer matrix that exhibit Newtonian flow (free-flowing liquid) at room temperature and become non-Newtonian (viscous liquid) at body temperature and are used for drug delivery at various body sites. Poly(caprolactone) based biocompatible, biodegradable and nontoxic polymeric matrix were synthesized by a polymerization reaction and was used to formulate in situ thermoresponsive hydrogel for bladder drug delivery (133). A combination of PDT and chemotherapy was tested against bladder cancer. In this study, doxorubicin and zinc phthalocyanine were loaded into in situ thermoresponsive copolymer hydrogel at 37°C. A tumor efficacy study in the 5637-cell xenograft model showed improved outcomes of combination therapy with delayed tumor growth and improved survival.

Protein nanocarriers

Protein-based nanocarriers have unique advantages of biocompatibility and nontoxicity unlike nanocarriers obtained from synthetic copolymers by different polymerization methods. Oxygen generating human serum albumin coated manganese dioxide, chlorin e6 loaded multifunctional nanocarriers (HSA-MnO₂-Ce6 NPs) were designed to overcome challenges of tumor microenvironment-associated tissue hypoxia and improve the efficacy of PDT in bladder cancer (134). Magnetic resonance (MR) imaging and In vivo near-infrared (NIR) fluorescence imaging confirmed the effective bladder tumor-targeting property of HSA-MnO₂-Ce6 NPs (administered via IV) in the orthotopic bladder model in C57BL/6 mice. The nanocarriers showed improved therapeutic efficacy and significantly improved life span compared with control.

Liposomes

Liposomes are phospholipid-based vesicular nanocarriers approved by the FDA for the delivery of anti-cancer agents. Both hydrophilic and lipophilic compounds can easily be loaded into liposomes. The availability of clinically approved liposomal formulations is one of the motivations for using liposomes for PSs. However, the leakage of encapsulated content is a major drawback of liposomes. Intravesical placebo liposomes (without drugs) show therapeutic effects on Interstitial cystitis (IC)/bladder pain syndrome (BPS) patients, due to their ability to form a protective lipid film on the urothelial surface. The effect of Intravesical liposomal delivery was studied in rats for hyperactive bladder. A transepidermal gradient of hydration across skin layers encourages liposome movement across the skin by virtue of its elastic, ultra-deformable nature and various morphologies.

PEGylated transferrin-conjugated liposomes of aluminum phthalocyanine tetrasulfonate (Tf-Lip-AlPcS4) improved specificity, sensitivity and cellular uptake when administered intravesically (135). The transferrin-conjugated liposomes, when studied in the AY-27 orthotopic rat bladder cancer model, showed higher intracellular accumulation compared with the unconjugated liposome and free photosensitizer (AlPcS4): 384.1, 52.7 and 3.7 μM (calculated as AlPcS4 concentration), respectively. Transferrin-conjugated liposomes seemed excellent for PDT of non-muscle invasive bladder cancer due to its receptor-mediated uptake in cancer cells.

Another class of unilamellar liposomes was loaded with hydrophobic tetramethyl hematoporphyrin to enhance PDT efficiency after intravesical instillation for bladder cancer (136). These liposomes were tested against two human bladder cancer cell lines, Waf and Rec. Fluorescence microscopy showed localization of tetramethyl hematoporphyrin in a perinuclear region. The liposomal formulated PS showed phototoxicity at 5 μg mL⁻¹ when illuminated with a 630 nm laser at 7.2 J cm⁻¹. Interestingly, both uptake and phototoxicity of the liposomal formulated PS were higher in Waf than in Rec cells.

The acidic environment of tumor cells was targeted with pH-sensitive liposomes using phosphatidylethanolamine, phosphatidylcholine and cholesteryl hemisuccinate loaded with 9-acetoxy-tetra-n-propylporphycene for bladder cancer PDT (137). A decrease in cell survival to 0.1% was observed at 2 μg/1.5 mL of 9-acetoxy-tetra-n-propyl porphycene. Bladder carcinoma cells irradiated with fluence rates from 1 to 48 J cm⁻² showed statistically significant photokilling by pH-sensitive liposomes than non-pH-sensitive liposomes.

Liposomal antibody conjugates and direct antibody conjugates of sulphonated aluminum phthalocyanine were compared for their selective photoimmunotherapy in bladder cancers (138). The liposomal antibody conjugates showed higher, 13-fold, phototoxicity at equimolar antibody doses against bladder carcinoma cell 647 V. Overall, the liposomal antibody conjugates seemed better than direct antibody conjugates of the PSs in terms of therapeutic ratio.

PS-glycoconjugates

Photosensitizer conjugated with the carbohydrates (galactose, mannose and glucose) shows higher cellular uptake for cancers overexpressed with galectin-1 protein and improved PDT of bladder cancer. PS like phthalocyanine, ruthenium phthalocyanines, chlorine and porphyrin conjugates were synthesized successfully and studied for bladder application. Phthalocyanine PS has limitations for PDT applications such as aggregation, photo instability, low fluorescence and low singlet oxygen generation capability. Silicon (IV) Phthalocyanine (SiPc) anchored galactose-dendritic novel PDT agent studied for bladder cancer PDT (139). PDT effect of these Phthalocyanine anchored galactose-dendritic agents studied in UM-UC-3 cell line. SiPc(OH)2 showed higher accumulation in the bladder cancer cells due to higher amphiphilicity compared with other phthalocyanine PS. In summary silicon phthalocyanine with several axial galactose groups provide a new platform for designing novel PS for PDT. Another strategy involved functionalizing Ruthenium phthalocyanines PS with axial carbohydrates (mannose, glucose and galactose) to improve the biocompatibility and targeting of bladder cancer cells (HT-1376). Biocompatibility and solubility of ruthenium phthalocyanines PS were improved by introducing polyethylene glycol polyether chain to either axial or peripheral position (140). Singlet oxygen generation quantum yield for all ruthenium phthalocyanines PS reported (ø_Δ = 0.08 to 0.46). The presence of peripheral polyether chain decreases quantum yield (ø_Δ = 0.08–0.2) in DMSO. The absence of peripheral substituents shows a higher phototoxic effect, higher cellular uptake and higher singlet oxygen generation yield against bladder cancer cells HT-1376.

Mitochondrial targeting of Chlorin conjugated galactodendritic also improved PDT performance for bladder PDT. Interaction of Chlorin conjugated galactose (ChlGal) with galactose-binding proteins achieved higher concentration in HT-1376 bladder cancer-resistant cells for phototoxicity (141). ChlGal₈ showed higher photostability, ability to generate singlet oxygen and ability to interact with galactose-binding proteins such as galectin-1 and human serum albumin. These Chlorin conjugated galactodendritic PS show higher water solubility with higher interaction with galactose-binding protein expressed in various cancers and a 50-fold increase in the extinction coefficient at the lowest-energy Q-band relative to the previously reported porphyrin galactose (PorGal₈). Conventional porphyrin-based PS in the clinic shows low tumor selectivity and off-target side effects on healthy tissue. To overcome these issues and improve the performance of porphyrin-based PS, porphyrin with dendritic units of galactose (PorGal₈) was developed to target galectin-1 (galactose-binding protein), overexpressed in many tumors. In vitro studies of PorGal₈ in two bladder cancer cell lines, HT-1376 and UM-UC-3 show concentration and time-dependent uptake with a plateau in approximately 2 h. In vivo PDT efficacy was validated in athymic nude mice inoculated with UM-UC-3luc⁺ bladder cancer xenografts with overexpressed galectin-1 protein (142). PorGal₈ was administered (5 lmol kg⁻¹ by intraperitoneal route) followed by light delivery (50.4 J cm⁻²) after 24 h. PorGal₈ PDT showed significant tumor growth reduction (**P < 0.01) compared with PorGal₈ dark toxicity. The potential effect of PorGal₈ depends on the expression of galectin-1 in bladder cancer cells.

In another report phthalocyanine surface anchored with a shell of sixteen galactose units arranged in a dendritic manner (PcGal₁₆) was studied for PDT against HT-1376 and UM-UC-3 bladder cancer cell lines (143). Here, the galactose unit allows the uptake of a PcGal₁₆ through interaction with GLUT1 (glucose transporter) and galectin-1. PcGal₁₆ incubated with HT-1376 and UM-UC-3 cells and then illuminated with a red light at 2.5 mW cm⁻² for 40 min shows increased phototoxicity. PcGal₁₆ is nontoxic and an efficient targeting agent for cancers with overexpressed GLUT1 (glucose transporter) and galectin-1, highly phototoxic against HT-1376 and UM-UC-3 bladder cancer cells, and induces high oxidative stress and reactive oxygen species.

Antibody-PS conjugates

Near-infrared photoimmunotherapy (NIR-PIT) uses a photosensitizer (phthalocyanine dye, IR700) directly conjugated to a monoclonal antibody (mAb) that targets cancer cells and gets activated when illuminated with near-infrared light (144, 145). While PDT only affects the treatment site, conventional photosensitizers can be absorbed by the skin and normal tissue around the treatment site and exert PDT-associated toxicity. mAb-based PSs can reduce off-target toxicity by selectively binding to the target molecule on cancer cells; hence mAb-IR700 was developed (146).

An anti-CD104 monoclonal antibody is bioconjugated with porphyrin PS to enhance specificity toward tumors that express more CD104. Furthermore, porphyrin PS conjugated with an amine group of bovine and human serum albumin nanocarriers provided improved pharmacokinetics. The phototoxicity of porphyrin PS bioconjugate with anti-CD104 monoclonal antibody was statistically significant (P < 0.05) in relation to untreated UM-UC-3 bladder cancer cells. IC₅₀ of porphyrin in UM-UC-3 bladder cancer cells after PDT were 0.09, 0.14 and 0.53 μm for porphyrin PS bioconjugate with anti-CD104, porphyrin bovine serum albumin and porphyrin human serum albumin, respectively (147).

Several monoclonal antibodies were conjugated with IR700 to treat bladder cancer. Epidermal growth factor receptor (EGFR) protein is expressed in 74% of the urothelial carcinomas with a comparatively lower expression in normal bladder urothelium (148). Panitumumab was conjugated to IR700. When activated with NIR light, it rapidly killed UM-UC-5 cells, a squamous cell carcinoma cell line, mostly due to necrosis (149). An In vivo study using canine anti-EGFR mAb conjugated to IR700 was conducted in mice and treatment significantly (P < 0.001) inhibited tumor growth of SC xenograft tumors and significantly prolonged the survival (150). Another evaluated target is an innate immune checkpoint, CD47, which is highly expressed in all bladder tumor cells compared with lower expression in the normal urothelium (151). Anti-CD47-IR700 was developed and treatment significantly increased the phagocytosis of tumor cells compared with the antibody alone (152). In vivo study found that targeted PIT could significantly decrease tumor growth (in the SC xenograft tumor model) compared with the control and repeat treatment improved overall survival compared with the control group. NIR-PIT can be a potential treatment option for not only bladder cancer but also other cancer types with improved targeting efficiency and deeper tissue penetration, which helps to overcome the light penetration issues with treatment with visible light.

LIGHT DELIVERY

Compared with other human tissues, bladder tissue is more translucent and readily accessible with a thin fiber optic cable, making bladder cancers an excellent choice for PDT. Typically, in bladder cancer PDT, the whole bladder is illuminated using an optical fiber. To achieve improved PDT efficacy, controlling light interaction with PSs is very important and depends on the duration of light treatment, laser power and light penetration depth (153). A homogeneous irradiance profile could be achieved using a cylindrical emitter with a nonuniform longitudinal emission profile (154). Much work has been done to improve the homogeneity of light distribution in the bladder. Miyazaki et al. (155) developed a homogeneous irradiation fiber probe that could irradiate 69% of the bladder area compared to 36% with the control fiber.

Source fiber positioning is very important too to avoid any localized abundance of the irradiance (156). Displacement of an isotropic emitter in a transparent cavity with a large volume does not significantly affect irradiance. Despite this, source fiber positioning is crucial for bladders with a volume <100 mL or a radius less than 3 cm. A small displacement of 1 cm can double the light dose in the bladder wall of the smaller bladder (volume 75 mL). Prior to treatment, an ultrasound system is usually used to verify the position of the source fiber. Preclinical In vivo ultrasound imaging devices (e.g. GE Logiq e) can be used to determine the positions of source fibers in rodent bladders. With the addition of multiple sensors, it becomes possible to detect the local increase or decrease in irradiance that indicates the movement of the source fiber. For the safety of the bladder, monitoring irradiance is crucial, as an emission power of 2.5 W or higher may cause thermal burns if the emitter touches the bladder wall (48).

Light scattering can reduce the overall irradiance by causing confinement of the fluence rate around the source (48). PDT efficacy on bladder tumors can vary based on the light wavelength. For instance, while a potential PDT effect can be up to 20 mm with 633 nm light, it can expand up to 28 mm with a 693 nm light (153). Accurate measurement of the light dose is critical for bladder illumination as variation in the light dose can greatly influence efficacy and toxicity. Also, it should be kept in mind while choosing the PS that the optical properties of the bladder tissue can vary based on the light wavelength. So, one should carefully consider the absorption and scattering coefficient of the bladder tissue for the specific wavelength of light used for PDT.

While increasing wavelength improves the penetration depth, it also increases the chance of muscle damage. The human bladder is about 3–4 mm thick (156, 157). In clinically approved PDT for bladder cancer in Canada, Photofrin is given IV and red (630 nm) light is used for whole bladder illumination. However, such treatment conditions could cause collateral damage to the whole bladder, particularly the muscle layer, causing functional damage to the bladder. By contrast, 5-ALA or HAL generating PpIX can be excited by a green light that does not penetrate more than 1 mm into the bladder, hence sparing the muscle layer but falls short in efficacy.

CONCLUSION AND FUTURE PERSPECTIVE

Although more advanced stages of bladder cancers could also be treated with PDT after further optimization, NMIBC is an excellent and imminent target for PDT. From the lesson with clinical experience with Photofrin-PDT for bladder cancers, it is clear that efficacy needs to be improved, in particular, with minimal side effects for PDT to be clinically successful. Multiple variables of PDT have been optimized, such as photosensitizer, route of administration, formulation and light condition to achieve selective targeting of cancers over normal bladder tissue, particularly the muscle layer.

There are some important points to remember when developing PDT for NMIBC. First, the dwell time of intravesically administered photosensitizers is limited to 1–2 h and photosensitizers reach tumor cells mostly via diffusion from the bladder lumen. In vitro studies should reflect limited dwelling time and diffusion of formulated photosensitizers. Spheroid or organoid models can be used to test the diffusion of photosensitizers In vitro. Second, to better mimic NMIBC In vivo, intravesical administration and whole bladder illumination, orthotopic bladder cancer models are more suitable than SC tumor models. Mice are too small for whole bladder illumination using commercially available fibers. Thus, typically, orthotopic rat bladder cancer models are commonly used. Finally, the efficiency of PDT and diffusion of formulated photosensitizers are highly dependent on the thickness of targets, tumors or bladder walls. Although the rat bladder (~0.5 mm) is thicker than the mouse bladder (~0.1 mm), it is still much thinner than the human bladder (~3–4 mm), which should be considered in clinical translation, and data with larger animal models may be necessary.

Since bladder cancer was the first clinically approved PDT, we are optimistic about developing a clinically successful PDT for NMIBC too, hopefully soon. A holistic approach should be taken by utilizing knowledge from clinical experiences, advanced optics and a deeper understanding of PDT and the pathophysiology of NMIBC.

Biographies

Kazi Md Mahabubur Rahman is a Ph.D. candidate at SUNY at Buffalo’s Department of Pharmaceutical Sciences. Before joining the Ph.D. program, he worked as a quality control executive in a pharmaceutical company for two years after receiving his bachelor of pharmacy from East West University, Bangladesh. In his Bachelor of Pharmacy, he was awarded Cum Laude and during his Ph.D., he was awarded the Allen Barnett Fellowship. His research interests include pharmacokinetic and pharmacodynamic analysis of drug molecules, photodynamic therapy, drug delivery, and light-activated prodrugs.

Dr. Prabhanjan Giram is a Post-doctoral Associate at the University of Buffalo in Dr. Youngjae You’s Lab. He earned a Ph.D. at the National Chemical Laboratory, India under Dr. Garnaik’s guidance. He qualified CSIR-NET in chemical science AIR-81. A Newton Bhabha fellowship was awarded to him during his PhD tenure at King’s College London under mentor Dr. Al-jamal Khuloud, and he also received the Eudragits^® prize of 1500 €. He became an Assistant professor at DPU Pharmacy after Ph.D. Electrospinning for biomedical application, Photodynamic therapy, Light-activated produgs, targeted drug delivery for cancer and design of polymeric and lipid nanoformulations are some of his research interests.

Dr. Barbara A. Foster is a Professor of Oncology at Roswell Park Comprehensive Cancer Center. She received a Ph.D. in Anatomy at UCSF. She completed her postdoctoral fellowship in the Department of Cell Biology at Baylor College Medicine. Among her research interests are pre-clinical testing of novel therapeutic approaches in vivo models, resistance mechanisms to therapeutic response, models of cancer with a focus on bladder cancer, prostate cancer, lung cancer, pediatric cancers, patient derived models (xenograft, organoids and spheroid). She is also the Director of Graduate Studies for the experimental therapeutics track in Roswell Park’s Cancer Sciences Graduate Program.

Dr. Youngjae You is a Professor of Empire Innovation at the Department of Pharmaceutical Sciences of the SUNY at Buffalo. He obtained his PhD from Chungnam National University, Korea in 2001 and was a postdoctoral fellow (Breast Cancer Research Program, DoD) and a Research Assistant Professor at the SUNY Buffalo. He became an Assistant Professor in South Dakota State University and then moved to OUHSC in 2010. He was an American Cancer Society research scholar. His interests are to develop bioactive small molecules and drug conjugates, light-controlled drug delivery systems, and strategies to activate immune system for cancer immunotherapy.