Seite - 41 - in Cancer Nanotheranostics - What Have We Learnd So Far?

Dawidczyket al. Nanomedicines for cancer therapy studies varied from0.4 to 11%ID/g (Soundararajan et al., 2009; Kheirolomoom et al., 2010; Hsu et al., 2012). The large varia- tion is likely due to the differences in xenograft cell line, tumor size, anddose. Similarly, tumoraccumulation in twopre-clinical studies of 30nm diameter micelles with different polymer for- mulationswere1.5%ID/g (Yokoyamaet al., 1999) and9.5%ID/g (Blanco et al., 2010). These two studies used different mod- els (orthotopic vs. xenograft), tumor cell line (A549 vs. C26), tumor size (200 vs. 100mm3), and injected dose (30–50mg/kg vs. 10mg/kg).Thesedifferences in experimental design limit the ability to compare the twodifferentmicelle formulations. These examples highlight the difficulty in comparing pre-clinical trials due to thevariability inexperimentaldesign. ACTIVETARGETING Targeted delivery systems used in quantitative pre-clinical stud- ies include silica (Benezra et al., 2011; Tang et al., 2012; Chen et al., 2013), gold (Melancon et al., 2008; Lu et al., 2009, 2010; Chanda et al., 2010;Choi et al., 2010;Morales-Avila et al., 2011; Chattopadhyay et al., 2012), liposomes (Iyer et al., 2011;Helbok etal.,2012;Petersenetal.,2012),micelles(Huetal.,2008;Penate Medinaetal.,2011;Zhangetal.,2011b;Fongeetal.,2012;Helbok etal.,2012)(Rossinetal.,2005;Khemtongetal.,2009;Zhanetal., 2010;Poonetal.,2011;Zhangetal.,2011a;Xiaoetal.,2012), iron oxide(Natarajanetal.,2008;Kumaretal.,2010;Yangetal.,2011), graphene (Hong et al., 2012; Cornelissen et al., 2013; Shi et al., 2013),gadolinium(Oyewumietal., 2004),polymernanocarriers (Kunjachan et al., 2014), nanoemulsions (Ohguchi et al., 2008), quantumdots (Gaoet al., 2010), andhybrid (Chenget al., 2014) (SupplementaryTableS2). Similar topassive targeting, fewstud- ies (3/34) report %ID rather than%ID/g. The most common targetingligandsareantibodybased(9/34studies), theRGDpep- tide sequence (10/34), and folate (5/34). Targeting efficiencies obtained using RGD peptides, folate, antibodies, and antibody fragments are typically between1and15%ID/g (Supplementary TableS2). Assessing the efficiency of a targeting ligand in increasing tumor accumulation is complicated by the different control experiments used in these studies. The contribution of pas- sive targeting was assessed by measuring tumor accumulation of the delivery systemwithout attachment of the targeting lig- and (20/34), with attachment of a non-specific ligand (2/34), pre-injectionwith a blockingmolecule or treatment (10/34), or with a xenograft formed from a cell line that did not express the targetmolecule (2/34). Several studies (4/34) did not report a control experiment. Each control experiment has advantages and disadvantages. For example, removing a targeting ligand from a delivery systemmay alter the physico-chemical proper- ties and hence change the pharmacokinetics. As described in more detail below, xenografts formed from different cell lines may have significantly different vascularization and hence the rate of extravasation to the tumor site by the EPR effect may be significantly different. Pre-injectionwith ablockingmolecule may not completely prevent binding to the target molecule or may reduce binding in normal tissue. To account for these potential complications, a few studies (3/34) used multiple controls. Of the 30 pre-clinical studies that reported control exper- iments, 33% (10/30) showed less than a two-fold increase in targeting compared to the control, and 50% (15/30) showed an increase in tumor accumulation ofmore than 2%ID/gwith the targeting ligand. For example, a tumor accumulation of 9%ID was reported for SPIONSwith anti-ChL6 2 days post-injection comparedto1%without the targetingantibody(Natarajanetal., 2008). A tumor accumulation of 7 ± 1%ID was reported for gadoliniumnanoparticleswith a folate targeting ligand, and9± 4%ID in the control with no targeting ligand (Oyewumi et al., 2004).While active targetingof adelivery systemtoa tumor site has the potential to reduce unwanted side effects, these studies highlightthedifficultiesinassessingtargetingefficiencyduetothe largedifferences inexperimentaldesignandtherangeofcontrols usedtoassess thecontributionofpassive targeting. TUMORACCUMULATION In general, the uptake of a delivery system in a tumor tends to increase post-injection but then decreases at longer times (SupplementaryTable S1 andS2). For example, tumor accumu- lationof radiolabeled liposomes increased to11.3%ID/gover the first24h,thendecreasedto6.1%ID/gafter72h(Hsuetal.,2012). Tumor accumulation of self-activating quantum dots increased to 13%ID/g over the first 24h, but decreased to 11%ID/g after 42h (Sun et al., 2014). Similarly, tumor accumulation of pegy- latedmicelles with a gelatinase binding peptidewas reported to increasetoalmost18%ID/goverthefirst6h,butdecreasedto2% ID/gafter24h(PenateMedinaetal.,2011).Tumoraccumulation of gold nanoparticles with theRGDpeptide increased to 3.65% ID/gover thefirst hour followedby adecrease by almost half to 1.94%ID/g 24hpost-injection (Morales-Avila et al., 2011). The detailsof thetimedependenceoftumoraccumulationare impor- tant inunderstanding thepharmacokinetics, theEPReffect, and the limitations toaccumulatingadrugat the tumorsite. Inmany studies, an insufficientnumberof timepointsprecludesdetailed analysisofpharmacokineticsandtumoraccumulation. The cell lineused in forming a xenograft canhave significant influenceon tumoraccumulationandefficacy. In the74quanti- tativepre-clinical trials reviewedhere,35differentcell typeswere used to form xenografts. Themost common cell lines were the 4T1 murine breast cancer cell line (10/71) and the C26 colon carcinoma cell line (10/71), both of which form highly vascu- larized tumors. Tumor accumulation ofmicelles with the RGD peptide was 6%ID/g in a mouse model with a C26 xenograft and3%ID/gwitha less leakyBxPC3xenograft (Kunjachanet al., 2014),highlighting theneedfor standardizationofcell lines. Tumor size can have a significant influence on tumor accu- mulation. For example, a study using radiolabeled liposomes compared targeting efficiency among tumors of different sizes using the KB cell line (Harrington et al., 2000). The tumor accumulation for small tumors (≤0.1g) was around 15%ID/g, whereas for larger tumors(≥1g)wasonly3%ID/g. GUIDELINESFORPRE-CLINICALSTUDIESOFDELIVERY SYSTEMS While the physico-chemical properties of delivery systems are expected to exert a significant influence on pharmacokinetics, www.frontiersin.org August2014 |Volume2 |Article69 | 41