New Frontier in Hypericin-Mediated Diagnosis of Cancer with Current Optical Technologies
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Photosensitizers (PSs) have shown great potentials as molecular contrast agents in photodynamic diagnosis (PDD) of cancer. While the diagnostic values of PSs have been proven previously, little efforts have been put into developing optical imaging and diagnostic algorithms. In this article, we review the recent development of optical probes that have been used in conjunction with a potent PS, hypericin (HY). Various fluorescence techniques such as laser confocal microscopy, fluorescence urine cytology, endoscopy and endomicroscopy are covered. We will also discuss about image processing and classification approaches employed for accurate PDD. We anticipate that continual efforts in these developments could lead to an objective PDD and complete surgical clearance of tumors. Recent advancements in nanotechnology have also opened new horizons for PSs. The use of biocompatible gold nanoparticles as carrier for enhanced targeted delivery of HY has been attained. In addition, plasmonic properties of nanoparticles were harnessed to induce localized hyperthermia and to manage the release of PS molecules, enabling a better therapeutic outcome of a combined photodynamic and photothermal therapy. Finally, we discuss how nanoparticles can be used as contrast agents for other optical techniques such as optical coherence tomography and surface-enhanced Raman scattering imaging.
KeywordsHypericin Photodynamic diagnosis Fluorescence Endoscopy Urine cytology Endomicroscopy Gold nanoparticle Photothermal therapy
Successful detection of cancer onset and surgical clearance of tumor certainly help to increase the rate of survival without recurrence.60,62 Current diagnosis and surgical procedures depend largely on visual inspection using a primitive white-light endoscope (WLE). However, the given diagnostic information about the tissue has always been inadequate for an objective assessment that has a decisive impact on the therapeutic outcome. Many optical techniques82,94,101,103 are capable of acquiring rich diagnostic information and signaling any subtle changes occurring in tissue optics and biochemistry. These imaging techniques, therefore, provide a means of visualizing the structural and molecular contrast between healthy and cancerous tissues. This visual aid can potentially be a useful adjunct to complete resection of multifocal tumors.
While these optical diagnostic techniques are typically used in research laboratories, some of which are being translated to clinical practice.4,28,59,88 Advancements in fiber optics, optoelectronics, and microactuators have provided cost-effective solutions to the integration of these optical diagnostics into an existing catheter or endoscope that is being used as a navigation aid in standard diagnosis. Regardless of the techniques used, the measured optical signals are essentially correlated with the pathological status of the tissues that can vary the light-tissue interactions by light absorption, scattering, and emission. The acquired data, in general, is overwhelming, and thus needs to be modeled and condensed into a set of concise diagnostic parameters from which a diagnostic algorithm can be derived to perform a light-guided biopsy and surgery.
Photosensitizers (PSs) are light-triggered drugs specially used in photodynamic therapy (PDT) to eradicate cancerous cells.16 PSs are also fluorescent compounds with preferential uptake by tumors. These attributes make PS a promising biomarker to diagnose cancer using fluorescence techniques. Such optical diagnoses are now commonly referred to as photodynamic diagnosis (PDD). PDD of cancer has found its way into widespread clinical use, and the diagnostic values of PDD agents have already been reviewed.33,34,44 While the photosensitizing and chemical properties of PSs have been much studied,37 less emphasis is given to the probe design and diagnostic algorithms that also have an impact on the diagnostic accuracy. A fair evaluation of these aspects among different groups is usually difficult because the diagnostic outcomes depend on several factors including cancer types, PS administration, and formulation. In this review article, we narrow our focus to discussion on PDD using hypericin (HY), a potent PS, which our group has been investigating with customized instruments and diagnostic algorithms developed to aid quantitative and objective analysis. In addition to the diagnostic values from HY fluorescence, our group also attempts to couple HY with nanoparticles to extend its use in other optical imaging techniques.
This review is divided up as follows: First, PDD of cancer using PSs is discussed and compared with autofluorescence diagnosis. Second, the fluorescence properties of HY and issues surrounding its solubility and targeted delivery are presented. Third, studies of HY-PDD used with assorted fluorescence diagnostic devices are detailed; this section highlights the recent development of optical instruments and diagnostic algorithms for directing biopsies and assessing resection margins for lesions. While HY is widely used in PDD and PDT, its use can be further extended to other regimes. The following section summarizes the underlying trend in nanotechnology that gradually reinforces the use of HY for combined therapeutics and multimodality imaging.
Fluorescence Diagnosis of Cancer
Fluorescence diagnosis of cancer utilizes the characteristic fluorescence signatures, either from inherent or exogenous fluorophores, to distinguish abnormal tissues from surrounding normal tissues. If the fluorescence signatures of inherent biomolecules are utilized, then the diagnostic technique is termed as Autofluorescence diagnosis, and Exogenous fluorescence diagnosis, if exogenous fluorophores are used.
Successful autofluorescence diagnosis has been previously reported to distinguish normal from malignant tissues. For instance, Koenig et al.49 conducted cancer diagnosis by comparing the autofluorescence spectra of the tissues excited at 337 nm. By calculating the fluorescence intensity ratios at 385 and 455 nm, the autofluorescence signal from collagen and reduced nicotinamide adenine dinucleotide (NADH), respectively, the study could discern normal and malignant lesions with sensitivity and specificity of 97 and 98%, respectively. Another autofluorescence study by Zaak et al.,108—using different intensity ratios at 335 and 430 nm, corresponding to signals from tryptophan-rich proteins and NADH, respectively—distinguished malignant from benign lesions with sensitivity and specificity of 95 and 77%, respectively. Similarly, fluorescence endoscopy has also been utilized in discerning normal from cancerous lesions, where the extent of loss in autofluorescence emission in the green region of the visible spectrum was considered the distinguishing parameter.51,70
However, most of these endogenous fluorophores weakly fluoresce even when excited optimally at UV-blue region (300–450 nm). In addition, the penetration depth of UV-blue light in tissue is often limited by high tissue scattering and strong absorption of hemoglobin in 400–500 nm region.69 The epithelial thickening, which typically occurs in tumors, can further reduce the amount of excitation light reaching the inner submucosa where most endogenous fluorophores are located. The failure in probing the fluorescence emission from deeper layers presented limitation in distinguishing carcinoma from dysplasia and carcinoma in situ (CIS).11,51
Exogenous Fluorescence Diagnosis
Exogenous fluorescence diagnosis uses synthetic fluorophores, which are excitable at longer wavelength, to visualize neoplastic lesions that cannot be seen using white-light imaging and autofluorescence imaging. Unlike autofluorescence, exogenous fluorescence diagnosis relies on positive fluorescence staining of malignant and pre-malignant lesions. This reflected in increased detection of dysplasia and CIS, and discerning of these from malignant tumors.34,102 Currently, the exogenous fluorophores used in clinical environment are PS. PSs are compounds that, along with fluorescing capability, have favorable diagnostic characteristics, such as tumor specificity, rapid pharmacokinetic elimination, low levels of dark-toxicity, etc.6 HY, 5-aminolevulinic acid (ALA) and its derivatives, like 5-ALA hexylester, are prominent among PSs that are being evaluated for its clinical utility. These PSs are traditionally used in PDT for its aforementioned properties, and when used for diagnostic purposes, the technique is termed as PDD. PDD, as an alternative diagnostic technique, has already been approved for bladder cancer in European Union (EU) and European Economic Area (EEA).11,109
Ideal Photodynamic Diagnosis Agent
5-aminolevulinic acid is a PS that induces and accumulates fluorescent protoporphyrin IX (PpIX), and was initially reported by Kennedy et al.45 and Kreigmair et al.34 Though 5-ALA was widely investigated, it faced shortcomings like fast photobleaching of induced PpIX and less tissue penetration due to low lipophilicity and lower specificity. Subsequently, various derivatives of 5-ALA were also investigated with little success. Then D’Hallewin et al.12 identified HY as a better choice of PS used for detecting bladder cancer. Though the diagnosis with 5-ALA (78–100%) has sensitivity comparable to HY (82–94%),75 it was shown to yield low specificity ranging from 41 to 66%,34 which implies many false positives. On the other hand, studies that investigated HY for fluorescence diagnosis reported excellent sensitivity along with superior specificity ranging from 9181 to 98%,12 which greatly reduces the incidence of false positive results. Later studies continue to report HY’s advantages over other drugs, in fluorescence diagnosis and PDT87 of various cancers.51,56,64,89,109,111
Photodynamic Properties of Hypericin
Hypericin is extracted from a plant, Hypericum perforatum L., commonly known as St. John’s Wort. HY consistently exhibited well-resolved spectral bands when dissolved in various organic solvents, with absorption maxima at around 540 and 590 nm, and fluorescence maxima at around 590 and 640 nm.11,97,100 Absorption maxima of HY being at longer wavelength allows excitation light to reach HY in deeper tumors whereas light that excites 5-ALA-induced PpIX with absorption maxima at around 400 nm can only penetrate shallowly into tissue. Another important property of HY making it a prime PS in PDD is its photostability, as reported by various studies, and its fluorescence could be detected for up to 16 h after instillation. Its photostability could be further enhanced when formulated with 40% N-methyl pyrrolidone (NMP).77
Tumor Selectivity of Hypericin
Despite the fact that HY exhibits tumor selectivity, the specific mechanism involving the cellular uptake is yet to be fully understood. The possible mechanism of preferential HY accumulation in cancerous cells has been reviewed elsewhere.78 In brief, it has been proposed that both diffusion and endocytosis contribute to the overall transport of HY into individual cancerous cells.78 Olivo et al.66 showed that HY indeed preferentially localizes in the tumor and the intensity of microscopic HY fluorescence increased with both stage and histological grade of bladder cancer. For instance, the HY intensity of grade 3 cancerous cells was shown to be nearly 3 times higher than of grade 1 cells.
In addition to the fact that intracellular uptake of HY is higher in cancerous cells, subsequent studies unraveled other possible contributing factors to its selective accumulation at tissue level.30,68 For instance, Huygens et al.30 studied human bladder cell carcinoma using a 3-dimensional (3D) spheroid model, and reported that the loss in intercellular adhesion due to the reduced expression of a transmembrane adhesion protein, E-cadherin, plays a pivotal role in the selective uptake of HY. Later, Olivo et al.68 conducted further studies to evaluate the expression of other cell-adhesion molecules (E-cadherin, α-catenin, β-catenin, and γ-catenin) in histologically graded biopsies obtained after intravesical instillation of HY. In particular, using immunofluorescence staining, E-cadherin expression was found to decrease in proportion to both the tumor grade as well as the depth of invasion. In addition, mRNA analysis showed that expression of E-cadherin and β-catenin was down regulated in high-grade urothelial cell carcinoma (UCC), modifying the histoarchitecture in bladder tumor and decreasing the epithelial paracellular barrier function. This eventually leads to enhanced paracellular diffusion of HY into the deeper layers of tissue before intracellular uptake. Hence, stronger HY fluorescence in the UCC was observed with increased grade and advanced stage. The fluorescence imaging showed that the HY intensity of grade 1, 2, and 3 tissues was approximately 5.4, 8.4, and 10.4 times greater than that of normal bladder tissues.68
Solubility and Delivery
Hypericin is a lipophilic compound that forms aggregates and sparingly dissolves in aqueous solution. Different solvents have been tried to overcome this hydrophobicity of HY. These include organic solvents, such as ethanol, tetrahydrofuran, and acetone, and also serum proteins which are reported to dissolve most hydrophobic compounds.34,74,76,100 Therefore, the focus of recent studies by research groups involved in fluorescence diagnosis of cancer is directed toward new formulations to be used with HY, like using Solketal as absorption enhancer,42 polyvinylpyrrolidone (PVP)93 and cyclodextrins as solubilizers,78 and NMP as penetration enhancer.76 For example, our lab investigated HY formulated with different concentrations of NMP and found effective solubilizing and delivery of HY to the target site, bladder urothelium in this study.77 Subsequently, our in vitro study with MGH cell line and in vivo studies showed that HY with 0.05% NMP yielded optimal uptake and cell survival. Its delivery efficacy was found to be superior to HY with human serum albumin, which is being clinically used for PDD.76,101 However, further studies are warranted to determine the optimal concentration of NMP, which solubilizes HY and enhances its tissue penetration without compromising uptake and cell survival.
Hypericin-Mediated PDD of Cancer with Vital Fluorescence Technologies
D’Hallewin et al.12 first reported the clinical use of HY-induced PDD in detecting flat CIS lesions of bladder. Thereafter, studies of HY mostly focused on the detection of bladder cancer.14,43,81 Apart from bladder screening, the applications of HY were also extended to diagnosing other types of cancers including oral cancer88 and stomach cancer.15,58 The following sub-sections will mainly discuss about the development of instrumentation and data processing for HY-PDD of bladder and oral cancer.
Microscopic Fluorescence Studies and Diagnosis
Ex Vivo Urine Cytology
Currently, oral cancer and bladder cancer are diagnosed by examination under WLE followed by random biopsies on suspicious-looking tissues and histological examination of the biopsies. Histopathological examination of biopsy tissue remains the gold standard for definitive diagnosis. However, this approach to make diagnosis presents at least two key limitations. First, early flat oral or bladder lesions are hardly visible, and it is difficult to distinguish between benign and early cancer.9,88 Poor visual contrast in WLE therefore necessitates a random sampling of tissues, but still risks missing the lesion and gives false negative results.3 Additional biopsies could also cause unnecessary suffering to patients. Needless to say, a complete surgical clearance would also be difficult. Second, tissue processing is laborious and dependent on the skill of the operator. The histopathological examination also relies on subjective visual impression from clinicians. It is therefore of interest to develop novel imaging techniques that can provide objective and accurate diagnosis without the need for tissue resection.
Following the earlier statement that PSs accumulate selectively in abnormal cells, the microscopic fluorescence analysis was extended to macroscopic endoscopic images. FE was used to highlight lesions from which the PS fluorescence is emitted when excited by light at appropriate wavelength. Several groups have explored PSs that were suitably used in fluorescence endoscopy in an attempt to overcome the shortcomings of WLE.73,80,106,112 To date, a number of review articles are available to compare the efficacy of different PSs for detecting bladder tumor.33,34,44,60 A comparison of their diagnostic performance is thus not reviewed here. In essence, the mean value of the sensitivity for FE is 93% compared to only 73% for WLE.34 The increase in diagnostic sensitivity (82% for HY vs. 62% for white-light) compared to WLE has led to use of HY in fluorescence-guided biopsies to minimize unnecessary biopsies.81
Real-Time 3D Endomicroscopy
To take this HY-mediated fluorescence endoscopy platform further, our group developed an embedded, real-time computing system interfaced with a confocal laser endomicroscopy (CLE) for 3D visualization of tumors.90 CLE has been applied for autofluorescence diagnostic imaging in gastroenterology,20,29,46,71 gynecology,85 urology,83 human oral, and oropharyngeal mucosa.23 A CLE offers imaging capabilities for in vivo fluorescence imaging of tissue structures from the surface to a few hundred micrometers beneath the surface.17,46 This sub-surface imaging has been found useful to detect malignancy by means of the altered tissue architecture and irregularities in blood vessels.23
Coupling with Nanotechnology for Combinatorial Therapeutic and Multimodality Imaging
Traditionally, PSs are solely used for PDT in cancer treatment, and their inherent fluorescence properties are utilized to aid diagnosis as discussed in previous section. However, the recent advancements in nanotechnology opens up new horizon for PSs to be applied in entirely different ways. A short review is presented here to highlight the possibility of developing nanoconstruct integrated with HY for combinatorial therapeutic and multimodality imaging.
Enhanced Targeted Delivery
Nanometer-sized gold particles are known to exhibit surface plasmon resonance (SPR) effect that is absent in bulk gold. This plasmonic feature makes GNPs efficient in generating heat in the presence of electromagnetic radiation.21 At SPR frequency, the strongly absorbed radiation is converted into heat by electron–phonon and phonon–phonon processes.26 The particles thus serve as heating elements that lead to elevated temperature of the surrounding medium and induce hyperthermia or irreversible thermal damage to the targeted tissue, thereby offering an alternative cancer treatment called photothermal therapy (PTT).32,95 A study showed that the heat conversion efficiency of GNPs increases with their size.21 In addition, aspect ratio of the GNRs can be tailored to operate PTT within the optical window between 700 and 900 nm, where light penetration in tissue is optimal due to minimal absorption by water and hemoglobin in the tissue95; consequently, the excitation light can reach deeper tissues and induce PTT. More importantly, recent studies suggest that PDT and PTT can potentially be merged for efficient cancer treatment.31,41,67
Controlled Drug Release for PDT
There are other features of gold nanostructures that are worthy of mention. These gold nanostructures often possess high scattering cross sections and are able to enhance the weak Raman signal of molecules. Though HY fluorescence is more commonly studied, its Raman characteristics should not be ignored as the Raman signal is detectable when the HY molecules are sufficiently close to the gold surface which induces surface-enhanced Raman scattering (SERS).24 A study has shown that a joint fluorescence and SERS measurement of HY can facilitate the understanding of its biological activity.54
While PSs are useful in giving molecular contrast in fluorescence and SERS imaging, the gold nanostructures serve as additional optical contrast agent in other structural imaging such as optical coherence tomography (OCT)27 and two-photon luminescence.113 In particular, OCT, a biopsy-free imaging, is being used for in vivo clinical applications.5,19,57 The advantage of OCT is its ability to provide cross-sectional images of tissue with high spatial resolution by measuring the optical echoes backscattered from tissue.27 Currently, much effort is put to further develop OCT toward acquisition of histologically equivalent images for definitive diagnosis without the need of biopsies. The contrast in OCT image is highly dependent on the microscopic differences in scattering properties across the tissue layers. However, the early onset of tissue abnormalities barely changes the tissue optics that result in poor morphological contrast between normal and abnormal tissues in OCT images.38,40 To overcome these limitations, gold nanostructures, along with being utilized as a PS carrier, are used to increase the diagnostic and analytic capabilities of OCT by site-specific labeling of tissue of interest. The plasmonically active nanostructures, such as GNPs,40,48 GNRs,92 nanoshell55, and nanocages,8 can be optically tuned to specifically scatter the OCT probe beam. The key idea is simply to use OCT to demarcate the abnormal sites at which these light-scattering particles are targeted. Though this concept was demonstrated using rigid OCT probe, there are several groups1,36,47,110 working on miniaturization of OCT system that can potentially be merged with existing fluorescence endoscopy. For example, Mu et al.98 have developed a microelectromechanical systems (MEMS)-based OCT catheters to perform a rapid 3D volumetric scanning. This MEMS-OCT probe has an outer diameter of only 4 mm and encloses a patented MEMS micromirror as the optical steerer. One can envision that such technique combined with molecular imaging is powerful for improved diagnosis and informative monitoring of tissue structures during PDT or PTT. Hence, combining advancements in PDD and nanotechnology offers the opportunity to significantly impact future strategies toward improved multimodality therapeutics and imaging.
We have investigated the combined use of HY and state-of-the-art optical techniques for detecting early cancer, with improved diagnostic accuracy in comparison with the standard clinical practice. The standard diagnosis, which relies on WLE, lacks the ability in identifying early and flat lesions, and thus runs the risk of giving false negative results. HY used with novel optical techniques provides an alternative to detect the subtle structural and molecular changes expressed during the development of cancer.
We began by discussing the increased uptake of HY in progressively invasive cancer cell lines and biopsies. The tumor selectivity of HY, especially in bladder cancer, makes it a suitable fluorescent contrast agent in differentiating the scattered cancerous sites from the surrounding healthy tissues. These fluorescence properties were first exploited in ex vivo fluorescence urine cytology. Unlike tissue sampling, the cell yield in urine cytology is rather limited, and the presence of contaminants in urine could complicate the fluorescence analysis. Image processing techniques were applied to get around the problem. Meanwhile, the diagnostic information from each single cell was also maximized by studying the HY intensity histogram. Subsequently, the microscopic diagnosis with HY was extended to macroscopic level using FE. FE was employed to diagnose bladder and oral cavity in situ. With the use of appropriate camera and optics elements, a single shot of fluorescence image was adequate to perform a real-time PDD without the need for a slow fluorescence spectral measurement. Fluorescence diagnosis could be automated and visual aid was provided to clinicians for a fluorescence-guided biopsy or resection of tumor. PDD was further developed by incorporating a CLE for probing the HY intensity at different tissue depths. In addition, fluorescence images collected at different focal planes were processed to render a 3D visualization from which more detailed information about the lesion morphology could be assessed.
While the fluorescence properties of the HY have been well exploited in PDD, we discuss about the underlying trend of coupling HY with the nanotechnology for combined therapeutics and multimodality imaging. The use of gold nanostructures not only enhances the targeted delivery of HY to cancer cells, but also serves as a heating element to trigger PTT as well as providing optical contrast for other optical imaging techniques. Meanwhile, the photoheating of GNPs was utilized to manage the release of HY from nanoconstruct, thus controlling the PDT efficacy; such approach may carry high potential for modulating PDT and PTT. Apart from enhancing molecular contrast with HY, the nanoparticles serve as complimentary contrast agent when used with other novel optical imaging modalities, such as two-photon luminescence imaging, dark-field microscopy, OCT, and SERS. With the continual effort in this field, we envision a successful integration of PSs and multifunctional nanoparticles to realize informative diagnosis followed by a multifaceted therapy in near future.
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