Project Three:
Nanoparticles for photodynamic therapy
Project References
Nancy L. Oleinick, Ph.D. Department of Radiation Oncology
Clemens Burda, Ph.D. Department of Chemistry
Malcolm E. Kenney, Ph.D. Department of Chemistry
Case Western Reserve University
Photodynamic therapy (PDT) is a novel treatment for cancer and certain non-cancerous conditions characterized by the overgrowth of abnormal or undesired tissue. PDT involves the systemic or topical administration of a photosensitizer, generally porphyrin-related, followed by the focused photoirradiation of the tumor with tissue-penetrating visible (red) light that is absorbed by the photosensitizer. The ensuing photochemistry generates the highly reactive singlet oxygen that kills the targeted tissue [1]. PDT is FDA-approved for treatment of lung and esophageal cancer, Barrett’s esophagus, age-related macular degeneration, and actinic keratoses [2]. Photosensitizer fluorescence is also being investigated for delineating the tumor margins, which is dependent upon a high specificity of photosensitizer uptake by malignant cells. Although tissue damage can be focused through delivery of light, none of the photosensitizers is delivered in a way that specifically targets tumors.
Our prototype photosensitizer is Pc 4, a phthalocyanine synthesized and developed at Case and now in clinical trials in the Case Comprehensive Cancer Center for dermal malignancies [3]. Pc 4 is taken up somewhat preferentially into tumor as compared to surrounding normal tissue, without any specifically designed targeting mechanism [4]. Nanoparticles bearing tissue-targeting ligands could improve the focused delivery of a photosensitizer payload to malignant cells, making PDT even more effective for both imaging and treating of, e.g., infiltrating cancers or those growing near sensitive normal structures. Recent attempts to deliver photosensitizers to cells using nanoparticles have produced limited photodynamic efficacy; e.g., in one case, sequestering of the photosensitizer within a stable nanoparticle kept it from the most sensitive cellular targets [5]. One of our goals is to develop a platform technology for delivery of PDT photosensitizers into cancer cells in a manner that maximizes phototoxicity by targeting sensitive cellular sites. The technology is based on our understanding of the mechanisms of photodynamic action and our identification of the anti-apoptotic proteins Bcl-2 and Bcl-xL, and possibly also cardiolipin, as immediate molecular targets of Pc 4-PDT [6-8].
Approach:
Aim 1. To develop nanoparticles for targeted delivery of photosensitizers to tumors. Pc 4 will be bonded to non-toxic nanoparticles that are capped with (a) ligands providing stability during transit from an injection site to the desired tissue and (b) a targeting ligand, such as an antibody to a cell-surface receptor. This construct will allow the photosensitizer to reach the target tissue with improved specificity and bind to its preferred sites that are already known to confer highly efficient photodynamic cell killing when the tissue is illuminated. The constructs will be characterized in the Materials Core and the Center for Chemical Dynamics and then tested for the ability to deliver the photosensitizer to cancer cells in vitro, to further deliver it to a known target (Bcl-2), and to sensitize cells to photodynamic cell killing such that the dose response is improved over delivery of Pc 4 directly. The best construct will then be tested in a PDT protocol in vivo for its stability, pharmacokinetics, and efficacy in tumor ablation.
Aim 2. To develop nanoparticles capable of serving as efficient tumor-targeted photosensitizers for PDT. Highly fluorescent nanoparticles should be ideal for both localization and focused treatment, because of the ability to tune them to any desired wavelength of visible light and to target them with ligands having known binding sites. This part of the project will thus be directed to developing nanoparticles with (a) optimum spectral properties, (b) structures that promote entry into cancer cells, and (c) ligands directing the nanoparticles to specified cellular sites. These will be tested in a manner similar those in Aim 1.
Aim 3. To develop a method for monitoring and optimizing the delivery of the photosensitizer to target cells and tissue. To improve the nanoparticle delivery system (Aims 1 and 2), we will develop a method for diagnosing the extent of release of Pc 4 from the nanoparticle in cells and tissues. The method will take advantage of FRET (fluorescence resonance energy transfer) between nanoparticles and photosensitizer [9]. As prepared, the complex should efficiently conduct FRET and display strong emission from Pc 4 when the nanoparticle is activated. Once Pc 4 has been released within the target cells, FRET should be markedly reduced and the emission properties of the system changed.
Expectations:
We envision that these technologies will yield a superior modality for imaging tumor margins and treating cancer in a PDT setting. It will also yield a platform for delivery of other photosensitizers and drugs. Thus, the therapeutic and imaging uses of this technology are interrelated.
Synergy with other projects:
This project will benefit from collaboration with others designing nanoparticles for small-molecule drug delivery (Projects 5 and 6) as well as those delivering imaging agents. This project will contribute methodology to target specific sites within cancer cells and to track delivery of a fluorescent payload.
Imaging agents as payloads:
The incredible advances in imaging of late only serve to remind us of greater possibilities. Imaging molecules can be applied to all three phases of biomedical disease modification, starting with localizing the disease, continuing through tracking therapeutic delivery, and concluding with monitoring therapeutic efficacy. For example, molecular-targeted imaging could provide information not only on tumor anatomy but also tumor biology, vastly enhance our ability to identify metastates at an early stage, gauge the localization and efficacy of tumor therapeutics, and assess long-term tumor viability. Moreover, nanoparticles offer the opportunity to combine diagnostic and therapeutic approaches: the diagnostic imaging probes may be combined with therapeutics in the same payload. In some cases the imaging molecules may enhance the therapeutic effect. Case is an ideal site to develop new imaging modalities because of a unique small animal imaging resource (partially supported by a prior BRTT for colon cancer, but mainly by UHC and Case, and grants from the NIH and other private sources). This extensive resource, directed by Jeff Duerk, professor and director of the new Center for Animal Imaging, combines a “service facility” and a “research facility” that allows investigators to test their products expeditiously and reengineer if necessary. All major imaging modalities are available along with support for image analysis and animal handling in a centralized facility (see below). Moreover, there is a nucleus of investigators dedicated to devising new imaging approaches using nanoparticles.
Drs. Fitzmaurice, Burda, Wilson, Rollins and Pagel are collaborating in developing targeted nanocrystals suitable for imaging in three different modalities: fluorescence, MR, and OCT. Clemens Burda brings to this group the expertise to design and synthesize customized nanocrystals. Maryann Fitzmaurice has expertise in optical spectroscopy and is responsible for biologic testing and fluorescence imaging of the particles. Andrew Rollins has expertise in OCT system design, fabrication and imaging and is one of the first to devise new OCT contrast agents. He will oversee SHG testing and imaging of the particles. Mark Pagel will oversee MR testing and imaging of the particles. He has also been developing targeting methods that rely on functional changes induced in diseases tissues. For example, one of his “smart” particles has a linker cleavable by matrix metalloproteinase-9, a protease released at inflammatory sites. When the linker is cleaved, the imaging moiety is released and tags the site. Dr. David Wilson is a biomedical engineer and expert in image analysis, which is critical for analyzing multi-modality image data. This work is in the early stages but almost surely will provide new probes that will help bring molecular-based optical, MR and dual modality imaging techniques to the bedside. These imaginative approaches to imaging are also prototypes for next-generation particles, which can be engineered to suit many different purposes. They could be incorporated along with other payloads into dual purpose nanoparticles. There is clear collaboration between the Gao lab and these proposal: Dr. Gao is coinvestigator on Project 8. Two projects are proposed in this area.