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Fluorescence cystoscopy is based on the intravesical administration of a drug, which will be incorporated to a higher degree into tumour cells and result in fluorescence, revealing the presence of cancerous lesions. The concept was first published in 1996, by Kriegmair et al.  who described a sensitivity of 97% after 5-aminolevulinic acid (ALA) administration. ALA is taken up by the cells and metabolized through the heme cycle into a fluorescent compound, protoporphyrin IX (PpIX). Shortly thereafter Lange et al.  showed that the use of an ALA ester (hexylaminolevulinate, h-ALA, Hexvix®), induced a more intense fluorescence with reduced drug concentration. As multiple large studies have reported the major benefits that can be obtained through fluorescence-guided resection, irrespective of the prodrug used [3,4] without differences between 5-ALA and Hexvix . An enhanced sensitivity of detection (85–95%), leads to a more complete resection, resulting in a reduced recurrence rate (90% tumour free at 1 year vs 70% with white light). Recently, a prospective randomised international study of 814 patients also showed a significantly improved detection rate . However, in two multicentre double-blind randomised placebo-controlled trials, the authors failed to recognise any long-term benefit in terms of recurrence [6,7]. Moreover, major drawbacks, are the lack of tumour selectivity, the very superficial fluorescence and the efflux of PpIX a few hours after instillation [8,9]. These limitations can be reduced by developments in optical techniques, e.g. narrow-band imaging, Raman spectroscopy and optical coherence tomography, but necessitating costly equipment . There is thus a real need for other fluorescent markers that are more specific and less prone to photodegradation.
A current focus of pharmaceutical research is the development of nanocarrier systems to enhance drug delivery and specificity. Dendrimers are hyperbranched polymers that have attracted considerable interest in oncology for drug delivery, owing to their ability to carry high drug-payloads and their well-defined macromolecular structure . Recently, a dendrimer containing 18 ALA molecules coupled through biodegradable ester linkages was synthesised and tested in vitro and in vivo[12,13]. Those studies indicated a more efficient and sustained PpIX production than ALA at drug equivalent doses after i.p. administration.
The aim of the present study was to compare the in vitro behaviour of ALA, h-ALA (Hexvix) and ALA dendrimers in a bladder cancer cell line. The second objective was to compare the impact of intravesical administration of ALA dendrimers and h-ALA (Hexvix) on tumour specificity and depth of penetration of PpIX in rats bearing orthotopic bladder tumours.
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Fluorescence cystoscopy guided by ALA has been shown to benefit patients and provide more life years, but the high false-positive rates remains a major drawback . This relative low specificity can be reduced when applying hexylaminolevulinate . In the present study, we investigated the possibility of using a macromolecular delivery system based on a dendrimer. We have investigated two dendritic ALA derivatives: the 6-ALA dendrimer is a first-generation dendrimer, bearing six molecules of ALA, conjugated to the building blocks through esters bonds. The ester bonds were expected to be hydrolysed by non-specific esterase enzymes to yield the free ALA in the tissue. 18-ALA dendrimer is a second-generation dendrimer, carrying 18 ALA molecules (Fig. 1A,B). The exterior of the dendrimer is relatively hydrophilic at physiological pH due to protonation of the amino groups, whereas the interior structure is hydrophobic with an aromatic core and polyamido hydrocarbon spacers.
Previous studies showed that this dendrimer was incorporated in cells via endocytosis and resulted in an enhanced and sustained synthesis of PpIX in vitro as well as in vivo after i.p. administration [12,13]. However, specificity towards the tumour was essentially identical to that for ALA and never exceeded a factor of two . Malik et al.  showed that similar non-ALA bearing dendrimers were rapidly cleared from the blood stream after i.p. or i.v. administration.
As a result, the amount of ALA delivered has to be increased when using dendrimers to reach an identical or enhanced PpIX production in vitro and in vivo (Fig. 3, Table 2). Slow hydrolysis of ALA from dendrimers (Fig. 4) also implies longer resting times to synthesise sufficient PpIX to visualize fluorescence in vivo (Table 2, Fig. 5). In our previous work, we showed that 1-h instillation with h-ALA in a rat orthotopic bladder tumour model followed by 2 h resting time provided an optimal PpIX fluorescence signal . When dealing with 18-ALA the resting period must be extended to 3 h, which can be considered to be a clinical drawback. However, as the fluorescence persists at least as long as 24 h (Table 2), this might be very beneficial in a clinical setting. Indeed, the time between instillation and endoscopic inspection is no longer crucial and allows for more flexibility. It has indeed been shown that starting from 3 h after instillation, there is a progressive efflux of PpIX from cells, resulting in reduced fluorescence . However, one has to consider that at 24 h, the specificity no longer exceeds the one observed with h-ALA. In a s.c. tumour model, Casas et al.  also reported sustained tumour fluorescence up to 24 h, progressively declining without however reaching basal levels at 48 h.
From an ultrastructural point of view, human and rat healthy bladder urothelium and carcinoma are comparable in many aspects such as for instance glycocalix layer and tight junctions [22,23]. It has been shown that changes occur in the junctional complex during carcinogenesis, resulting in enhanced permeability of the urothelium making it from tight to leaky. Furthermore, alterations in the glycocalix and the formation of microvilli on the luminal cell surface are seen, thus enhancing contact possibilities with intraluminal compounds. These conditions are partly responsible for the higher PpIX synthesis seen in bladder tumours. ALA and h-ALA, being small molecules with low molecular weight (Table 2), are capable of penetrating between normal urothelial cells, thus enhancing contact possibilities and internalisation, and potentially reducing tumour selectivity . Small molecules have been shown to be more prone to enter the vasculature after intravesical administration  and to pass across fetal membranes after ex vivo application as opposed to dendrimers .
Thus, the size of an intravesically administered molecule appears to be essential to optimise tumour selectivity and depth of penetration into the tumour. Small molecules will tend to enter normal urothelium as well as the tumour, with loss of specificity. Penetration into the tumour depth would be facilitated but this would also potentially implicate enhanced penetration into the tumour vasculature. This would in turn result in reduced fluorescence in the deeper tumour layers as well as potential generalised toxicity due to systemic reabsorption, as previously shown .
We therefore investigated three compounds with different size/molecular weight; h-ALA 251.8 g/mol, 6-ALA 1716 g/mol and 18-ALA 4317.5 g/mol. At comparable ALA molar equivalents, tumour selectivity as compared with normal urothelium was the highest for 18-ALA and was identical for h-ALA or 6-ALA (1.7 vs 1.2, Table 2).
There were no differences between tumour/muscle ratios (±2.5) or depth of fluorescence visualisation (150 µm) at equimolar concentrations of 8–9 mm ALA When increasing the concentration, both parameters were significantly better for the largest molecules (Table 2). Tumour to muscle ratio increases to 3.4 with a mean penetration depth of 250 µm, reaching up to 750 µm. This can probably be attributed to the dendritic structure itself. Steric hindrance is responsible for the slow hydrolysis seen. However, by increasing the quantity of intracellular dendrimers, access to esterases is multiplied, resulting in enhanced PpIX production and fluorescence [26,27]. In case of h-ALA, increasing the concentration results in a saturation of the enzymatic pathway and reduced PpIX production as we have previously shown .
To further confirm the importance of size, we assessed free ALA, ALA esters and PpIX in plasma after instillation in normal and tumour-bearing bladders (Table 3). PpIX levels were always below detection limits of our set up (data not shown), although PpIX has been described in human plasma after h-ALA instillation . ALA and esters were found in the highest quantities for h-ALA, less so for intermediate size dendrimers and were completely undetected for large size dendrimers. This observation also accounts for the deeper fluorescence seen after sensitisation with 18-ALA. Indeed, in the absence of systemic reabsorption, the dendrimers are in prolonged contact with the deeper tumour cell layers. Absence of systemic reabsorption of ALA is also an important factor when considering potential toxicity. Oral administration of ALA has been shown to induce temporary (<48 h) high plasma levels of ALA/PpIX, without a significant skin sensitisation effect but some cardiovascular effects, e.g. hypotension and tachycardia .
False-positive fluorescence signals emanating from non-transformed epithelium are a major drawback in fluorescence cystoscopy. This relative poor specificity decreases with increased experience with the technique as well as by using an esterified derivative. False-positive results have been attributed to several conditions, e.g. inflammation, hyperplasia or immunotherapy [1,30,31]. The fluorescence intensity from orthotopic rat bladder tumours never exceeds a factor of two compared with normal urothelium, irrespective of the prodrug used . This can be attributed to the previous intravesical interventions for tumour induction. However, the present study achieves fluorescence ratios of three. This suggests that 18-ALA could be a valuable alternative prodrug to enhance sensitivity as well as specificity for tumour detection. Clinical trials are thus mandatory.
In conclusion, dendrimers bearing 18 ALA molecules can be instilled intravesically and induce sustained production of PpIX that can still be seen after 24 h. However, due to the slow hydrolysis of ALA, the resting time after instillation is longer (3–4 vs 2 h) after instillation. The concentration of the prodrug needed is significantly smaller than for 5-ALA (0.7 vs 180 mm) and h-ALA (0.7 vs 8 mm). Tumour specificity vs muscle and normal urothelium is significantly higher compared with h-ALA, the currently approved ALA derivative for fluorescence bladder cancer detection and the fluorescence signal can be seen at greater depths. This can be attributed to the molecular weight/size of the prodrug. There is no systemic reabsorption thus minimising generalised toxicity. 18-ALA dendrimers could be an alternative to h-ALA in fluorescence-guided cystoscopy.