Article Published in the Author Account of

Diana M Tacelosky

Calcium Phosphosilicate Nanoparticles for Imaging and Photodynamic Therapy of Cancer

Abstract: Photodynamic therapy (PDT) has emerged as an alternative modality for cancer treatment. PDT works by initiating damaging oxidation or redox-sensitive pathways to trigger cell death. PDT can also regulate tumor angiogenesis and modulate systemic antitumor immunity. The drawbacks to PDT -- photosensitizer toxicity, a lack of selectivity and efficacy of photosensitizers, and a limited penetrance of light through deep tissues -- are the same pitfalls associated with diagnostic imaging. Developments in the field of nanotechnology have generated novel platforms for optimizing the advantages while minimizing the disadvantages of PDT. Calcium phosphosilicate nanoparticles (CPSNPs) represent an optimal nano-system for both diagnostic imaging and PDT. In this review, we will discuss how CPSNPs can enhance optical agents and serve as selective, non-toxic, and functionally stable photosensitizers for PDT. We will also examine novel applications of CPSNPs and PDT for the treatment of leukemia to illustrate their potential utility in cancer therapeutics.



Photodynamic Therapy

Photodynamic therapy (PDT) has emerged as an alternative strategy for treating cancer. PDT consists of three main components: a photosensitizer, light, and oxygen. PDT takes advantage of an appropriate wavelength of light that excites a photosensitizer to a triplet energy state (Juarranz et al., 2008; Ortel et al., 2009; Wainwright, 2008). In the presence of molecular oxygen, energy is transferred to relax the excited state of the photosensitizer (Figure 1). This energy transfer in turn excites molecular oxygen to form excited, singlet state oxygen (Figure 2). Singlet oxygen induces cell death via damaging oxidation or redox-sensitive cellular signaling pathways, thus mediating the effects of PDT (Dolmans et al., 2003; Huang et al., 2008; Juarranz et al., 2008). Intriguingly, PDT has also been shown to regulate processes beyond tumor cell death including tumor angiogenesis and modulation of the immune system (Dolmans et al., 2003; Gollnick et al., 2010; Juarranz et al., 2008; Reddy et al., 2006). It has been speculated that PDT, when effective, can actually restrict tumor nutrient supply by the ablation of tumor blood vessels and by triggering an immune response that can afford systemic antitumor immunity (Gollnick et al., 2010; Hu et al., 2010; 2011; Mroz et al., 2010; 2011; Tammela et al., 2011). For these reasons, PDT continues to garner considerable interest, as technologies develop to overcome potential limitations. Disadvantages of current PDT include photosensitizer toxicity, a lack of efficacious and selective photosensitizers, and an inability of light to sufficiently penetrate through tissues to reach tumors deep within the body (Chatterjee et al., 2008; Donnelly et al., 2008; Huang et al., 2006; Sibani et al., 2008; Wainwright, 2008). For these reasons, PDT is primarily utilized to treat cancers of the skin and esophagus (Ficheux, 2009; Gross et al., 2010; Kotimaki, 2009; Nyst et al., 2009). Interestingly, the pitfalls of PDT are the same as those associated with diagnostic imaging. Advances in the field of nanotechnology may therefore overcome the hurdles associated with both diagnostic imaging and PDT. In this review, we will focus on the development of our novel nanotechnology and its application for both diagnostic imaging and PDT. It is important to note that although we focus on our technology, other considerable advances within the fields of nanotechnology and photobiology are also reinvigorating the field of PDT research. In particular, various polymeric and nonpolymeric nanoparticles have been reported to encapsulate photosensitizers and have demonstrated efficacy in both in vitro and in vivo models while decreasing toxicity (Cheng et al., 2011; Hocine et al., 2010; Lee et al., 2011a; Lee et al., 2011b; Ohulchanskyy et al., 2007; Qin et al., 2011; Reddy et al., 2006; Rungta et al., 2011).

Calcium Phosphosilicate Nanoparticles

Figure 1.

Figure 1. Jablonski diagram depicting changes in molecular electronic states associated with photodynamic therapy. Energy from light of a specific wavelength is absorbed by the photosensitizer indocyanine green (ICG), which excites the molecule from a ground singlet state (S0) to an excited singlet state (S1). ICG in this excited state can return to a ground state via fluorescence or non-radioactive decay, or can undergo intersystem crossing and enter an excited triplet state (T1). Molecular oxygen exists in a ground triplet state, and so an energy transfer can occur between the triplet state of ICG and molecular oxygen. This energy transfer results in the generation of singlet oxygen.

Calcium phosphosilicate nanoparticles (CPSNPs) were initially developed to improve the optical and quantum properties of encapsulated dyes (Altinoglu et al., 2008; Kester et al., 2008; Morgan et al., 2008). CPSNPs were also engineered to protect agents during systemic delivery, ensuring a pH-dependent release following endocytosis (Kester et al., 2008; Morgan et al., 2008). CPSNPs are very small (approximately 20 nm diameter) (Adair et al., 2010; Tabakovic et al., 2012). They offer an advantage over other nanoparticles, because dyes, drugs, or other molecules of interest are encapsulated within a calcium phosphosilicate nanomatrix. This is in contrast to surface decoration of the nanoparticle, which can be the case for a variety of other nanoparticles, including other calcium phosphate-based systems (Graham et al., 1973). Calcium and phosphate are also advantageous materials for nanoparticles because they are both abundant in physiological systems and pose no inherent toxicity (Tabakovic et al., 2012). This is in stark contrast to other nanoparticles composed of cadmium, selenium, heavy metals, or hydrocarbons that are significantly toxic and cannot be used effectively in biomedical applications (Adair et al., 2010; Tabakovic et al., 2012). Additionally, the calcium phosphosilicate nanomatrix is stable at physiological pH, allowing these nanoparticles to protect their “payloads” during systemic circulation. CPSNPs are engulfed by cells during endocytosis and ultimately trafficked via the endosomal-lysosomal pathway (Altinoglu et al., 2008; Barth et al., 2010; 2011; Kester et al., 2008; Morgan et al., 2008; Muddana et al., 2009; Tabakovic et al., 2012). Local acidic pH changes cause the CPSNPs to degrade and release their contents. This controlled release ensures that free-dye, or free-drug, is not exposed in systemic circulation where the free-dye/free-drug can do considerable harm. Improvements to CPSNPs, such as selective targeting, further ensure that these nanoparticles only go where they are intended, thus protecting the body’s tissues from unintended and off-target effects (Barth et al., 2010; 2011). Our design of CPSNPs also includes surface functionalization with polyethylene glycol (PEG) (Altinoglu et al., 2008; Barth et al., 2010; 2011; Kester et al., 2008; Morgan et al., 2008). PEGylation of nanoparticles, such as CPSNPs, helps to permit longer systemic retention and reduce nanoparticle clearance by the immune system (stealth characteristic). Additionally, PEGylated nanoparticles have the ability to accumulate within solid tumors via a process known as the enhanced permeation and retention (EPR) (Altinoglu et al., 2008). This effect is due to the unique and disorganized vasculature of tumors, in addition to poor lymphatic drainage, which allows small nano-sized materials to accumulate within tumors (Fang et al., 2011; Hirsjarvi et al., 2011). In a diagnostic imaging trial using CPSNPs, we showed that the accumulation of CPSNPs within tumors is dependent upon PEGylation of the nanoparticles; because non-PEGylated CPSNPs failed to accumulate and retain within breast cancer tumors in vivo (Altinoglu et al., 2008). This imaging study also revealed that CPSNPs primarily leave the body via hepatobiliary clearance, further minimizing toxicity. Altogether, CPSNPs represent an optimal nano-system for biomedical applications because of eight key achievements: (1) CPSNPs are made of non-toxic materials, (2) CPSNPs are truly nano-sized, (3) CPSNPs completely encapsulate payloads, (4) CPSNPs are colloidally stable in physiological conditions, (5) CPSNPs have increased retention times due to surface PEGylation, (6) CPSNPs clear via hepatobiliary excretion, (7) CPSNPs offer controlled pH-mediated release of payloads, and (8) CPSNPs can be functionalized to selectively target specific cells (Adair et al., 2010).

CPSNPs for Diagnostic Imaging of Cancer

Figure 2.

Figure 2. Schematic of the energy transfer process between indocyanine green and oxygen during photodynamic therapy. Left panel: indocyanine green (ICG) is in a ground singlet energy state (S0), while molecular oxygen exists in a ground triplet energy state (T1). Center panel: following absorption of an appropriate wavelength of light, the photosensitizer ICG enters an excited singlet energy state (S1). Right panel: excited ICG (S1 state) undergoes intersystem crossing to enter an excited triplet energy state (T1), which allows an energy transfer to molecular oxygen to occur and generate singlet oxygen (ground S0 state). This schematic also depicts simplified molecular orbital diagrams for molecular oxygen indicating the changes in the spin of the electrons in the two degenerate antibonding πθ orbitals.

One of the distinct goals of CPSNP development was to improve the optical properties of fluorescent dyes to allow for better imaging for cancer diagnostics. We have developed CPSNPs that encapsulate a range of fluorescent dyes, and we have tested these in various experimental settings including cell and animal imaging. For whole animal imaging, we chose to encapsulate the near-infrared fluorescent dye indocyanine green (ICG) (Altinoglu et al., 2008; Barth et al., 2010; 2011). For biological tissues, light scattering is a serious drawback to effective imaging. Near-infrared fluorescing probes, such as ICG, offer an advantage in that their excitation and emission wavelengths are long enough to pass through biological tissues with minimal to no light scattering. As mentioned, the optical properties of fluorescent dyes, such as ICG, are vastly improved by encapsulation within the nanomatrix of CPSNPs (Altinoglu et al., 2008). ICG is currently used as an FDA-approved contrast agent in imaging applications (Bennett et al., 2009). However, the use of ICG suffers considerably from a short plasma half-life (2-4 minutes), photobleaching, and nonspecific quenching due to binding with serum proteins (Desmettre et al., 2000). Encapsulation of ICG within CPSNPs overcame these drawbacks and vastly improved its optical properties. In our initial diagnostic imaging trial we compared the ability of free (non-encapsulated) ICG and CPSNPs loaded with ICG to image mice implanted with breast cancer tumors (Altinoglu et al., 2008). In this study, free ICG was not able to effectively image tumors, whereas ICG-loaded CPSNPs (PEGylated) effectively accumulated in tumors and imaged tumors up to 96 hours following systemic injection (Altinoglu et al., 2008). The improvements to ICG-mediated imaging are directly reflective of the enhanced fluorescence quantum efficiency (ΦF) and photostability of ICG when encapsulated within CPSNPs. In a subsequent study, the ΦF of free ICG and nano-encapsulated ICG were directly compared (Russin et al., 2010). It was demonstrated that the ΦF in PBS for free ICG was 0.027 +/- 0.001, a typical CPSNP loaded with ICG was 0.053 +/- 0.003, and ICG molecules encapsulated within CPSNPs were 0.066 +/- 0.004 (Russin et al., 2010). This study also found that 6 +/- 2 ICG molecules were encapsulated per typical CPSNP (Russin et al., 2010). The specific optical improvements described for ICG-loaded CPSNPs also potentially expand the use of ICG beyond imaging to therapeutic applications such as PDT.

Our original diagnostic imaging study relied on the EPR effect to allow for effective imaging of implanted breast cancer tumors (Altinoglu et al., 2008). This allowed for good CPSNP accumulation, but we questioned if this would be the most effective imaging modality. Furthermore, more selective targeting was of interest to improve the potential for therapeutic delivery to a cell population. In a subsequent study, we improved in vivo imaging by targeting CPSNPs specifically to CD71 on breast cancer tumors or to the gastrin receptor on pancreatic cancer tumors (Barth et al., 2010). The transferrin receptor (CD71) is found predominately on proliferating cells with elevated metabolic levels, including many cancerous cells (e.g., breast cancer cells), brain capillary endothelial cells, and hematopoietic cells (Daniels et al., 2006a; 2006b; Gosk et al., 2004; Shindelman et al., 1981; Sutherland et al., 1981). Similarly, gastrin receptors have prevalence within particular tissues within the gastrointestinal and central nervous systems (Wank et al., 1992). These G-protein-coupled receptors, also known as the cholecystokinin-2 (CCK2 or CCK-B) receptor family, are often increased in many cases of gastrointestinal cancer (Smith et al., 1996; 1998). In pancreatic cancer, there can be a particular increase in the expression of a specific splice variant (CCK2i4sv or CCK-C) of the receptor (Smith et al., 1994; 1995; 2002). Our study evaluated different methods for attaching ligands, fragments of ligands, or antibodies to CPSNPs to permit targeting of either CD71 or gastrin receptors (Barth et al., 2010). We found that targeting CD71 improved the imaging capability of CPSNPs for subcutaneously implanted breast cancer tumors. Furthermore, and more profoundly, targeting CPSNPs to gastrin receptors permitted robust imaging of orthotopically implanted pancreatic cancer in vivo. This was particularly exciting because these tumors are diffuse and occur in regions with poor blood supply to allow for nanoparticle accumulation. While non-targeted CPSNPs were able to image orthotopic pancreatic tumors, gastrin receptor-targeted CPSNPs were able to robustly image the entire extent of the orthotopic tumor (Figure 3). Owing to the prevalence of gastrin receptors in the central nervous system, these targeted CPSNPs were also able to cross the blood brain barrier and image the brain (Barth et al., 2010; Wank et al., 1992). Other groups have described actively targeted nanoparticles that cross the blood brain barrier, suggesting that targeted CPSNPs could be engineered to diagnose and treat tumors of the central nervous system (Madhankumar et al., 2009).

Photodynamic Therapy Utilizing Indocyanine Green-loaded CPSNPs

Figure 3. Imaging of pancreatic cancer with indocyanine green-loaded calcium phosphosilicate nanoparticles is improved by gastrin receptor targeting. Orthotopic human BxPC-3 pancreatic cancer tumors were established in athymic nude mice. Indocyanine green (ICG)-loaded calcium phosphosilicate nanoparticles (CPSNPs) were systemically injected and tumor imaging was performed using a Kodak In Vivo FX Imager 24 hours later. A, Non-targeted ICG-loaded CPSNPs (PEGylated). B, Gastrin receptor-targeted ICG-loaded CPSNPs (gastrin-10 peptide covalently linked to PEGylation). Note targeting of the brain in panel B due to the presence of gastrin (cholecystokinin) receptors in the central nervous system.

Figure 3. Imaging of pancreatic cancer with indocyanine green-loaded calcium phosphosilicate nanoparticles is improved by gastrin receptor targeting. Orthotopic human BxPC-3 pancreatic cancer tumors were established in athymic nude mice. Indocyanine green (ICG)-loaded calcium phosphosilicate nanoparticles (CPSNPs) were systemically injected and tumor imaging was performed using a Kodak In Vivo FX Imager 24 hours later. A, non-targeted ICG-loaded CPSNPs (PEGylated). B, gastrin receptor-targeted ICG-loaded CPSNPs (gastrin-10 peptide covalently linked to PEGylation). Note targeting of the brain in panel B due to the presence of gastrin (cholecystokinin) receptors in the central nervous system.

The limitations of the photosensitizers used for PDT are similar to those of optical agents used for diagnostic imaging. Furthermore, ICG has previously been evaluated as a photosensitizer for PDT in various models of cancer (Abels et al., 2000; Baumler et al., 1999; Bozkulak et al., 2009; Crescenzi et al., 2004; Fickweiler et al., 1997; Mamoon et al., 2009; Rungta et al., 2011; Tseng et al., 2003; Urbanska et al., 2002). Therefore, we hypothesized that the improvements we achieved for diagnostic imaging using ICG-loaded CPSNPs could translate to utility as a photosensitizer for PDT of cancer. Both improvements in optical properties and the ability to selectively target CPSNPs were essential to the development of ICG-loaded CPSNPs as effective photosensitizers (Altinoglu et al., 2008; Barth et al., 2010; Russin et al., 2010). As described below, our initial and published work validated the utility of ICG-loaded CPSNPs for PDT of leukemia (Barth et al., 2011). Moreover, we were able to selectively target our CPSNPs to leukemia stem cells (LSCs), eradicating the specific cells responsible for the maintenance and progression of the disease. Finally, we will discuss briefly our unpublished work that has started to evaluate PDT utilizing ICG-loaded CPSNPs in solid tumors. This work is more specifically related to our initial imaging studies and has centered on the utility of ICG-loaded CPSNPs as “theranostic” agents based on the ability to simultaneously diagnose and treat.

Photodynamic therapy of leukemia

Leukemia, a cancer of the blood and bone marrow, is a rather broad term used to describe many different hematological malignancies (Perrotti et al., 2010; Radhi et al., 2010; Roboz et al., 2009). Typically, leukemia is sub-classified by cell origin (myeloid or lymphoid), and by disease onset or progression (acute or chronic). Even a given subtype, such as acute myeloid leukemia, characterizes many different hematological malignancies that can be further described based on cytogenetic or molecular markers. The treatment of leukemia typically consists of high dose chemotherapy, which in itself can pose significant risk to the patient. These risks are more profound when considering that leukemia is both one of the most common pediatric cancers and one of the more common cancers in the elderly (Perrotti et al., 2010; Radhi et al., 2010; Roboz et al., 2009). In these populations, these aggressive treatments can lead to serious complications or even be fatal. Additionally, high dose chemotherapeutics also carry the risk of being carcinogens that may possibly lead to therapy-induced leukemia in the patient’s future. Therefore, better therapeutics for the treatment of leukemia, both with improved efficacy and lower toxicity, are needed.

The potential treatment of leukemia with PDT is an intriguing idea, in part because the side effects of PDT are modest in comparison to current leukemia chemotherapeutics. Due to the short lifetime of singlet oxygen, it is thought that little opportunity exists during PDT for carcinogenic transformation to occur, leaving cell death as the only outcome (Juarranz et al., 2008; Robertson et al., 2009). Unfortunately, PDT has never been used clinically to treat leukemia. This is likely due to the nature of leukemia as a systemic disease with little opportunity for photosensitizers to accumulate in circulating leukemic cells. Furthermore, the inability to penetrate appropriate light throughout the body to activate photosensitizers is another limitation. Generally, the published laboratory reports of PDT utility for leukemia have almost exclusively been performed in cell culture (Feuerstein et al., 2009; Furre et al., 2005; Gamaleia et al., 2008; Pluskalova et al., 2006).

To our knowledge, there are currently three published studies where PDT was studied in vivo for the treatment of leukemia (Huang et al., 2006; Barth et al., 2011; Wen et al., 2011). The first was a hybrid study which reported the utility of PDT to purge leukemic cells from autologous hematopoietic stem cells ex vivo prior to transplant in animal models (Huang et al., 2006). The other two studies, including our study, evaluated more traditional PDT, occurring exclusively in vivo (Barth et al., 2011; Wen et al., 2011). Simultaneous with our publication, Wen et al. published a report of in vivo PDT in a murine model of B cell lymphoma. In that study, the photosensitizer Photodithazine was injected systemically into the tail vein and then photofibers were inserted into the tail vein, via the same needle, to perform PDT using a diode laser. There is some question, from our perspective, as to whether or not the experimental methods may have attributed to the reported therapeutic efficacy. Namely, “successful” PDT was performed only one day following the initial injection of leukemic cells. In contrast, PDT was unsuccessful when it was performed five days following initial injection of the leukemic cells. This raises an important question as to whether or not the leukemia had actually engrafted in the study by Wen et al. Furthermore, the method of inserting photofibers into the tail vein of mice for over an hour of laser irradiation treatment implies an invasive procedure that may be necessitated by the limitation of light penetration. Indeed, this is similar to laboratory animal trials and human clinical trials of PDT for abdominal and other internal malignancies where laser fiber optics are introduced into the body via a surgical procedure (Allison et al., 2009; Friedberg, 2009; Huang et al., 2008; Moore et al., 2009; Ortner, 2009).

The third instance of PDT being used in vivo for the treatment of leukemia is our recently published study evaluating ICG-loaded CPSNPs (Barth et al., 2011). We studied selectively targeted CPSNPs compared to non-targeted CPSNPs. We evaluated several physical properties of the nanoparticles, including size, charge, and the ability to label and internalize into cells of interest. We also studied the specific ability of ICG-loaded CPSNPs to generate reactive oxygen species (singlet oxygen and superoxide) in cellular models in response to laser treatment. Additionally, we evaluated the therapeutic efficacy of our ICG-loaded CPSNP version of PDT in cellular models of murine chronic myeloid leukemia and in human samples from patients with acute myeloid leukemia. For our in vivo work, we used a murine model of blast crisis chronic myeloid leukemia (32D-P210-GFP cells engrafted in C3H/HeJ mice), which behaves similarly to acute myeloid leukemia when in blast crisis (Barth et al., 2011; Daley et al., 1990; Greenberger et al., 1983; Keasey et al., 2010; Ling et al., 2006). We confirmed engraftment of the disease via flow cytometry before treatments were commenced. In our model, laser treatment was performed completely outside of the body with the laser directed at the spleen (Barth et al., 2011). In addition to a profound extension of life, using targeted CPSNPs, we also observed disease-free survival of some treated animals. Importantly, we were also able to monitor disease progression by analyzing blood for GFP+ leukemic cells.

Figure 4. Photodynamic therapy utilizing indocyanine green-loaded calcium phosphosilicate nanoparticles prolongs the survival of leukemic mice. Myelomonocytic leukemia was established in BALB/cJ mice by systemic injection of the WEHI-3B-GFP cell line. One week following engraftment indocyanine green (ICG)-loaded calcium phosphosilicate nanoparticles (CPSNPs) were systemically injected. Near-infrared laser treatment was given to the spleen 24 hours following nanoparticle injection. Treatments were repeated once per week for four weeks. Controls consisted of empty (Ghost) CPSNPs. All nanoparticles were PEGylated. Average survival was significantly extended from 29 to 39 days (Logrank test, p=0.021).

Figure 4. Photodynamic therapy utilizing indocyanine green-loaded calcium phosphosilicate nanoparticles prolongs the survival of leukemic mice. Myelomonocytic leukemia was established in BALB/cJ mice by systemic injection of the WEHI-3B-GFP cell line. One week following engraftment indocyanine green (ICG)-loaded calcium phosphosilicate nanoparticles (CPSNPs) were systemically injected. Near-infrared laser treatment was given to the spleen 24 hours following nanoparticle injection. Treatments were repeated once per week for four weeks. Controls consisted of empty (Ghost) CPSNPs. All nanoparticles were PEGylated. Average survival was significantly extended from 29 to 39 days (Logrank test, p=0.021).

The success of our study using the 32D-P210-GFP model was due in large part to the ability to target CPSNPs (described in more detail below). We have also recently duplicated this work in a different animal model of myelomonocytic leukemia, in which we implanted WEHI-3B-GFP cells into BALB/cJ mice. In this model, the disease does not progress robustly in the blood but rather forms masses throughout the abdominal cavity (Keasey et al., 2010). Although the tumor growth characteristics are similar to those reported in the model used by Wen et al. (and differ from the tumors in our original work) (Wen et al., 2011; Barth et al., 2011), we were able to significantly extend the survival of mice given PDT using non-targeted ICG-loaded CPSNPs and laser light directed only at the spleen (Figure 4). The anti-leukemia efficacy that we have observed with our nanotechnology is a reflection of the enhanced optical properties associated with encapsulating the near-infrared fluorescing probe ICG within CPSNPs. Moreover, despite laser treatment of only the spleen, the efficacy of these studies may also have been due to the idea that PDT can often trigger systemic anticancer immune responses. This idea has been put forward by many PDT researchers and presents another distinct advantage of PDT over traditional anticancer therapies; since an immune response to cancer can be both highly effective and minimally destructive to normal tissues.

Targeting leukemia stem cells by CPSNP-mediated photodynamic therapy

Recently, the presence of small populations of cells within solid and non-solid malignancies with stem cell-like properties has been characterized (Misaghian et al., 2009; Perl et al., 2011; Roboz et al., 2009; Sloma et al., 2010). These cancer stem cells exhibit markers resembling embryonic and adult stem cells, and they are fully capable of initiating malignant growth in vivo (Misaghian et al., 2009; Roboz et al., 2009). Interestingly, these distinct cell populations are associated with multidrug resistance and are believed to be primarily responsible for relapse due to their inherent resistance to conventional chemotherapeutics (Misaghian et al., 2009). Normal hematopoietic stem cells contain unique cellular surface markers, and these same markers, CD34+ and CD38-, are also present on LSCs. In addition, other surface markers have been identified that specifically define the LSC, such as CD96 for the identification of CD34+CD38-CD96+ LSCs in acute myeloid leukemia (Hosen et al., 2007). Likewise, studies have also indicated that LSCs can be defined by the presence of CD117 in patients and animal models of chronic myeloid leukemia (Chen et al., 2009; 2010; Gerber et al., 2011). Importantly, these surface features offer the opportunity to specifically target therapeutics, such as CPSNPs, to LSCs.

In our recently published study, we evaluated the utility of ICG-loaded CPSNPs for PDT of leukemia. We engineered CPSNPs targeted to either CD96 or CD117 (Barth et al., 2011). Both CPSNPs preparations were verified by an increase in size as determined by dynamic light scattering and transmission electron microscopy. Our CPSNP preparations were also neutral-charged. We evaluated CPSNPs targeted to CD96 in human patient samples of acute myeloid leukemia. Our LSC-targeted nanoparticles were necessary to achieve cell death in an in vitro assay whereas non-targeted nanoparticles showed no utility. We further evaluated CD117-targeted CPSNPs both in vitro and in vivo using the 32D-P210-GFP murine model of chronic myeloid leukemia. We initially demonstrated that these CD117-targeted CPSNPs had increased uptake kinetics, owing in large part to the internalization of CD117/c-kit upon receptor binding. In both in vitro and in vivo models, we achieved therapeutic efficacy. In vivo this was reflected by a decreased (or non-existent) peripheral blood GFP count (measured via flow cytometry) or by profound extension of survival (Barth et al., 2011). Monitoring blood GFP counts also allowed us to assess normal myeloid populations of our treated animals. CD117 is expressed on normal hematopoietic cells, thus off-target effects from therapy leading to myelosuppression were considered. Although modest myelosuppression was observed following initial treatment, myelosuppression never became complete and resolved in most mice (Barth et al., 2011). We additionally utilized the imaging capabilities of the ICG-loaded CPSNPs to show that the major targeting in normal leukemia-free mice was confined to the spleen, where many normal CD117+ cells reside (Fossati et al., 2010; Pelayo et al., 2005).

Taken together, our study shows that PDT utilizing ICG-loaded CPSNPs may be used as an effective non-invasive therapy for leukemia. We found that the efficacy of PDT was most robust by targeting the CPSNPs to LSCs (Barth et al., 2011). It is our opinion that this study is the first successful in vivo demonstration of PDT for leukemia. The development of ICG-loaded CPSNPs targeted to LSCs was essential for this success.

Photodynamic therapy of solid tumors

Figure 5. Hypothesized mechanism(s) of action mediating photodynamic therapy of solid tumors utilizing indocyanine green-loaded calcium phosphosilicate nanoparticles. Photodynamic therapy (PDT) occurs following injection of indocyanine green (ICG)-loaded calcium phosphosilicate nanoparticles (CPSNPs). ICG-loaded CPSNPs are allowed to circulate and target tumors (active or passive) prior to near-infrared laser irradiation of the tumor. This triggers two pathways. Pathway I results in direct tumor cell death via oxidative pathways triggered by damaging singlet oxygen. Pathway II is initiated by the generation of a bioactive mediator that both blocks immunosuppressive cells and promotes the expansion of immune effector cells from hematopoietic progenitor cells. Immune effectors are then hypothesized to mediate an antitumor immune response.

Figure 5. Hypothesized mechanism(s) of action mediating photodynamic therapy of solid tumors utilizing indocyanine green-loaded calcium phosphosilicate nanoparticles. Photodynamic therapy (PDT) occurs following injection of indocyanine green (ICG)-loaded calcium phosphosilicate nanoparticles (CPSNPs). ICG-loaded CPSNPs are allowed to circulate and target tumors (active or passive) prior to near-infrared laser irradiation of the tumor. This triggers two pathways. Pathway I results in direct tumor cell death via oxidative pathways triggered by damaging singlet oxygen. Pathway II is initiated by the generation of a bioactive mediator that both blocks immunosuppressive cells and promotes the expansion of immune effector cells from hematopoietic progenitor cells. Immune effectors are then hypothesized to mediate an antitumor immune response.

As mentioned, we have started to assess the utility of ICG-loaded CPSNPs for PDT in solid tumor models. Our ultimate goals are to demonstrate therapeutic efficacy and to fully describe the mechanisms associated with solid tumor PDT using ICG-loaded CPSNPs. To date, we have tested our nanoparticles in models of breast cancer, pancreatic cancer, and metastatic osteosarcoma. We have begun an in-depth analysis of immune regulation by PDT utilizing ICG-loaded CPSNPs. The idea that PDT can initiate a systemic antitumor immune response is a concept that persists among PDT researchers (Gollnick et al., 2010; Mroz et al., 2010; 2011). We have embraced this concept, and are now investigating regulation of tumor immunology whereby PDT may regulate both immunosuppressive cells and immune effector cells. Immunosuppresssive cells are a normal part of the functioning immune system and usually prevent aberrant autoimmune reactions (Frumento et al., 2006; Gabrilovich et al., 2009; Leao et al., 2008; Ostrand-Rosenberg, 2010; Whiteside, 2010a; 2010b; Yang et al., 2010). Unfortunately, tumor-driven factors, such as inflammation, can prevent the antitumor immune response from occurring (Ostrand-Rosenberg et al., 2009). Immunosuppressive cells can interfere with immune effectors, such as T cells, dendritic cells, natural killer cells, and B cells, via a variety of direct and indirect mechanisms. Immunosuppression is becoming increasingly recognized as a hallmark of many advanced malignancies and as a serious hurdle to effective therapy. Our studies conducted in solid tumor models demonstrated that PDT utilizing ICG-loaded CPSNPs triggers a therapeutic response. Following PDT, we observed a decrease in the immunosuppressive milieu and a concomitant increase in immune effector cells (Figure 5). We speculate that this mechanism of therapeutic efficacy occurs due to traditional singlet oxygen-mediated cell death. The generation of singlet oxygen is likely responsible for a therapeutic effect following even a single treatment regime. We hypothesize that this immune-modulating effect of PDT using ICG-loaded CPSNPs is due to singlet oxygen-mediated production of specific bioactive immune modulators. We are currently in the process of verifying this hypothesis. We are also working to identify the unique bioactive mediators involved and to reinforce the currently held position in the field of PDT that immune regulation is an essential component of effective treatment. ICG-loaded CPSNPs can successfully overcome previous limitations of PDT, such as non-specific targeting and poor optical properties of photosensitizers. In our opinion, the development ICG-loaded CPSNPs will enable detailed mechanistic research and novel cancer therapeutics.

Acknowledgments

The authors would like to thank James M. Kaiser, Erhan I. Altinoglu, and Christopher McGovern for assistance with the initial development of ICG-loaded CPSNPs for PDT of cancer, as well as during the completion of the original published research studies.

Disclosure

J.H.A. and M.K. serve as CSO and CMO, respectively, of Keystone Nano, Inc. The Pennsylvania State University Research Foundation has licensed CPSNPs, and PDT utilizing CPSNPs, to Keystone Nano, Inc.

Corresponding Author

Brian M. Barth, Ph.D., Departments of Pharmacology and Medicine and Penn State Hershey Cancer Institute, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.

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[Discovery Medicine; ISSN: 1539-6509; Discov Med 13(71):275-285, April 2012. Copyright © Discovery Medicine. All rights reserved.]

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