calcium phosphosilicate nanoparticles for imaging and photodynamic therapy of cancer

Downloaded/ordered from Discovery Medicine on May 07, 2014.Distribution of this article in any form is not permitted. © Discovery Medicine.

275

Discovery Medicine, volume 13, Number 71, Pages 275-285, April 2012

Abstract: Photodynamic therapy (PDT) hasemerged as an alternative modality for cancer treat-ment. PDT works by initiating damaging oxidationor redox-sensitive pathways to trigger cell death.PDT can also regulate tumor angiogenesis and mod-ulate systemic antitumor immunity. The drawbacksto PDT -- photosensitizer toxicity, a lack of selectivi-ty and efficacy of photosensitizers, and a limitedpenetrance of light through deep tissues -- are thesame pitfalls associated with diagnostic imaging.Developments in the field of nanotechnology havegenerated novel platforms for optimizing the advan-tages while minimizing the disadvantages of PDT.Calcium phosphosilicate nanoparticles (CPSNPs)represent an optimal nano-system for both diagnos-tic imaging and PDT. In this review, we will discusshow CPSNPs can enhance optical agents and serveas selective, non-toxic, and functionally stable pho-

tosensitizers for PDT. We will also examine novelapplications of CPSNPs and PDT for the treatmentof leukemia to illustrate their potential utility in can-cer therapeutics. [Discovery Medicine 13(71):275-285, April

2012]

Photodynamic Therapy

Photodynamic therapy (PDT) has emerged as an alter-native strategy for treating cancer. PDT consists ofthree main components: a photosensitizer, light, andoxygen. PDT takes advantage of an appropriate wave-length of light that excites a photosensitizer to a tripletenergy state (Juarranz et al., 2008; Ortel et al., 2009;Wainwright, 2008). In the presence of molecular oxy-gen, energy is transferred to relax the excited state ofthe photosensitizer (Figure 1). This energy transfer inturn excites molecular oxygen to form excited, singletstate oxygen (Figure 2). Singlet oxygen induces celldeath via damaging oxidation or redox-sensitive cellu-lar signaling pathways, thus mediating the effects ofPDT (Dolmans et al., 2003; Huang et al., 2008;Juarranz et al., 2008). Intriguingly, PDT has also beenshown to regulate processes beyond tumor cell deathincluding tumor angiogenesis and modulation of theimmune system (Dolmans et al., 2003; Gollnick et al.,2010; Juarranz et al., 2008; Reddy et al., 2006). It hasbeen speculated that PDT, when effective, can actuallyrestrict tumor nutrient supply by the ablation of tumorblood vessels and by triggering an immune responsethat can afford systemic antitumor immunity (Gollnicket al., 2010; Hu et al., 2010; 2011; Mroz et al., 2010;2011; Tammela et al., 2011). For these reasons, PDTcontinues to garner considerable interest, as technolo-gies develop to overcome potential limitations.Disadvantages of current PDT include photosensitizertoxicity, a lack of efficacious and selective photosensi-tizers, and an inability of light to sufficiently penetratethrough tissues to reach tumors deep within the body

Calcium�Phosphosilicate�Nanoparticles�for

Imaging�and�Photodynamic�Therapy�of�Cancer

DIANA M. TAceloSky, AMy e. creecy, SrIrAM S. ShANMugAvelANDy, JIll P.

SMITh, DAvID F. clAxToN, JAMeS h. ADAIr, MArk keSTer, AND BrIAN M. BArTh

Diana M. Tacelosky, Sriram S. Shanmugavelandy,Mark Kester, and Brian M. Barth are at theDepartment of Pharmacology, The Pennsylvania StateUniversity College of Medicine, Hershey, Pennsylvania17033, USA.Amy E. Creecy is at the Department of BiomedicalEngineering, University of Texas, Austin, Texas 78712,USA.Jill P. Smith and David F. Claxton are at theDepartment of Medicine, The Pennsylvania StateUniversity College of Medicine, Hershey, Pennsylvania17033, USA.James H. Adair is at the Department of MaterialsScience and Engineering, The Pennsylvania StateUniversity College of Medicine, Hershey, Pennsylvania16802, USA.Drs. Claxton, Kester, and Barth are also at the PennState Hershey Cancer Institute, The PennsylvaniaState University College of Medicine, Hershey,Pennsylvania 17033, USA.

Corresponding author: Brian M. Barth, Ph.D.([email protected]).

© Discovery�Medicine. All rights reserved.

DISCOVERY MEDIC INE ®

www.discoverymedicine.comISSN: 1539-6509; eISSN: 1944-7930

http://www.discoverymedicine.com





276

(Chatterjee et al., 2008; Donnelly et al., 2008; Huang etal., 2006; Sibani et al., 2008; Wainwright, 2008). Forthese reasons, PDT is primarily utilized to treat cancersof 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 withdiagnostic imaging. Advances in the field of nanotech-nology may therefore overcome the hurdles associatedwith both diagnostic imaging and PDT. In this review,we will focus on the development of our novel nan-otechnology and its application for both diagnosticimaging and PDT. It is important to note that althoughwe focus on our technology, other considerableadvances within the fields of nanotechnology and pho-tobiology are also reinvigorating the field of PDTresearch. In particular, various polymeric and nonpoly-meric nanoparticles have been reported to encapsulatephotosensitizers and have demonstrated efficacy in bothin 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

Calcium phosphosilicate nanoparticles (CPSNPs) wereinitially developed to improve the optical and quantumproperties of encapsulated dyes (Altinoglu et al., 2008;Kester et al., 2008; Morgan et al., 2008). CPSNPs werealso engineered to protect agents during systemic deliv-ery, ensuring a pH-dependent release following endocy-tosis (Kester et al., 2008; Morgan et al., 2008). CPSNPsare very small (approximately 20 nm diameter) (Adairet al., 2010; Tabakovic et al., 2012). They offer anadvantage over other nanoparticles, because dyes,drugs, or other molecules of interest are encapsulatedwithin a calcium phosphosilicate nanomatrix. This is incontrast to surface decoration of the nanoparticle,which can be the case for a variety of other nanoparti-cles, including other calcium phosphate-based systems(Graham et al., 1973). Calcium and phosphate are alsoadvantageous materials for nanoparticles because they

Discovery Medicine, volume 13, Number 71, April 2012

cPSNPs for Imaging and Photodynamic Therapy of cancer

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 excitesthe molecule from a ground singlet state (S0) to an excited singlet state (S1). ICG in this excited state can return toa ground state via fluorescence or non-radioactive decay, or can undergo intersystem crossing and enter an excitedtriplet state (T1). Molecular oxygen exists in a ground triplet state, and so an energy transfer can occur between thetriplet state of ICG and molecular oxygen. This energy transfer results in the generation of singlet oxygen.



277



are both abundant in physiological systems and pose noinherent toxicity (Tabakovic et al., 2012). This is instark contrast to other nanoparticles composed of cad-mium, selenium, heavy metals, or hydrocarbons that aresignificantly toxic and cannot be used effectively inbiomedical applications (Adair et al., 2010; Tabakovicet al., 2012). Additionally, the calcium phosphosilicatenanomatrix is stable at physiological pH, allowing thesenanoparticles to protect their “payloads” during sys-temic circulation. CPSNPs are engulfed by cells duringendocytosis and ultimately trafficked via the endoso-mal-lysosomal pathway (Altinoglu et al., 2008; Barth etal., 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 degradeand release their contents. This controlled releaseensures that free-dye, or free-drug, is not exposed insystemic circulation where the free-dye/free-drug cando considerable harm. Improvements to CPSNPs, suchas selective targeting, further ensure that these nanopar-ticles only go where they are intended, thus protectingthe body’s tissues from unintended and off-targeteffects (Barth et al., 2010; 2011). Our design of

CPSNPs also includes surface functionalization withpolyethylene glycol (PEG) (Altinoglu et al., 2008;Barth et al., 2010; 2011; Kester et al., 2008; Morgan etal., 2008). PEGylation of nanoparticles, such asCPSNPs, helps to permit longer systemic retention andreduce nanoparticle clearance by the immune system(stealth characteristic). Additionally, PEGylatednanoparticles have the ability to accumulate withinsolid tumors via a process known as the enhanced per-meation and retention (EPR) (Altinoglu et al., 2008).This effect is due to the unique and disorganized vascu-lature of tumors, in addition to poor lymphatic drainage,which allows small nano-sized materials to accumulatewithin tumors (Fang et al., 2011; Hirsjarvi et al., 2011).In a diagnostic imaging trial using CPSNPs, we showedthat the accumulation of CPSNPs within tumors isdependent upon PEGylation of the nanoparticles;because non-PEGylated CPSNPs failed to accumulateand retain within breast cancer tumors in vivo(Altinoglu et al., 2008). This imaging study alsorevealed that CPSNPs primarily leave the body viahepatobiliary clearance, further minimizing toxicity.Altogether, CPSNPs represent an optimal nano-system

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


https://www.researchgate.net/publication/44572514_Fang_J_Nakamura_H_Maeda_H_The_EPR_effect_Unique_features_of_tumor_blood_vessels_for_drug_delivery_factors_involved_and_limitations_and_augmentation_of_the_effect_Adv_Drug_Deliv_Rev_63_136-151?el=1_x_8&enrichId=rgreq-0335763d-ca2f-42f1-917c-23fd9646c974&enrichSource=Y292ZXJQYWdlOzIyNDg2NTQ2MTtBUzoyMTc4NjAzNjMyOTY3NjhAMTQyODk1MzM2NjIwNw==


278



for biomedical applications because of eight keyachievements: (1) CPSNPs are made of non-toxicmaterials, (2) CPSNPs are truly nano-sized, (3)CPSNPs completely encapsulate payloads, (4) CPSNPsare colloidally stable in physiological conditions, (5)CPSNPs have increased retention times due to surfacePEGylation, (6) CPSNPs clear via hepatobiliary excre-tion, (7) CPSNPs offer controlled pH-mediated releaseof payloads, and (8) CPSNPs can be functionalized toselectively target specific cells (Adair et al., 2010).

CPSNPs for Diagnostic Imaging of Cancer

One of the distinct goals of CPSNP development was toimprove the optical properties of fluorescent dyes toallow for better imaging for cancer diagnostics. Wehave developed CPSNPs that encapsulate a range offluorescent dyes, and we have tested these in variousexperimental settings including cell and animal imag-ing. For whole animal imaging, we chose to encapsu-late 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 draw-back to effective imaging. Near-infrared fluorescingprobes, such as ICG, offer an advantage in that theirexcitation and emission wavelengths are long enoughto pass through biological tissues with minimal to nolight scattering. As mentioned, the optical properties offluorescent dyes, such as ICG, are vastly improved byencapsulation within the nanomatrix of CPSNPs(Altinoglu et al., 2008). ICG is currently used as anFDA-approved contrast agent in imaging applications(Bennett et al., 2009). However, the use of ICG suffersconsiderably from a short plasma half-life (2-4 min-utes), photobleaching, and nonspecific quenching dueto binding with serum proteins (Desmettre et al., 2000).Encapsulation of ICG within CPSNPs overcame thesedrawbacks and vastly improved its optical properties.In our initial diagnostic imaging trial we compared theability of free (non-encapsulated) ICG and CPSNPsloaded with ICG to image mice implanted with breastcancer tumors (Altinoglu et al., 2008). In this study,free ICG was not able to effectively image tumors,whereas ICG-loaded CPSNPs (PEGylated) effectivelyaccumulated in tumors and imaged tumors up to 96hours following systemic injection (Altinoglu et al.,2008). The improvements to ICG-mediated imaging aredirectly reflective of the enhanced fluorescencequantum efficiency (ΦF) and photostability of ICGwhen encapsulated within CPSNPs. In a subsequentstudy, the ΦF of free ICG and nano-encapsulated ICGwere directly compared (Russin et al., 2010). It wasdemonstrated that the ΦF in PBS for free ICG was0.027 +/- 0.001, a typical CPSNP loaded with ICG was0.053 +/- 0.003, and ICG molecules encapsulated with-

in CPSNPs were 0.066 +/- 0.004 (Russin et al., 2010).This study also found that 6 +/- 2 ICG molecules wereencapsulated per typical CPSNP (Russin et al., 2010).The specific optical improvements described for ICG-loaded CPSNPs also potentially expand the use of ICGbeyond imaging to therapeutic applications such as PDT.

Our original diagnostic imaging study relied on theEPR effect to allow for effective imaging of implantedbreast cancer tumors (Altinoglu et al., 2008). Thisallowed for good CPSNP accumulation, but we ques-tioned if this would be the most effective imagingmodality. Furthermore, more selective targeting was ofinterest to improve the potential for therapeutic deliv-ery to a cell population. In a subsequent study, weimproved in vivo imaging by targeting CPSNPs specif-ically to CD71 on breast cancer tumors or to the gastrinreceptor on pancreatic cancer tumors (Barth et al.,2010). The transferrin receptor (CD71) is found pre-dominately on proliferating cells with elevated meta-bolic 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; Sutherlandet al., 1981). Similarly, gastrin receptors have preva-lence within particular tissues within the gastrointesti-nal and central nervous systems (Wank et al., 1992).These G-protein-coupled receptors, also known as thecholecystokinin-2 (CCK2 or CCK-B) receptor family,are often increased in many cases of gastrointestinalcancer (Smith et al., 1996; 1998). In pancreatic cancer,there can be a particular increase in the expression of aspecific splice variant (CCK2i4sv or CCK-C) of thereceptor (Smith et al., 1994; 1995; 2002). Our studyevaluated different methods for attaching ligands, frag-ments of ligands, or antibodies to CPSNPs to permittargeting of either CD71 or gastrin receptors (Barth etal., 2010). We found that targeting CD71 improved theimaging capability of CPSNPs for subcutaneouslyimplanted breast cancer tumors. Furthermore, and moreprofoundly, targeting CPSNPs to gastrin receptors per-mitted robust imaging of orthotopically implanted pan-creatic cancer in vivo. This was particularly excitingbecause these tumors are diffuse and occur in regionswith poor blood supply to allow for nanoparticle accu-mulation. While non-targeted CPSNPs were able toimage orthotopic pancreatic tumors, gastrin receptor-targeted CPSNPs were able to robustly image the entireextent of the orthotopic tumor (Figure 3). Owing to theprevalence of gastrin receptors in the central nervoussystem, these targeted CPSNPs were also able to crossthe blood brain barrier and image the brain (Barth et al.,2010; Wank et al., 1992). Other groups have describedactively targeted nanoparticles that cross the blood



279



brain barrier, suggesting that targeted CPSNPs could beengineered to diagnose and treat tumors of the centralnervous system (Madhankumar et al., 2009).

Photodynamic Therapy Utilizing IndocyanineGreen-loaded CPSNPs

The limitations of the photosensitizers used for PDTare similar to those of optical agents used for diagnos-tic imaging. Furthermore, ICG has previously beenevaluated as a photosensitizer for PDT in various mod-els 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 etal., 2011; Tseng et al., 2003; Urbanska et al., 2002).Therefore, we hypothesized that the improvements weachieved for diagnostic imaging using ICG-loadedCPSNPs could translate to utility as a photosensitizerfor PDT of cancer. Both improvements in optical prop-erties and the ability to selectively target CPSNPs wereessential to the development of ICG-loaded CPSNPs aseffective photosensitizers (Altinoglu et al., 2008; Barthet al., 2010; Russin et al., 2010). As described below,our initial and published work validated the utility ofICG-loaded CPSNPs for PDT of leukemia (Barth et al.,2011). Moreover, we were able to selectively target ourCPSNPs to leukemia stem cells (LSCs), eradicating thespecific cells responsible for the maintenance and pro-gression of the disease. Finally, we will discuss brieflyour unpublished work that has started to evaluate PDTutilizing ICG-loaded CPSNPs in solid tumors. Thiswork is more specifically related to our initial imagingstudies and has centered on the utility of ICG-loadedCPSNPs as “theranostic” agents based on the ability tosimultaneously diagnose and treat.

Photodynamic therapy of leukemia

Leukemia, a cancer of the blood and bone marrow, is arather broad term used to describe many differenthematological malignancies (Perrotti et al., 2010;Radhi et al., 2010; Roboz et al., 2009). Typically,leukemia is sub-classified by cell origin (myeloid orlymphoid), and by disease onset or progression (acuteor chronic). Even a given subtype, such as acutemyeloid leukemia, characterizes many different hema-tological malignancies that can be further describedbased on cytogenetic or molecular markers. The treat-ment of leukemia typically consists of high dosechemotherapy, which in itself can pose significant riskto the patient. These risks are more profound when con-sidering that leukemia is both one of the most commonpediatric cancers and one of the more common cancersin the elderly (Perrotti et al., 2010; Radhi et al., 2010;Roboz et al., 2009). In these populations, these aggres-

sive treatments can lead to serious complications oreven be fatal. Additionally, high dose chemotherapeu-tics also carry the risk of being carcinogens that maypossibly lead to therapy-induced leukemia in thepatient’s future. Therefore, better therapeutics for thetreatment of leukemia, both with improved efficacy andlower toxicity, are needed.

The potential treatment of leukemia with PDT is anintriguing idea, in part because the side effects of PDTare modest in comparison to current leukemiachemotherapeutics. Due to the short lifetime of singletoxygen, it is thought that little opportunity exists dur-ing PDT for carcinogenic transformation to occur, leav-ing cell death as the only outcome (Juarranz et al.,2008; Robertson et al., 2009). Unfortunately, PDT hasnever been used clinically to treat leukemia. This islikely due to the nature of leukemia as a systemic dis-

Figure 3. Imaging of pancreatic cancer with indocya-nine green-loaded calcium phosphosilicate nanoparti-cles is improved by gastrin receptor targeting.Orthotopic human BxPC-3 pancreatic cancer tumorswere established in athymic nude mice. Indocyaninegreen (ICG)-loaded calcium phosphosilicate nanoparti-cles (CPSNPs) were systemically injected and tumorimaging was performed using a Kodak In Vivo FXImager 24 hours later. A, non-targeted ICG-loadedCPSNPs (PEGylated). B, gastrin receptor-targetedICG-loaded CPSNPs (gastrin-10 peptide covalentlylinked to PEGylation). Note targeting of the brain inpanel B due to the presence of gastrin (cholecystokinin)receptors in the central nervous system.



280



ease with little opportunity for photosensitizers to accu-mulate in circulating leukemic cells. Furthermore, theinability to penetrate appropriate light throughout thebody to activate photosensitizers is another limitation.Generally, the published laboratory reports of PDT util-ity for leukemia have almost exclusively been per-formed in cell culture (Feuerstein et al., 2009; Furre etal., 2005; Gamaleia et al., 2008; Pluskalova et al., 2006).

To our knowledge, there are currently three publishedstudies where PDT was studied in vivo for the treatmentof leukemia (Huang et al., 2006; Barth et al., 2011; Wenet al., 2011). The first was a hybrid study which report-ed the utility of PDT to purge leukemic cells from autol-ogous hematopoietic stem cells ex vivo prior to trans-plant in animal models (Huang et al., 2006). The othertwo studies, including our study, evaluated more tradi-tional PDT, occurring exclusively in vivo (Barth et al.,2011; Wen et al., 2011). Simultaneous with our publica-tion, Wen et al. published a report of in vivo PDT in amurine model of B cell lymphoma. In that study, thephotosensitizer Photodithazine was injected systemical-ly into the tail vein and then photofibers were insertedinto the tail vein, via the same needle, to perform PDTusing a diode laser. There is some question, from ourperspective, as to whether or not the experimental meth-ods may have attributed to the reported therapeutic effi-cacy. Namely, “successful” PDT was performed onlyone day following the initial injection of leukemic cells.In contrast, PDT was unsuccessful when it was per-formed five days following initial injection of theleukemic cells. This raises an important question as towhether or not the leukemia had actually engrafted inthe study by Wen et al. Furthermore, the method ofinserting photofibers into the tail vein of mice for overan hour of laser irradiation treatment implies an inva-sive procedure that may be necessitated by the limita-tion of light penetration. Indeed, this is similar to labo-ratory animal trials and human clinical trials of PDT forabdominal and other internal malignancies where laserfiber optics are introduced into the body via a surgicalprocedure (Allison et al., 2009; Friedberg, 2009; Huanget al., 2008; Moore et al., 2009; Ortner, 2009).

The third instance of PDT being used in vivo for thetreatment of leukemia is our recently published studyevaluating ICG-loaded CPSNPs (Barth et al., 2011). Westudied selectively targeted CPSNPs compared to non-targeted CPSNPs. We evaluated several physical prop-erties of the nanoparticles, including size, charge, andthe ability to label and internalize into cells of interest.We also studied the specific ability of ICG-loadedCPSNPs to generate reactive oxygen species (singletoxygen and superoxide) in cellular models in response

to laser treatment. Additionally, we evaluated the thera-peutic efficacy of our ICG-loaded CPSNP version ofPDT in cellular models of murine chronic myeloidleukemia and in human samples from patients withacute myeloid leukemia. For our in vivo work, we useda murine model of blast crisis chronic myeloid leukemia(32D-P210-GFP cells engrafted in C3H/HeJ mice),which behaves similarly to acute myeloid leukemiawhen 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 dis-ease via flow cytometry before treatments were com-menced. In our model, laser treatment was performedcompletely outside of the body with the laser directed atthe spleen (Barth et al., 2011). In addition to a profoundextension of life, using targeted CPSNPs, we alsoobserved disease-free survival of some treated animals.Importantly, we were also able to monitor disease pro-gression by analyzing blood for GFP+ leukemic cells.

The success of our study using the 32D-P210-GFPmodel was due in large part to the ability to targetCPSNPs (described in more detail below). We have alsorecently duplicated this work in a different animalmodel of myelomonocytic leukemia, in which weimplanted WEHI-3B-GFP cells into BALB/cJ mice. Inthis model, the disease does not progress robustly in theblood but rather forms masses throughout the abdomi-nal cavity (Keasey et al., 2010). Although the tumorgrowth characteristics are similar to those reported inthe model used by Wen et al. (and differ from thetumors in our original work) (Wen et al., 2011; Barth etal., 2011), we were able to significantly extend the sur-vival of mice given PDT using non-targeted ICG-loadedCPSNPs and laser light directed only at the spleen(Figure 4). The anti-leukemia efficacy that we haveobserved with our nanotechnology is a reflection of theenhanced optical properties associated with encapsulat-ing the near-infrared fluorescing probe ICG withinCPSNPs. Moreover, despite laser treatment of only thespleen, the efficacy of these studies may also have beendue to the idea that PDT can often trigger systemic anti-cancer immune responses. This idea has been put for-ward by many PDT researchers and presents anotherdistinct advantage of PDT over traditional anticancertherapies; since an immune response to cancer can beboth highly effective and minimally destructive to nor-mal tissues.

Targeting leukemia stem cells by CPSNP-mediatedphotodynamic therapy

Recently, the presence of small populations of cellswithin solid and non-solid malignancies with stem cell-like properties has been characterized (Misaghian et al.,



281



2009; Perl et al., 2011; Roboz et al., 2009; Sloma et al.,2010). These cancer stem cells exhibit markers resem-bling embryonic and adult stem cells, and they are fullycapable of initiating malignant growth in vivo(Misaghian et al., 2009; Roboz et al., 2009).Interestingly, these distinct cell populations are associ-ated with multidrug resistance and are believed to beprimarily responsible for relapse due to their inherentresistance to conventional chemotherapeutics(Misaghian et al., 2009). Normal hematopoietic stemcells contain unique cellular surface markers, and thesesame markers, CD34+ and CD38-, are also present onLSCs. In addition, other surface markers have beenidentified that specifically define the LSC, such asCD96 for the identification of CD34+CD38-CD96+

LSCs in acute myeloid leukemia (Hosen et al., 2007).Likewise, studies have also indicated that LSCs can bedefined by the presence of CD117 in patients and ani-mal models of chronic myeloid leukemia (Chen et al.,2009; 2010; Gerber et al., 2011). Importantly, these sur-face features offer the opportunity to specifically targettherapeutics, such as CPSNPs, to LSCs.

In our recently published study, we evaluated the utili-ty of ICG-loaded CPSNPs for PDT of leukemia. Weengineered CPSNPs targeted to either CD96or CD117 (Barth et al., 2011). Both CPSNPspreparations were verified by an increase insize as determined by dynamic light scatter-ing and transmission electron microscopy.Our CPSNP preparations were also neutral-charged. We evaluated CPSNPs targeted toCD96 in human patient samples of acutemyeloid leukemia. Our LSC-targetednanoparticles were necessary to achieve celldeath in an in vitro assay whereas non-target-ed nanoparticles showed no utility. We fur-ther evaluated CD117-targeted CPSNPs bothin vitro and in vivo using the 32D-P210-GFPmurine model of chronic myeloid leukemia.We initially demonstrated that these CD117-targeted CPSNPs had increased uptake kinet-ics, owing in large part to the internalizationof CD117/c-kit upon receptor binding. Inboth in vitro and in vivo models, we achievedtherapeutic efficacy. In vivo this was reflect-ed by a decreased (or non-existent) peripher-al blood GFP count (measured via flowcytometry) or by profound extension of sur-vival (Barth et al., 2011). Monitoring bloodGFP counts also allowed us to assess normalmyeloid populations of our treated animals.CD117 is expressed on normal hematopoiet-ic cells, thus off-target effects from therapy

leading to myelosuppression were considered.Although modest myelosuppression was observed fol-lowing initial treatment, myelosuppression neverbecame complete and resolved in most mice (Barth etal., 2011). We additionally utilized the imaging capabil-ities of the ICG-loaded CPSNPs to show that the majortargeting in normal leukemia-free mice was confined tothe spleen, where many normal CD117+ cells reside(Fossati et al., 2010; Pelayo et al., 2005).

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

Photodynamic therapy of solid tumors

As mentioned, we have started to assess the utility ofICG-loaded CPSNPs for PDT in solid tumor models.Our ultimate goals are to demonstrate therapeutic effi-cacy and to fully describe the mechanisms associated

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



282



with solid tumor PDT using ICG-loaded CPSNPs. Todate, we have tested our nanoparticles in models ofbreast cancer, pancreatic cancer, and metastaticosteosarcoma. We have begun an in-depth analysis ofimmune regulation by PDT utilizing ICG-loadedCPSNPs. The idea that PDT can initiate a systemic anti-tumor immune response is a concept that persistsamong PDT researchers (Gollnick et al., 2010; Mroz etal., 2010; 2011). We have embraced this concept, andare now investigating regulation of tumor immunologywhereby PDT may regulate both immunosuppressivecells and immune effector cells. Immunosuppresssivecells are a normal part of the functioning immune sys-tem and usually prevent aberrant autoimmune reactions(Frumento et al., 2006; Gabrilovich et al., 2009; Leaoet al., 2008; Ostrand-Rosenberg, 2010; Whiteside,2010a; 2010b; Yang et al., 2010). Unfortunately, tumor-driven factors, such as inflammation, can prevent theantitumor immune response from occurring (Ostrand-Rosenberg et al., 2009). Immunosuppressive cells caninterfere with immune effectors, such as T cells, den-dritic cells, natural killer cells, and B cells, via a varietyof direct and indirect mechanisms. Immunosuppression

is becoming increasingly recognized as a hallmark ofmany advanced malignancies and as a serious hurdle toeffective therapy. Our studies conducted in solid tumormodels demonstrated that PDT utilizing ICG-loadedCPSNPs triggers a therapeutic response. FollowingPDT, we observed a decrease in the immunosuppres-sive milieu and a concomitant increase in immuneeffector cells (Figure 5). We speculate that this mecha-nism of therapeutic efficacy occurs due to traditionalsinglet oxygen-mediated cell death. The generation ofsinglet oxygen is likely responsible for a therapeuticeffect following even a single treatment regime. Wehypothesize that this immune-modulating effect of PDTusing ICG-loaded CPSNPs is due to singlet oxygen-mediated production of specific bioactive immunemodulators. We are currently in the process of verifyingthis hypothesis. We are also working to identify theunique bioactive mediators involved and to reinforcethe currently held position in the field of PDT thatimmune regulation is an essential component of effec-tive treatment. ICG-loaded CPSNPs can successfullyovercome previous limitations of PDT, such as non-specific targeting and poor optical properties of photo-

Figure 5. Hypothesized mechanism(s) of action mediating photodynamic therapy of solid tumors utilizing indocya-nine green-loaded calcium phosphosilicate nanoparticles. Photodynamic therapy (PDT) occurs following injection ofindocyanine green (ICG)-loaded calcium phosphosilicate nanoparticles (CPSNPs). ICG-loaded CPSNPs are allowedto circulate and target tumors (active or passive) prior to near-infrared laser irradiation of the tumor. This triggers twopathways. 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 pro-motes the expansion of immune effector cells from hematopoietic progenitor cells. Immune effectors are then hypoth-esized to mediate an antitumor immune response.



283



sensitizers. In our opinion, the development ICG-loaded CPSNPs will enable detailed mechanisticresearch and novel cancer therapeutics.

Acknowledgments

The authors would like to thank James M. Kaiser,Erhan I. Altinoglu, and Christopher McGovern forassistance with the initial development of ICG-loadedCPSNPs for PDT of cancer, as well as during the com-pletion 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 StateUniversity Research Foundation has licensed CPSNPs,and PDT utilizing CPSNPs, to Keystone Nano, Inc.

References

Abels C, Fickweiler S, Weiderer P, Baumler W, Hofstadter F, LandthalerM, Szeimies RM. Indocyanine green (ICG) and laser irradiation inducephotooxidation. Arch Dermatol Res 292(8):404-411, 2000.

Adair JH, Parette MP, Altinoglu EI, Kester M. Nanoparticulate alterna-tives for drug delivery. ACS Nano 4(9):4967-4970, 2010.

Allison RR, Zervos E, Sibata CH. Cholangiocarcinoma: an emergingindication for photodynamic therapy. Photodiagnosis Photodyn Ther6(2):84-92, 2009.

Altinoglu EI, Russin TJ, Kaiser JM, Barth BM, Eklund PC, Kester M,Adair JH. Near-infrared emitting fluorophore-doped calcium phosphatenanoparticles for in vivo imaging of human breast cancer. ACS Nano2(10):2075-2084, 2008.

Barth BM, Altinoglu EI, Shanmugavelandy SS, Kaiser JM, Crespo-Gonzalez D, DiVittore NA, McGovern C, Goff TM, Keasey NR, AdairJH, Loughran TP, Jr, Claxton DF, Kester M. Targeted indocyanine-green-loaded calcium phosphosilicate nanoparticles for in vivo photo-dynamic therapy of leukemia. ACS Nano 5(7):5325-5337, 2011.

Barth BM, Sharma R, Altinoglu EI, Morgan TT, Shanmugavelandy SS,Kaiser JM, McGovern C, Matters GL, Smith JP, Kester M, Adair JH.Bioconjugation of calcium phosphosilicate composite nanoparticles forselective targeting of human breast and pancreatic cancers in vivo. ACSNano 4(3):1279-1287, 2010.

Baumler W, Abels C, Karrer S, Weiss T, Messmann H, Landthaler M,Szeimies RM. Photo-oxidative killing of human colonic cancer cellsusing indocyanine green and infrared light. Br J Cancer 80(3-4):360-363, 1999.

Bennett TJ, Barry CJ. Ophthalmic imaging today: an ophthalmic pho-tographer’s viewpoint - a review. Clin Experiment Ophthalmol 37(1):2-13, 2009.

Bozkulak O, Yamaci RF, Tabakoglu O, Gulsoy M. Photo-toxic effects of809-nm diode laser and indocyanine green on MDA-MB231 breast can-cer cells. Photodiagnosis Photodyn Ther 6(2):117-121, 2009.

Chatterjee DK, Fong LS, Zhang Y. Nanoparticles in photodynamic ther-apy: an emerging paradigm. Adv Drug Deliv Rev 60(15):1627-1637,2008.

Chen Y, Hu Y, Zhang H, Peng C, Li S. Loss of the Alox5 gene impairsleukemia stem cells and prevents chronic myeloid leukemia. Nat Genet41(7):783-792, 2009.

Chen Y, Peng C, Sullivan C, Li D, Li S. Critical molecular pathways incancer stem cells of chronic myeloid leukemia. Leukemia 24(9):1545-1554, 2010.

Cheng Y, Meyers JD, Broome AM, Kenney ME, Basilion JP, Burda C.Deep penetration of a PDT drug into tumors by noncovalent drug-goldnanoparticle conjugates. J Am Chem Soc 133(8):2583-2591, 2011.

Crescenzi E, Varriale L, Iovino M, Chiaviello A, Veneziani BM,Palumbo G. Photodynamic therapy with indocyanine green comple-ments and enhances low-dose cisplatin cytotoxicity in MCF-7 breastcancer cells. Mol Cancer Ther 3(5):537-544, 2004.

Daley GQ, Van Etten RA, Baltimore D. Induction of chronic myeloge-nous leukemia in mice by the P210bcr/abl gene of the Philadelphia chro-mosome. Science 247(4944):824-830, 1990.

Daniels TR, Delgado T, Helguera G, Penichet ML. The transferrinreceptor part II: targeted delivery of therapeutic agents into cancer cells.Clin Immunol 121(2):159-176, 2006a.

Daniels TR, Delgado T, Rodriguez JA, Helguera G, Penichet ML. Thetransferrin receptor part I: Biology and targeting with cytotoxic antibod-ies for the treatment of cancer. Clin Immunol 121(2):144-158, 2006b.

Desmettre T, Devoisselle JM, Mordon S. Fluorescence properties andmetabolic features of indocyanine green (ICG) as related to angiogra-phy. Surv Ophthalmol 45(1):15-27, 2000.

Dolmans DE, Fukumura D, Jain RK. Photodynamic therapy for cancer.Nat Rev Cancer 3(5):380-387, 2003.

Donnelly RF, McCarron PA, Morrow DI, Sibani SA, Woolfson AD.Photosensitiser delivery for photodynamic therapy. Part 1: Topical car-rier platforms. Expert Opin Drug Deliv 5(7):757-766, 2008.

Fang J, Nakamura H, Maeda H. The EPR effect: Unique features oftumor blood vessels for drug delivery, factors involved, and limitationsand augmentation of the effect. Adv Drug Deliv Rev 63(3):136-151,2011.

Feuerstein T, Schauder A, Malik Z. Silencing of ALA dehydrataseaffects ALA-photodynamic therapy efficacy in K562 erythroleukemiccells. Photochem Photobiol Sci 8(10):1461-1466, 2009.

Ficheux H. Photodynamic therapy: principles and therapeutic indica-tions. Ann Pharm Fr 67(1):32-40, 2009.

Fickweiler S, Szeimies RM, Baumler W, Steinbach P, Karrer S, GoetzAE, Abels C, Hofstadter F, Landthaler M. Indocyanine green: intracel-lular uptake and phototherapeutic effects in vitro. J PhotochemPhotobiol B 38(2-3):178-183, 1997.

Fossati V, Kumar R, Snoeck HW. Progenitor cell origin plays a role infate choices of mature B cells. J Immunol 184(3):1251-1260, 2010.

Friedberg JS. Photodynamic therapy as an innovative treatment formalignant pleural mesothelioma. Semin Thorac Cardiovasc Surg21(2):177-187, 2009.

Frumento G, Piazza T, Di Carlo E, Ferrini S. Targeting tumor-relatedimmunosuppression for cancer immunotherapy. Endocr Metab ImmuneDisord Drug Targets 6(3):233-237, 2006.

Furre IE, Shahzidi S, Luksiene Z, Moller MT, Borgen E, Morgan J,Tkacz-Stachowska K, Nesland JM, Peng Q. Targeting PBR by hexam-inolevulinate-mediated photodynamic therapy induces apoptosis



through translocation of apoptosis-inducing factor in human leukemiacells. Cancer Res 65(23):11051-11060, 2005.

Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regula-tors of the immune system. Nat Rev Immunol 9(3):162-174, 2009.

Gamaleia NF, Shishko ED, Gluzman DF, Sklyarenko LM. Sensitivity ofnormal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage. Exp Oncol 30(1):65-69, 2008.

Gerber JM, Qin L, Kowalski J, Smith BD, Griffin CA, Vala MS,Collector MI, Perkins B, Zahurak M, Matsui W, Gocke CD, Sharkis SJ,Levitsky HI, Jones RJ. Characterization of chronic myeloid leukemiastem cells. Am J Hematol 86(1):31-37, 2011.

Gollnick SO, Brackett CM. Enhancement of anti-tumor immunity byphotodynamic therapy. Immunol Res 46(1-3):216-226, 2010.

Gosk S, Vermehren C, Storm G, Moos T. Targeting anti-transferrinreceptor antibody (OX26) and OX26-conjugated liposomes to braincapillary endothelial cells using in situ perfusion. J Cereb Blood FlowMetab 24(11):1193-1204, 2004.

Graham FL, van der Eb AJ. A new technique for the assay of infectivi-ty of human adenovirus 5 DNA. Virology 52(2):456-467, 1973.

Greenberger JS, Sakakeeny MA, Humphries RK, Eaves CJ, Eckner RJ.Demonstration of permanent factor-dependent multipotential (ery-throid/neutrophil/basophil) hematopoietic progenitor cell lines. ProcNatl Acad Sci U S A 80(10):2931-2935, 1983.

Gross SA, Wolfsen HC. The role of photodynamic therapy in the esoph-agus. Gastrointest Endosc Clin N Am 20(1):35-53, vi, 2010.

Hirsjarvi S, Passirani C, Benoit JP. Passive and active tumour targetingwith nanocarriers. Curr Drug Discov Technol 8(3):188-196, 2011.

Hocine O, Gary-Bobo M, Brevet D, Maynadier M, Fontanel S, RaehmL, Richeter S, Loock B, Couleaud P, Frochot C, Charnay C, Derrien G,Smaihi M, Sahmoune A, Morere A, Maillard P, Garcia M, Durand JO.Silicalites and mesoporous silica nanoparticles for photodynamic thera-py. Int J Pharm 402(1-2):221-230, 2010.

Hosen N, Park CY, Tatsumi N, Oji Y, Sugiyama H, Gramatzki M,Krensky AM, Weissman IL. CD96 is a leukemic stem cell-specificmarker in human acute myeloid leukemia. Proc Natl Acad Sci U S A104(26):11008-11013, 2007.

Hu Z, Rao B, Chen S, Duanmu J. Selective and effective killing ofangiogenic vascular endothelial cells and cancer cells by targeting tissuefactor using a factor VII-targeted photodynamic therapy for breast can-cer. Breast Cancer Res Treat 126(3):589-600, 2011.

Hu Z, Rao B, Chen S, Duanmu J. Targeting tissue factor on tumour cellsand angiogenic vascular endothelial cells by factor VII-targetedverteporfin photodynamic therapy for breast cancer in vitro and in vivoin mice. BMC Cancer 10:235, 2010.

Huang HF, Chen YZ, Wu Y, Chen P. Purging of murine erythroblasticleukemia by ZnPcS2P2-based-photodynamic therapy. Bone MarrowTransplant 37(2):213-217, 2006.

Huang Z, Xu H, Meyers AD, Musani AI, Wang L, Tagg R, Barqawi AB,Chen YK. Photodynamic therapy for treatment of solid tumors–poten-tial and technical challenges. Technol Cancer Res Treat 7(4):309-320,2008.

Juarranz A, Jaen P, Sanz-Rodriguez F, Cuevas J, Gonzalez S.Photodynamic therapy of cancer. Basic principles and applications. ClinTransl Oncol 10(3):148-154, 2008.

Keasey N, Herse Z, Chang S, Liggitt DH, Lay M, Fairman J, ClaxtonDF. A non-coding cationic lipid DNA complex produces lasting anti-leukemic effects. Cancer Biol Ther 10(6):625-631, 2010.

Kester M, Heakal Y, Fox T, Sharma A, Robertson GP, Morgan TT,Altinoglu EI, Tabakovic A, Parette MR, Rouse SM, Ruiz-Velasco V,Adair JH. Calcium phosphate nanocomposite particles for in vitro imag-ing and encapsulated chemotherapeutic drug delivery to cancer cells.Nano Lett 8(12):4116-4121, 2008.

Kotimaki J. Photodynamic therapy of eyelid basal cell carcinoma. J EurAcad Dermatol Venereol 23(9):1083-1087, 2009.

Leao IC, Ganesan P, Armstrong TD, Jaffee EM. Effective depletion ofregulatory T cells allows the recruitment of mesothelin-specific CD8 Tcells to the antitumor immune response against a mesothelin-expressingmouse pancreatic adenocarcinoma. Clin Transl Sci 1(3):228-239, 2008.

Lee SJ, Koo H, Jeong H, Huh MS, Choi Y, Jeong SY, Byun Y, Choi K,Kim K, Kwon IC. Comparative study of photosensitizer loaded and con-jugated glycol chitosan nanoparticles for cancer therapy. J ControlRelease 152(1):21-29, 2011a.

Lee YE, Kopelman R. Polymeric nanoparticles for photodynamic ther-apy. Methods Mol Biol 726:151-178, 2011b.

Ling X, Wang Y, Dietrich MF, Andreeff M, Arlinghaus RB. Vaccinationwith leukemia cells expressing cell-surface-associated GM-CSF blocksleukemia induction in immunocompetent mice. Oncogene 25(32):4483-4490, 2006.

Madhankumar AB, Slagle-Webb B, Wang X, Yang QX, Antonetti DA,Miller PA, Sheehan JM, Connor JR. Efficacy of interleukin-13 receptor-targeted liposomal doxorubicin in the intracranial brain tumor model.Mol Cancer Ther 8(3):648-654, 2009.

Mamoon AM, Gamal-Eldeen AM, Ruppel ME, Smith RJ, Tsang T,Miller LM. In vitro efficiency and mechanistic role of indocyaninegreen as photodynamic therapy agent for human melanoma.Photodiagnosis Photodyn Ther 6(2):105-116, 2009.

Misaghian N, Ligresti G, Steelman LS, Bertrand FE, Basecke J, LibraM, Nicoletti F, Stivala F, Milella M, Tafuri A, Cervello M, Martelli AM,McCubrey JA. Targeting the leukemic stem cell: the Holy Grail ofleukemia therapy. Leukemia 23(1):25-42, 2009.

Moore CM, Pendse D, Emberton M. Photodynamic therapy for prostatecancer–a review of current status and future promise. Nat Clin PractUrol 6(1):18-30, 2009.

Morgan TT, Muddana HS, Altinoglu EI, Rouse SM, Tabakovic A,Tabouillot T, Russin TJ, Shanmugavelandy SS, Butler PJ, Eklund PC,Yun JK, Kester M, Adair JH. Encapsulation of organic molecules in cal-cium phosphate nanocomposite particles for intracellular imaging anddrug delivery. Nano Lett 8(12):4108-4115, 2008.

Mroz P, Hashmi JT, Huang YY, Lange N, Hamblin MR. Stimulation ofanti-tumor immunity by photodynamic therapy. Expert Rev ClinImmunol 7(1):75-91, 2011.

Mroz P, Szokalska A, Wu MX, Hamblin MR. Photodynamic therapy oftumors can lead to development of systemic antigen-specific immuneresponse. PLoS One 5(12):e15194, 2010.

Muddana HS, Morgan TT, Adair JH, Butler PJ. Photophysics of Cy3-encapsulated calcium phosphate nanoparticles. Nano Lett 9(4):1559-1566, 2009.

Nyst HJ, Tan IB, Stewart FA, Balm AJ. Is photodynamic therapy a goodalternative to surgery and radiotherapy in the treatment of head and neckcancer? Photodiagnosis Photodyn Ther 6(1):3-11, 2009.

284





Ohulchanskyy TY, Roy I, Goswami LN, Chen Y, Bergey EJ, PandeyRK, Oseroff AR, Prasad PN. Organically modified silica nanoparticleswith covalently incorporated photosensitizer for photodynamic therapyof cancer. Nano Lett 7(9):2835-2842, 2007.

Ortel B, Shea CR, Calzavara-Pinton P. Molecular mechanisms of photo-dynamic therapy. Front Biosci 14:4157-4172, 2009.

Ortner MA. Photodynamic therapy for cholangiocarcinoma: overviewand new developments. Curr Opin Gastroenterol 25(5):472-476, 2009.

Ostrand-Rosenberg S. Myeloid-derived suppressor cells: more mecha-nisms for inhibiting antitumor immunity. Cancer Immunol Immunother59(10):1593-1600, 2010.

Ostrand-Rosenberg S, Sinha P. Myeloid-derived suppressor cells: link-ing inflammation and cancer. J Immunol 182(8):4499-4506, 2009.

Pelayo R, Welner R, Perry SS, Huang J, Baba Y, Yokota T, Kincade PW.Lymphoid progenitors and primary routes to becoming cells of theimmune system. Curr Opin Immunol 17(2):100-107, 2005.

Perl A, Carroll M. BCR-ABL kinase is dead; long live the CML stemcell. J Clin Invest 121(1):22-25, 2011.

Perrotti D, Jamieson C, Goldman J, Skorski T. Chronic myeloidleukemia: mechanisms of blastic transformation. J Clin Invest120(7):2254-2264, 2010.

Pluskalova M, Peslova G, Grebenova D, Halada P, Hrkal Z.Photodynamic treatment (ALA-PDT) suppresses the expression of theoncogenic Bcr-Abl kinase and affects the cytoskeleton organization inK562 cells. J Photochem Photobiol B 83(3):205-212, 2006.

Qin M, Hah HJ, Kim G, Nie G, Lee YE, Kopelman R. Methylene bluecovalently loaded polyacrylamide nanoparticles for enhanced tumor-tar-geted photodynamic therapy. Photochem Photobiol Sci 10(5):832-841,2011.

Radhi M, Meshinchi S, Gamis A. Prognostic factors in pediatric acutemyeloid leukemia. Curr Hematol Malig Rep 5(4):200-206, 2010.

Reddy GR, Bhojani MS, McConville P, Moody J, Moffat BA, Hall DE,Kim G, Koo YE, Woolliscroft MJ, Sugai JV, Johnson TD, Philbert MA,Kopelman R, Rehemtulla A, Ross BD. Vascular targeted nanoparticlesfor imaging and treatment of brain tumors. Clin Cancer Res12(22):6677-6686, 2006.

Robertson CA, Evans DH, Abrahamse H. Photodynamic therapy (PDT):a short review on cellular mechanisms and cancer research applicationsfor PDT. J Photochem Photobiol B 96(1):1-8, 2009.

Roboz GJ, Guzman M. Acute myeloid leukemia stem cells: seek anddestroy. Expert Rev Hematol 2(6):663-672, 2009.

Rungta P, Bandera YP, Roeder RD, Li Y, Baldwin WS, Sharma D,Sehorn MG, Luzinov I, Foulger SH. Selective imaging and killing ofcancer cells with protein-activated near-infrared fluorescing nanoparti-cles. Macromol Biosci 11(7):927-937, 2011.

Russin TJ, Altinoglu EI, Adair JH, Eklund PC. Measuring the fluores-cent quantum efficiency of indocyanine green encapsulated in nanocom-posite particulates. J Phys Condens Matter 22(33):334217, 2010.

Shindelman JE, Ortmeyer AE, Sussman HH. Demonstration of thetransferrin receptor in human breast cancer tissue. Potential marker foridentifying dividing cells. Int J Cancer 27(3):329-334, 1981.

Sibani SA, McCarron PA, Woolfson AD, Donnelly RF. Photosensitiserdelivery for photodynamic therapy. Part 2: systemic carrier platforms.Expert Opin Drug Deliv 5(11):1241-1254, 2008.

Sloma I, Jiang X, Eaves AC, Eaves CJ. Insights into the stem cells ofchronic myeloid leukemia. Leukemia 24(11):1823-1833, 2010.

Smith JP, Fantaskey AP, Liu G, Zagon IS. Identification of gastrin as agrowth peptide in human pancreatic cancer. Am J Physiol 268(1 Pt2):R135-R141, 1995.

Smith JP, Liu G, Soundararajan V, McLaughlin PJ, Zagon IS.Identification and characterization of CCK-B/gastrin receptors inhuman pancreatic cancer cell lines. Am J Physiol 266(1 Pt 2):R277-R283, 1994.

Smith JP, Shih AH, Wotring MG, McLaughlin PJ, Zagon IS.Characterization of CCK-B/gastrin-like receptors in human gastric car-cinoma. Int J Oncol 12(2):411-419, 1998.

Smith JP, Stock EA, Wotring MG, McLaughlin PJ, Zagon IS.Characterization of the CCK-B/gastrin-like receptor in human coloncancer. Am J Physiol 271(3 Pt 2):R797-805, 1996.

Smith JP, Verderame MF, McLaughlin P, Martenis M, Ballard E, ZagonIS. Characterization of the CCK-C (cancer) receptor in human pancre-atic cancer. Int J Mol Med 10(6):689-694, 2002.

Sutherland R, Delia D, Schneider C, Newman R, Kemshead J, GreavesM. Ubiquitous cell-surface glycoprotein on tumor cells is proliferation-associated receptor for transferrin. Proc Natl Acad Sci U S A78(7):4515-4519, 1981.

Tabakovic A, Kester M, Adair JH. Calcium phosphate-based compositenanoparticles in bioimaging and therapeutic delivery applications. WileyInterdiscip Rev Nanomed Nanobiotechnol 4(1):96-112, 2012.

Tammela T, Saaristo A, Holopainen T, Yla-Herttuala S, Andersson LC,Virolainen S, Immonen I, Alitalo K. Photodynamic ablation of lymphat-ic vessels and intralymphatic cancer cells prevents metastasis. Sci TranslMed 3(69):69ra11, 2011.

Tseng WW, Saxton RE, Deganutti A, Liu CD. Infrared laser activationof indocyanine green inhibits growth in human pancreatic cancer.Pancreas 27(3):e42-5, 2003.

Urbanska K, Romanowska-Dixon B, Matuszak Z, Oszajca J, Nowak-Sliwinsk P, Stochel G. Indocyanine green as a prospective sensitizer forphotodynamic therapy of melanomas. Acta Biochim Pol 49(2):387-391,2002.

Wainwright M. Photodynamic therapy: the development of new photo-sensitisers. Anticancer Agents Med Chem 8(3):280-291, 2008.

Wank SA, Pisegna JR, de Weerth A. Brain and gastrointestinal cholecys-tokinin receptor family: structure and functional expression. Proc NatlAcad Sci U S A 89(18):8691-8695, 1992.

Wen LY, Bae SM, Chun HJ, Park KS, Ahn WS. Therapeutic effects ofsystemic photodynamic therapy in a leukemia animal model using A20cells. Lasers Med Sci 27(2):445-452, 2011.

Whiteside TL. Immune responses to malignancies. J Allergy ClinImmunol 125(2 Suppl 2):S272-S283, 2010a.

Whiteside TL. Inhibiting the inhibitors: evaluating agents targeting can-cer immunosuppression. Expert Opin Biol Ther 10(7):1019-1035,2010b.

Yang Z, Zhang B, Li D, Lv M, Huang C, Shen GX, Huang B. Mast cellsmobilize myeloid-derived suppressor cells and Treg cells in tumormicroenvironment via IL-17 pathway in murine hepatocarcinomamodel. PLoS One 5(1):e8922, 2010.

285




calcium phosphosilicate nanoparticles for imaging and photodynamic therapy of cancer

Documents