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Journal of Controlled Release
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Anticancer nanomedicine and tumor vascular permeability; Where is themissing link?
Sebastien Taurin a, Hayley Nehoff a, Khaled Greish a,b,⁎a Department of Pharmacology & Toxicology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealandb Department of Oncology, Faculty of Medicine, Suez Canal University, Egypt
Article history:Received 30 March 2012Accepted 8 July 2012Available online 16 July 2012
Keywords:Enhanced permeability and retention (EPR)NanomedicineDrug targetingVascular permeabilityTumor vesselPharmacokinetics
Anticancer nanomedicine was coined to describe anticancer delivery systems such as polymer conjugates, li-posomes, micelles, and metal nanoparticles. These anticancer delivery platforms have been developed withthe enhanced permeability and retention (EPR) effect as a central mechanism for tumor targeting. EPRbased nanomedicine has demonstrated, beyond doubt, to selectively target tumor tissues in animal models.However, over the last two decades, only nine anticancer agents utilizing this targeting strategy have beenapproved for clinical use. In this review, we systematically analyze various aspects that explain the limitedclinical progress yet achieved. The influence of nanomedicine physicochemical characteristics, animaltumor models, and variations in tumor biology, on EPR based tumor targeting is closely examined. Further-more, we reviewed results from over one hundred publications to construct patterns of factors that can influ-ence the transition of EPR based anticancer nanomedicine to the clinic.
The discovery of the EPR effect originates from pioneering workthat started more than 100 years ago. In 1907, E. Goldman describedtumor vasculature as “The normal blood vessels of the organs inwhich the tumor is developing are disturbed by chaotic growth,there is a dilatation and spiraling of the affected vessels, marked cap-illary budding and new vessel formation, particularly at the advancedborder” [1]. The immunological hypothesis developed by Paul Ehrlichat the beginning of the 20th century which evolved to what becameknown as ‘Ehrlich's magic bullet’ wherein a drug would specificallytarget the organism responsible for a disease [2]. Later, JudahFolkman demonstrated the ability of tumors to stimulate their ownvascularization [3]. Subsequent studies established that these tumorblood vessels harbored higher permeability compared to normalblood vessels [4–7]. These newly formed vessels usually have irregu-lar and incomplete structures in conjunction with modified physio-logical responses [8]. Further work led to the identification of thetumor vascular permeability factor (VPF) which was later found tobe identical to vascular endothelial growth factor (VEGF), a major de-terminant for enhanced vessel permeability [9]. The growth factors ofthe VEGF family are endothelial mitogens as well as potent mediatorsof vascular permeability. Ultrastructural studies have providedinformation on the mechanisms by which endothelial cells respondto VEGF [10]. Through alternative splicing, tumors generally
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overexpress multiple isoforms of VEGF [11] and these are known tobind VEGFR-1/Flt, VEGFR-2/Flk and some to neuropilin. Each isoformcan induce vascular permeability but as they differ in their ability tointeract with matrix proteins, this results in structural aberrations ofblood vessels (for review see [12]). Further research revealed someof the mechanisms by which VEGF influences vascular permeabilityin pathological conditions (for review see [13]). Finally, the observa-tion that macromolecules and lipids selectively permeate the tumorvasculature and remain in the tumor interstitium for an extended pe-riod of time led to the characterization of the tumor-selective EPR ef-fect [14]. The EPR effect explains the distinctive increase in vascularpermeability, both in tumor vasculature [15] and in inflammatory tis-sues [16]. The discovery of this unique phenomenon in solid tumors isconsidered a landmark for anticancer nanomedicine development.In the early 1980s, Maeda invented the first known anticancernanomedicine, styrene co-maleic acid conjugated neoCarzinostatin(SMANCS) based on the EPR concept [17]. Following the pioneeringwork of Maeda, many investigators have developed EPR based anti-cancer nanosystems. The main advantage of EPR-based anticancernanomedicines is their altered pharmacokinetics caused by their hy-drodynamic diameter that can reach 7 nm in size. A diameter superi-or to 7 nm will escape renal filtration and urinary excretion [18], dueto the slit diaphragms at the level of the podocyte foot of theglomerula which prevent the filtration of globular plasma proteinsabove this size [19]. Therefore, high nanometer sized particles can ex-hibit prolonged circulatory half-life, high area under concentration/time curve (AUC) and higher partition into tumor tissues [20]. Suc-cess of an effective drug delivery system is thus dependent on thesize [21] as well as the shape [22] of the nanomedicine to evade
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renal filtration and to limit clearance. Furthermore, nanodrug carrierscan be functionalized by a variety of moieties to prolong their circula-tory half-life in the blood beyond the advantage of size [23]. Aprolonged circulation will favor high plasma AUC compared to thefree drug [20,24,25] and significantly increase drug accumulation intumor tissue [21].
The Food and Drug Administration (FDA) and other internationalequivalents have approved over 30 nano-therapeutics for clinicaluse, of which 11 are used for the treatment or detection of various can-cers (Table 1). EPR effect has certainly influenced the design, develop-ment and improvement of different delivery platforms. Nevertheless,the rate of transition of EPR principles into the clinic has been up tonow disappointingly slow. In this article, we will discuss the currentstatus of EPR based drugs in the clinic; followed by a review of the cur-rent literature to identify factors that might adversely influence thedevelopment of EPR effect based anticancer nanomedicine.
2. EPR in the clinic
Proof of the EPR effect concept was clearly demonstrated byMaeda, 26 years ago, using Evans blue dye (EBD) injected intrave-nously into rodents. The dye was bound to albumin and selectivelyconcentrated in tumor tissue [15]. The EPR effect is a time dependentphenomenon. It usually takes more than 6 h of high plasma concen-tration of the drug to achieve accumulation in tumor tissue that lastfor an extended period of time ranging from hours to several days[38,39]. Maeda's work demonstrated that the rate of accumulationof macromolecules and lipids in tumor was inversely proportionalto their clearance rate. Based on these findings, several nano-sizedrug carriers were developed. SMANCS was the first clinical polymer-ic conjugate which was approved in Japan in 1993 for the treatmentof hepatocellular carcinoma (Table 1). Following this landmarkapplication of the EPR principle for tumor targeting, severalnanomedicines were developed for anticancer chemotherapy in thefollowing years. Liposomes have achieved significant success com-pared to other nanomedicines (see Table 1). For example, Doxilwhich is a PEGylated (polyethylene glycol coated) liposome that en-capsulates doxorubicin, was approved for the treatment of Kaposisarcoma and late stage ovarian cancer in 1995 [40]. Other polymericor micellar second or third generation drugs are currently beingassessed in clinical trials (phase I–III).
The size and shape of nanoparticles are critical for nanomedicineapplications. Extensive studies have shown that both criteria willdetermine their longevity in the blood circulation and theirbiodistribution when administrated to a patient. To be effective, in-travascularly administrated particles must avoid recognition by thereticulo-endothelial system (RES) composed of macrophages pres-ent in the liver, spleen, bone marrow and lymph nodes [23]. Gener-ally, particles larger than 100 nm are rapidly eliminated from the
Table 1Clinically approved nanomedicines for cancer treatment or imaging.
circulation by the RES [41,42]. To avoid this, several strategies havebeen developed, for instance, the addition of chemical modificationsby synthetic polymers such as polyethylene glycol (PEG) sterically hin-der interaction with plasma proteins [43] and reduced opsonization[44]. Gold nanorods have a prolonged circulation time and are lessphagocytosed compared to their spherical counterparts, howeverthe biodistribution of PEGylated gold nanorods and spheres ofequivalent sizes showed a similar accumulation profiles in the liverand spleen [22].
The advantages of nanomedicine over conventional medicinesrely essentially on the EPR effect and their improved pharmacokinet-ics, loading capacity, release rates, lower systemic toxicity and abilityto bypass multidrug resistance mechanisms that involve cell-surfaceprotein pumps.
3. The advantages and limitations of the EPR effect
Unlikemost normal tissues, tumor vasculature is heterogeneous anddoes not conform to standard morphology (i.e., arterioles, capillariesand venules which form a well-organized network with dichotomousbranching and hierarchic order) [45]. The tumor microenvironment iscomposed of a variety of cell types such as tumors cells, stromal cellsand inflammatory cells which all influence the angiogenic process [3].Newly formed blood vessels are dilated, saccular, poorly aligned andheterogeneous [46]. Tumor vasculature is characterized by defectiveendothelial cells with wide fenestration ranging from 300 to 4700 nm,lack of a smooth muscle layer or innervation, a wide lumen, and im-paired functional receptors for angiotensin II [47]. Furthermore, lym-phatic drainage of tumor tissues is usually deficient [3,38]. Thesefactors alter the molecular and fluid transport dynamics in the tumorand increase the retention of nano-sized drugs. While being above therenal filtration threshold, macromolecules larger than 40 kDa, selec-tively leak from tumor vessels and accumulate in tumor tissues forprolonged periods [47]. Arnida et al. also showed that geometry (sizeand shape) as well as surface characteristics influence theirbiodistribution, circulation time and tumor uptake [22]. Additionalstudies demonstrate that the EPR effect is further accentuated by ex-travasation factors such as VEGF, bradykinin, nitric oxide (NO), prosta-glandins, peroxynitrite andmatrix metalloproteinases [48,49]. All thesefactors increase blood flow and promote diffusion and retention ofnanomedicines inside tumors.
The recognition of the EPR effect among researchers in the field ofdrug development has resulted in a considerable momentum tothe field of nanomedicine. The EPR effect served as a basis for thedevelopment of anticancer macromolecular drugs to improve its thera-peutic efficacy by targeting tumor tissues and decreasing systemic ad-verse effects compared to free drug. Most low molecular weightanticancer drugs lack specific targeting and can traverse blood vesselsfreely; the consequent distribution of the drug to healthy tissues usually
Approval (year) Treatment References
FDA (1995) Late-stage ovarian cancer [26]Advanced HIV-associated Kaposi's sarcoma [27]
FDA (1996) Advanced HIV-associated Kaposi's sarcoma [28]FDA (1999) Malignant lymphomatous meningitis [29]Europe (2000) Metastatic breast cancer in combination
with cyclophosphamide[30]
Canada (2001)Europe (2004) Osteosarcoma [31]FDA (2006) Acute lymphoblastic leukemia [32]South-Korea (2006) Metastatic breast cancer [33]Japan (1993) Hepatocellular carcinoma [34]FDA (2005) Metastatic breast cancer [35]FDA (1996) MRI contrast agent [36]FDA (1996) MRI contrast agent [37]
Fig. 1. Differences between normal and tumor tissue in relation to targeting ofnanomedicines by the enhanced permeability and retention (EPR) effect. (A) Normaltissue contains tightly connected endothelial cells which prevents the diffusion of thenanomedicine outside the blood vessel. (B) Tumor tissue contains large fenestrationbetween the endothelial cells allowing the nanomedicines to reach the matrix andthe tumor cells by the EPR effect. VEGF secreted by tumor cells, stroma cells and mac-rophages increases permeability and stimulates angiogenesis and the migration of en-dothelial cells towards the tumor. A considerable proportion of the nanomedicinenever reaches the tumor either due to entrapment or nonspecific interaction withcollagen composing the matrix or removal through macrophage endocytosis.Nanomedicines tend to concentrate at the periphery of the tumor, only a small propor-tion will diffuse to the center of the tumor.
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results in adverse effects [50]. The EPR effect was predicted in the ma-jority of human solid tumors with the exception of poorly vascularisedtumor such as prostate and pancreatic cancer [50]. The expectation thatnanotechnology would result in the development of selective antican-cer drugs utilizing the EPR effect was widespread. However, with theexception of the nine nanomedicines listed in Table 1, the transition ofnanomedicines from the laboratory to an effective clinical anticancerdrug has been disappointingly slow. A major drawback in the special-ized field of nanomedicine arises from a dissociation between differentfacets of science. Currently the ability to design and develop anticancernanomedicine far surpasses the understanding of the EPR effect and itsbiological principles. Anticancer nanomedicine has so far not succeededin fully exploiting the EPR phenomenon for optimum cancer manage-ment. The following describes some of the commonly overlooked fac-tors that could account for the slow transition of EPR based anticancerdrugs towards clinical application.
3.1. Influence of EPR based drug design on internalization, release rate,and biocompatibility
Integral to utilization of the EPR effect is the design of specific drugdelivery systemwith amolecular size larger than 7 nm. A hydrodynam-ic diameter of a nanoparticle lower than 7 nm is rapidly cleared fromthe body via renal filtration and urinary excretion [18]. The physico-chemical characteristics of engineered nanosystems should be consid-ered critically from the biological perspective in order to optimize itsefficacy. In many instances, the nature of the nanomedicine itself hasbeen a limiting factor that negatively impacted its chance of clinical suc-cess. The loading of active drug into the delivery system can result inpractical limitations to the dose needed to be delivered to tumor tissue.For example, the HPMA copolymer-paclitaxel conjugate showed insuf-ficient drug loading (≤10%) with a particle size in the range of 12–15 nm [51], and lacked ester linker stability [52]. Consequentlyinsufficient tumor tissue accumulation of the drug was evident inphase I clinical trials [53]. Another factor limiting the efficacy of thenanomedicines is a fast release rate of drug in the circulation. Forinstance, the low molecular weight HPMA copolymer-camptothecinconjugate showed a rapid release of drug and quick renal filtrationand consequent bladder toxicity in phase I clinical trials [54]. Thenanomedicinewas designedwith a labile ester linker, decreasing its sta-bility and therefore its tumor accumulation [55]. In this example, a lowmolecularweight (below renal excretory threshold) coupled to the tox-icity associated with a fast release rate resulted in the drug failing toachieve EPR based pharmacokinetics.
Following are a few considerations inherent to the design ofnanomedicine that may significantly influence the outcome of EPRbased drug targeting.
3.1.1. Intercellular internalizationThe concentration of drug inside tumor resulting from the EPR ef-
fect in a subset of highly vascularized tumors does not guarantee theefficient internalization of the drug within the cytoplasmic or nuclearcompartments of the tumor cells. Multiple factors can influence thecellular internalization process of the nanomedicine. Usually,nanoparticles and polymer-based drug delivery systems are internal-ized by endocytosis, a multistep process that culminates in the forma-tion of a late endosome which finally fuses with a lysosome [56].Malignant cells have an accelerated metabolism, a high glucose re-quirement and an increase glucose uptake characterized by the ele-vated expression of glucose transporter proteins (GLUT) [57].However, recent studies have shown that many cancer cell lines ex-hibit limited capacity for endocytosis compare to normal cells [58,59].
In addition, nanomedicine can be frequently recognized as foreignbodies resulting in its rapid uptake and elimination by specializedcells of the reticulo-endothelial system (RES).
Compared to tumor cells, macrophages usually exhibit a higher up-take of nano-sizedmolecules [22,60] as they can recognize nanomedicineeither through their Toll-like receptor 4 (TLR-4) [61] or through scaven-ger receptors [62]. Much work has therefore been devoted to the devel-opment of nanoparticles which can evade macrophage recognition,resulting in longer circulatory time and increased interaction with targettissue. On this basis, polyethylene glycol (PEG) is the polymermost com-monly used to enhance in vivo circulatory half-life [63,64]. Coatingnanoparticles with PEG results in the formation of a polymeric layerwhich sterically hinders the interaction of nanoparticleswith plasmapro-teins and cell membranes [65] preventing opsonization and phagocytosisby components of the RES [66,67]. PEG-liposome-incorporated doxorubi-cin (Doxil®) is approved by the FDA for the treatment of ovarian cancer(see Table 1). Additional polymers such as N-(2-hydroxypropyl)methylacrylamide (HPMA), polyacrylamide or poly(vinyl pyrrolidone)have also been used to improve the circulation time and steric hindranceof nanomedicines [68,69]. The main disadvantage of this strategy limitsthe interaction of (stealth) nanoconstructs with the tumor cell mem-brane and subsequently reduced internalization and uptake by tumorcells. To improve specific uptake by endocytosis, several nanoparticleshave been coated with a ligand of folate [70] or transferrin [71] to inducereceptor mediated endocytosis. These coatings increased the accumula-tion of drug inside tumor cells. However, the practical advantages in themanagement of human tumors in the clinic remain to be proven. Follow-ing intracellular internalization, active drug should be liberated from thelysosomal compartment to reach its cellular target. Mechanisms to es-cape the lysosomal compartment and improve intracellular targeted de-livery have been described by Breunig, Bauer and Goepferich [72].Another consideration relevant to relatively large sized macromolecularnanomedicine is their nonspecific interactions with the extracellular ma-trix: to reach tumor cells, nanoconstructsmustmove through thematrix,a highly interconnected network of collagen fibers that intermingle withproteins such as proteoglycans and glycosaminoglycans. This semi-solidbarrier could significantly reduce the amount of nanomedicine reachingtumor cells through nonspecific interaction (Fig. 1) or simply by imped-ing convection movements of relatively large size of nanoconstructs[73]. This could lead to nanomedicine being locally concentrated in
Fig. 2. Variation of nanocarrier release rate and accumulation profile (A) Determinationof the release rate over a 24 h period of different nanocarriers across several studies[166–175]. (B) Comparison of the ratio of free to nanoconstruct associated drugs invarious tissues normalized across studies as % of injected dose per gram of tissue.[176–191]. (C) Comparison of the ratio of free to nanoconstruct associated drugs intumor tissue normalized across studies as % of injected dose per gram of tissue relativeto the animal model used [176,177,184,189,192–195].
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proximity to the capillary that it leaked fromwithout reaching the targettumor cells.
Recently, several studies have developed methods to circum-vent these limitations such as using of the tumor penetrating pep-tide, iRGD which has been shown to increase the delivery ofnanomedicines in solid tumors by improving its interstitial trans-port [74]. Additional therapeutic strategies aiming to normalizethe tumor vasculature and extracellular matrix in order to improvetumoral penetration of the drug have been described (for review[75,76]).
3.1.2. Release rateConjugates can be synthesized through covalent linking of drugs
to polymeric carriers such as SMANCS (Table 1) [17]. In comparison,entrapment of drug inside a micellar structure requires either cova-lent or non-covalent bonds (ionic, hydrogen bonds or hydrophobic)and involves a block polymer or copolymer. Various chemical bondssuch as amide, ester, azide, imine, hydrazone, thioether and urethaneare currently used to prepare nanomedicines [77,78]. Based on the na-ture of these chemical bonds, the release of drug from its carrier can de-pend on either pH, usually acidic pH of the lysosome [77], temperature[43] or on enzymatic cleavage [79]. Furthermore, the nature of this bondwill determine the release rate; for example an ester bond ensures arapid release of drug due to an abundance of esterases in plasmawhere-as an amide bond will show a slower release profile [54,78].
The comparison of release rates between polymer conjugates, li-posomes and micelles nanomedicines after 24 h incubation (Fig. 2A)in different studies showed a distinctive profile. Overall, polymer con-jugates and micelles have a comparable release rate which is higherthan that of liposomes (Fig. 2A). The release profile of liposomes ap-pears relatively homogeneous with a mean value of 24% (3–39%)while the release profile of conjugates and micelles appears heteroge-neous across several studies with a mean value of 39% (2.5–100%)and 41% (2.5–100%) respectively.
In order for a nanocarrier to provide tumor targeting, the carriershould have stable chemical bond with the cargo drug while in circu-lation. This prevents the rapid release of free drug and permits a ther-apeutic effect at the site of action. A rapid drug release from itsdelivery system in plasma can result in a biodistribution and toxicityprofile comparable to its related free drug. In contrast, engineering astable linkage between the drug and its carrier can result in a slow re-lease rate at the target site and inability to reach the critical therapeu-tic concentration. The release rate of nanoconstructs needs to betailored for the treatment of a specified tumor doubling time (seeTable 2). Thus, the choice of a specific linker is critical for a favorableanticancer outcome of EPR targeted nanosystems.
3.1.3. BiocompatibilityWith success of EPR based drug targeting, accumulation of active
drug inside the tumor rarely exceeds 5% of the total dose ofnanomedicine administrated by i.v. injection. The majority of injecteddose accumulates in various organs such as liver and spleen and to aminor extent kidneys and lungs [104]. Surface modification of thenanomedicine, such as PEGylation, may increase their retention inthe systemic circulation and favor tumor accumulation. However,more than 90% of PEGylated nanoparticles will still be removedthrough liver clearance within several hours of administration. Stud-ies have demonstrated that, as little as ~2% of the total i.v. adminis-tered dose was found in the tumor after 4 h [104]. Thus there is alegitimate safety concern regarding the off target accumulation ofthe drug delivery system. Ideally, the drug carrier should be eliminat-ed after drug release. But, unless the nanocarrier is biodegradable, itwill remain in the body and be dealt with as a foreign body. Theinnate elements of the immune system could be stimulated non-specifically by these foreign bodies through TLR-4 [105]. Activatedmacrophages will phagocytose and attempt to degrade the nanocarrier
in its lysosomal compartment. Failure to do so may lead to the forma-tion of foreign body giant cells caused by fusion of multiple macro-phages or monocytes [106] and ultimately to the formation of lesionsresembling granulomas [107]. This can potentially result in the patho-logical formation of a dense fibrous capsule replacing the originalfunctional tissue. Another concern in relation to the accumulation ofnon-degradable materials is the induction of malignancy resultingfrom frustrated phagocytosis and prolonged inflammation [108](Fig. 3).
Fig. 3. Schematic representation of the variables that can influence the outcome of EPRbased nanomedicine. (A) Interpretation of data obtained from animal model needscareful consideration of the immune status of the animal (1), the type of tumor cell or-igin (2), as well as the site of injection (3). (B) In translation of specific nanomedicinetowards clinical use, tumor doubling time which is usually related to high vascular den-sity and elevated expression of VEGF and its receptor, need to be considered. Hepatocel-lular carcinoma (1) and prostate cancer (2) are two extremes; hepatocellular carcinomahas a rapid tumor doubling time (42 days for themost aggressive) and a highmicrovas-cular density of 127.2. In contrast, prostate cancer is characterized by a slow tumor dou-bling time, low microvascular density of 39.2 as well as large variation in VEGFexpression. Off target accumulation (3) mainly to liver and spleen. Concern of chronictoxicity (4) associated with non-biodegradable nanoparticles and the presence ofnanoparticles in the brain such as silver (5).
Table 2Expression of VEGFR isoforms and ligands in various cancer cell lines and tumors.
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To address these issues, recent work has focused on the develop-ment of biodegradable and non-immunologic drug delivery systemscontaining either enzymatically or reductively degradable spacerssuch as poly-(-D,L-lactide-co-glycoside) (PLGA) [109] or the starHPMA polymer carrier which enable a controlled degradation of thedrug carrier [110,111]. Some of these carriers demonstrate prolongedblood circulation and tumor drug accumulation but difficulties in thereproducibility of their synthesis could hamper further clinical devel-opment [110].
3.2. Validations of animal models
The EPR effect has been repeatedly proven in animal modelsthrough the use of EBD. EBD binds instantly to plasma albuminwhich results in a large molecular weight complex of about 7 nm di-ameter that can simulate the effect of a nanomedicine. After 6 h, thereis usually a distinct accumulation in tumor lesions compared to sur-rounding tissues. Similarly, many nanomedicines have been observedto accumulate in tumor tissue up to 27-fold more than free drugs(Fig. 2B). The question of whether the results of EPR-based drugtargeting in animal models can be faithfully translated to the clinic re-mains unanswered.
Production of VEGF bymacrophages and its role in cancer develop-ment is well documented [112,113]. To test the anticancer propertiesof a given nanomedicine, it is commonpractice to utilize immunocom-promisedmice to enable the use of human tumor xenografts. Howeverhuman cancer patients are rarely immunocompromised. A change inmacrophage activity in immunocompromised mice [114] can resultin less VEGF leading to a tumor with reduced vascular density,which in turn limits the access of the nanoconstruct to the tumor. Fur-thermore, results obtained from immunocompromised models differfrom results obtained in immunocompetent mice. Reviewing 22 stud-ies involving various drug delivery systems (conjugates, liposomesandmicelles), revealed an approximate two-fold increase in tumor ac-cumulation of nanoconstructs in immunocompetent mice relative toimmunocompromised ones Fig. 2C. Moreover, immunocompetentmice bear murine tumors and not human cancer cell lines which fur-ther complicate interpretation of in vivo animal data and jeopardizeits value in predicting the performance of new drugs in clinical trials.Table 2 compares the expression of VEGF and its receptors betweencommonly used human tumor cell lines and their clinically isolatedvariants. It is clearly evident that tumor cell lines have pronounced ex-pression of VEGF and its receptors with far less variability in compar-ison to clinical tumors. Relevant to this is the design of targeted
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nanomedicine to tumors which relies on the conjugation of target li-gands that bind strongly to tumor cell surface receptors to increasecell recognition and cellular uptake. Galactosamine [115], transferrin[116] and folate [117] have been incorporated in nanomedicinebased on the preferential expression of these molecules by cancercells. Despite promising in vitro studies, these targeted nanomedicinefailed to demonstrate significant benefit at the preclinical or clinicallevel [118]. The discrepancy between the results obtained from testingspecific tumor cell lines in tumor model and the clinical trials pointsfurther to the sampling errors in generalizing the results of from spe-cific cell line to that of relevant tumors [119].
A substantial difference between tumormodels in animals and thoseof human patients is the progression rate. Animals usually develop alarge, clinically relevant tumor (>5 mm) one week following subcuta-neous (SC) tumor cell inoculation, while such a tumor volume can takeyears to develop in a human (Table 3). This rapid progression rate in an-imalmodels results in the overestimation of the targeting role of EPR ef-fect. Animal tumors developing quickly presumably produce a largequantity of VEGF and vascular mediators to support their rapid growth.In addition a one-gram tumor mass in a thirty-gram mouse is about 3%of its total weight. In humans, a comparable tumorwouldweigh 2–5 kg,which is an advanced tumor stage that is not ideal for utilization of an-ticancer nanomedicine. Finally, tumors are usually implanted SC in an-imal models, which allow the developing tumors to take advantage ofthe extensive cutaneous vascular network for extending their bloodsupply. A condition that is rarely encountered in human malignancy.
Data collected from available literature to date show that althoughthere is a trend towards higher concentration of nanoconstructs in SCmodels, the results are however not conclusive given the limitednumber of studies. Whether site of tumor development can influencethe efficacy of the EPR effect remains an unanswered question.
3.3. Tumor biology diversity
3.3.1. Tumor doubling time (TDT)Tumor doubling time (TDT) is an important factor to consider
when designing EPR based anticancer nanomedicine. In general, fast
Table 3Tumor doubling time of various types of cancers.
doubling times (DTs) provide a selection trait that is exploited bychemotherapeutics that target processes necessary for mitosis suchas DNA synthesis and cytoskeleton remodeling. Many chemothera-peutic agents fail to cope with rapidly dividing tumors as the amountof drug necessary to kill a given number of cells will double with eachtumor doubling. However, the dose that will elicit dose limiting tox-icity will remain the same. A short TDT is well known to be associatedwith an unfavorable survival prognosis [138–143].
TDT is a highly heterogeneous, both within and between differenttumor types, stages and grades. There is a large degree of variation ofTDT between tumors of different tissue origins. Pituitary adenoma, forexample, has an extremely long DT of 506–5378 days [133] whilestomach cancer may have a minimal DT of as little as 12 days [137].
Within tumor types variation is also pronounced. For instance, lungadenocarcinoma has an extremely high variation in TDT of 964-fold[120] followed by breast cancer with a variation of 117.5-fold [122](see Table 3). TDT can also differ according to the specific cellular originwithin a given tissue. Bronchoalveolar cancer, for example, has an ex-tremely varied TDT of 36–1092 days, a variation of 30.3 fold [120]while small cell lung cancer has a TDT of 61.9–120.4, amere 1.9-fold dif-ference [136]. In addition, TDT can range depending on tumor grade.Poorly differentiated hepatocellular carcinoma is highly invasive and,predictably, has a DT of 20–78 days [128] while moderately differenti-ated hepatocellular carcinoma has a significantly extended DT of 94–380 days [129]. Astrocytoma also follows this trend with DT ofhigh-grade astrocytoma at 60–78 days and the TDT of low-grade astro-cytoma at 108–330 days. The primary or metastatic status of a tumorcan also cause large fluctuations. For example primary melanoma mayhave a DT of 50–377 days [126] while metastatic melanoma may havea DT of 8–212 days [127] (see Table 3).
Scientists designing EPR based anticancer nanomedicine shouldconsider doubling time variation when planning the release mecha-nism of active chemotherapeutic agents from its nanocarrier, as wellas the internalization rate of macromolecular complexes into tumorcells. For example, a slow releasing amide bond between the polymerback bone and the drug, or slowly internalized liposome, could bothbe a good choice for tumors with a slow DT.
In contrast a fast releasing micelle or an ester bond linkage can bea better fit for rapidly dividing tumors. Generally, EPR basednanomedicine has a wider therapeutic window [144], an advantagethat can be exploited to shape dose regimens based on individual pa-tient conditions. Tumor inherent sensitivity to specific chemothera-peutic agents as well as TDT is of the upmost importance indesigning EPR based anticancer nanomedicine.
3.3.2. Variation in tumor vascular densityThe EPR effect is a phenomenon that strictly depends on the vascula-
ture of the tumor with theoretical assumption that all tumors indepen-dently of their origin, stage and organswill behave identically. However,this conception is drastically challenged by a number of reports thatshow a high diversity in angiogenesis behavior [76,145–147]. Nagy etal. have identified six structurally and functionally distinct types ofblood vessels in human cancers [147]. Vascular density may relate totumor progression. As shown in Table 4, vascular density is largely de-pendent on the type of cancer and varies largely within each tumortype. For instance, renal cell carcinoma is highly vascularized [148]while the density of micro-vessels appears low in head and neck squa-mous cell carcinoma [149] or in ovarian carcinoma [150]. In addition,higher stages of cancer are well correlated with higher microvasculardensity as observed in non-small cell lung cancer [151] while in othertypes of tumors such as renal cell carcinoma no direct correlation canbe established between tumor stage and vascular density [152]. Further-more, metastatic tumors tend to possess higher vascular densitycompared to non-metastatic tumors [153–156]. Another element re-garding the EPR effect is the secretion of VEGF by the tumor. Vascularpermeability can be altered by VEGF as well as a wide array of
Table 4Microvessel vascular density of various types of cancers.
Malignant melanoma metastatic 35.8 (20.3–51.3) 20 Factor VIII [155]
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inflammatory mediators [10], which can affect the extent ofnanomedicine accumulation driven by the EPR effect and the penetra-tion of nanodrug inside the tumor. As shown in Table 2, there is a largeheterogeneity in the expression of VEGF seen when comparing prostateand lung cancers.
When designing the nanocarrier, the properties of the targetedtumor tissue such as the cancer type, the microvascular density,and the secretion of permeability factors such as VEGF should thereforebe taken into account in order to take full advantage of the EPRphenomenon.
3.3.3. EPR related pharmacokineticsIn classical pharmacology, the tissue concentration of a certain
drug cannot exceed its plasma concentration. With respect to tradi-tional pharmacokinetics, any administered drug is equally distributedto different body compartments indiscriminately forming the volumeof distribution. Conventional chemotherapeutic agents usually exhib-it large volume of distribution. For example, doxorubicin has avolume of distribution of ~680 L [161] indicating that it is indiscrim-inately partitioned to various organ tissues.
As drugs reach sizes of 7 nm, classical pharmacokinetics roles can-not be accurately applied due to the acquisition of two drasticchanges. Firstly, nano-sized drugs cannot be eliminated by renal glo-meruli as they exceed the renal threshold of excretion dictated by thepore size in the glomeruli [19,162]. Secondly, their organ distributionis limited to tissues that have capillaries with large enough endothe-lial fenestrations to allow macromolecular drugs to pass through[19,20]. The EPR effect utilizes the unique characteristic of largegaps between endothelial cells that makes up tumor vessels. Usuallythese gaps can vary from few nanometers to up to 400 nm in size[151,163]. At this large size, nanomedicines can preferentially accu-mulate in tumor tissues. However, tumors are not the only organswith such large fenestration as the spleen and liver show similarcharacteristics. Liver sinusoid can have fenestration of around100 nm in humans [164] whereas the spleen has large sinusoidlumina of 5 μm that can support extravasations of aged red bloodcells [19]. With such a large fenestration size, great amounts ofnano-sized drugs are filtered by these organs. As shown if Fig. 2Bmeta-analysis of 73 studies over the last 10 years revealed that withall the EPR effect-based nanoconstructs that were used, liver andspleen were the two major organs competing with tumors for thenanoconstructs (conjugate, micelles, and liposomes). While spleenfunction can be compensated for by other lymphatic organs, liverdamage due to the concentration of cytotoxic nanomedicine remainsa potential challenge to successful anticancer drug targeting. For ex-ample, nanoconstructs of cis-platinum have reduced toxicity in thekidney compared to the free drug, but results in a dose limiting
liver toxicity [165]. To summarize, EPR related pharmacokinetic pa-rameters such as long circulatory half-life, reduced elimination, andaltered distribution could be a double edge sword. Careful consider-ation of these parameters is essential for effective and more personal-ized cancer treatments and the prevention of unexpected toxicity.
4. Conclusion
The EPR concept has been the corner stone in the developmentof a new class of anticancer drugs aiming at increased anticanceractivity and lower toxicity. Contrary to the high expectations ofEPR effect, only a few drugs based on this principle have reachedthe clinic over the last 20 years. Unjustified generalization of resultsobtained from subcutaneous animal tumor models to human coun-terparts, might have in part contributed to the slow progressionof anticancer nanomedicine. Human cancer diversity, characteristicsof commonly used animal tumor models, biodistribution of nano-sized molecules, variable release rates, slow intracellular internali-zation and biocompatibility all are important factors to take into ac-count when designing optimal nanomedicines. Wise considerationof these variables will ensure a prosperous future for EPR-basedcancer chemotherapy.
Acknowledgment
KG gratefully acknowledges the support of Professor Hiroshi Maeda.The EPR effect was first described and extensively studied by ProfessorMaeda's group in the department ofMicrobiology, Kumamoto University,Japan. This work has been supported by departmental fund no.;(PL. 108403.01.S. LM) to KG and HRC Emerging Researcher First Grant(PL. 108360.01.P.LM), UORG and AG305 for ST from the Department ofPharmacology and Toxicology, Otago University. KG thanks Dr. FlorianeImhoff, Ms Céline Bourdon and Mr SimranMaggo for proof reading thearticle.
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