Nanotechnology for breast cancer therapy

Takemi Tanaka & Paolo Decuzzi &Massimo Cristofanilli & Jason H. Sakamoto &

Ennio Tasciotti & Fredika M. Robertson & Mauro Ferrari

Published online: 29 July 2008# Springer Science + Business Media, LLC 2008

Abstract Breast cancer is the field of medicine with thegreatest presence of nanotechnological therapeutic agents inthe clinic. A pegylated form of liposomally encapsulateddoxorubicin is routinely used for treatment against meta-static cancer, and albumin nanoparticulate chaperones ofpaclitaxel were approved for locally recurrent and meta-static disease in 2005. These drugs have yielded substantialclinical benefit, and are steadily gathering greater beneficial

impact. Clinical trials currently employing these drugs incombination with chemo and biological therapeutics exceed150 worldwide. Despite these advancements, breast cancermorbidity and mortality is unacceptably high. Nanotech-nology offers potential solutions to the historical challengethat has rendered breast cancer so difficult to contain anderadicate: the extreme biological diversity of the diseasepresentation in the patient population and in the evolution-ary changes of any individual disease, the multiple path-ways that drive disease progression, the onset of ‘resistance’to established therapeutic cocktails, and the gravity of theside effects to treatment, which result from generally verypoor distribution of the injected therapeutic agents in thebody. A fundamental requirement for success in thedevelopment of new therapeutic strategies is that breastcancer specialists—in the clinic, the pharmaceutical andthe basic biological laboratory—and nanotechnologists—engineers, physicists, chemists and mathematicians—optimize their ability to work in close collaboration. Thisfurther requires a mutual openness across cultural andlanguage barriers, academic reward systems, and many other‘environmental’ divides. This paper is respectfully submit-ted to the community to help foster the mutual interactionsof the breast cancer world with micro- and nano-technol-ogy, and in particular to encourage the latter community todirect ever increasing attention to breast cancer, where anextraordinary beneficial impact may result. The paperinitiates with an introductory overview of breast cancer,its current treatment modalities, and the current role ofnanotechnology in the clinic. Our perspectives are thenpresented on what the greatest opportunities for nanotech-nology are; this follows from an analysis of the role ofbiological barriers that adversely determine the biologicaldistribution of intravascularly injected therapeutic agents.Different generations of nanotechnology tools for drug

Biomed Microdevices (2009) 11:49–63DOI 10.1007/s10544-008-9209-0

T. Tanaka : P. Decuzzi : J. H. Sakamoto : E. Tasciotti :M. Ferrari (*)Brown Institute of Molecular Medicine,Department of Biomedical Engineering,University of Texas Houston Health Science Center,1825 Herman Pressler Street, Suite 537D,Houston, TX 77030, USAe-mail: mauro.ferrari@uth.tmc.edu

P. DecuzziCenter of Bio-/Nanotechnology and Bio-/Engineeringfor Medicine, University of Magna Graecia,Viale Europa, LOC. Germaneto,88100 Catanzaro, Italy

M. Cristofanilli : F. M. Robertson :M. FerrariDepartment of Experimental Therapeutics,University of Texas M.D. Anderson Cancer Center,1515 Holcombe Boulevard,Houston, TX 77030, USA

M. FerrariDepartment of Bioengineering, Rice University,Houston, TX 77005, USA

P. DecuzziSchool of Health Information Sciences,University of Texas Houston Health Science Center,7000 Fannin St.,Houston, TX 77030, USA

delivery are reviewed, and our current strategy for address-ing the sequential bio-barriers is also presented, and isaccompanied by an encouragement to the community todevelop even more effective ones.

Keywords Nanotechnology . Breast cancer .

Biological barrier . Porous silicon . Drug delivery

1 Breast cancer

In 2005, cancer overcame cardiovascular disease as theleading cause of death in individuals under age 85 in theUS (Jemal et al. 2007). The global incidence and mortalityof breast cancer remains high despite extraordinary prog-ress in understanding the molecular mechanisms underlyingcarcinogenesis, tumor promotion, and the establishment ofmolecular targeted therapies. Worldwide, 1,301,867 newcases of breast cancer were diagnosed, 464,854 deaths werecaused by breast cancer, and more than 4.4 million womenwere diagnosed with breast cancer in 2007 (www.cancer.org). The estimated national number of newly diagnosedcases in the United States in 2008 is 182,460 with anexpected death toll of 40,480 (Jemal et al. 2007). Since1990, there has been an overall increase in breast cancerincidence rates of about 1.5% annually. Breast cancerdetection involves self and clinical examination andradiography (including mammography positron emissiontomography and magnetic resonance imaging) followed byinvasive biopsy for the histological confirmation of inva-sive disease. The development of mammography hasgreatly increased the likelihood of early detection of breastcancer, and randomized clinical trials have demonstrated a30% reduction in breast cancer mortality in women age 50–69, who are screened annually with mammography(Elwood et al. 1993; Kerlikowske 1997). Although earlydetection of breast cancer by mammography is associatedwith less invasive surgical procedures and may increasesurvival, the 5-year survival rate of metastatic breast cancer(stage IV) is still below 15% (www.cancer.org). Thus, thedevelopment of effective therapies against invasive breastcancer and particularly highly metastatic disease stillremains a significant priority. The treatment of primarybreast cancer has mainly relied upon initial surgicalintervention (including lumpectomy, or partial or totalmastectomy) followed by radiation and various forms ofsystemic adjuvant therapy including cytotoxic chemother-apy, hormonal therapy, and most recently immunotherapy(e.g. trastuzumab). Generally, breast tumors are categorizedinto four different stages based upon their size, location,and evidence of metastasis (www.cancer.org). Treatmentoptions are also determined by the stage, hormone andhuman epidermal growth factor receptor 2 (HER-2/neu)

status of breast tumors. Over the past 30 years, many noveldrugs have been developed for controlling breast cancergrowth, and these drugs have shown significant clinicalbenefits in some cases of breast cancer. Approximately 65%of breast tumors demonstrate hormone receptor positivityand therefore the most common breast cancer therapiestoday are hormonal therapies (e.g. selective estrogenreceptor modulators (SERMs), and aromatase inhibitors).Additional therapies include chemotherapy (e.g. anthracy-clines and taxanes), often used in combinations andimmunotherapies (e.g. trastuzumab).

2 Conventional breast cancer therapy

2.1 Hormone therapy

Estrogen receptors (ER) are known to regulate breast cellgrowth in response to estrogen. The estrogen-dependentbreast cancer growth was first demonstrated by the fact thata removal of the ovaries of premenopausal women wasassociated with the regression of advanced breast tumors.ER is a transcription factor that belongs to a member of thenuclear hormone receptors superfamily, which initiates orenhances the transcription of genes containing specifichormone response elements (estrogen response element,ERE) (Umesono and Evans 1989). The human ER proteinhas a molecular weight of 66 kDa and consists of 595amino acids (Green et al. 1986) that form six differentfunctional domains, including a ligand binding domain forestrogen and a DNA binding domain (Fig. 1) (Kumar et al.1986, 1987). Estrogen, a ligand for ER, is produced by theovary, diffuses through the plasma membranes of cellswhere it binds to the ER (Rao 1981). Once the ER bindsestrogen, it dimerizes, translocates to the nucleus, and bindsto ERE in the promoter region of genes, thereby activatingdownstream gene expression (Fig. 1). Selective estrogenreceptor modulators, SERMs (tamoxifen, raloxifene andarzoxifene) have been established to antagonize the effectsof ER activation through the AF2 domain (Fig. 1) (Aapro2001). Among all breast cancer cases, hormone receptorpositive breast cancer accounts for 75%, and hormonaltherapy has been shown to significantly reduce the risk ofbreast cancer recurrence and increase the 10-year survivalof women with ER+ breast tumors (Aapro 2001). Fiveyears of adjuvant tamoxifen treatment reduces the annualbreast cancer death rate by 31% (2005).

2.2 Immunotherapy

Human epidermal growth factor receptor 2, a receptortyrosine kinase, is upregulated in 25% of breast tumor dueto abnormal gene amplification and overexpression of

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which clinically correlates with reduced survival andreduced time to relapse compared to patients with normalreceptor levels (Slamon et al. 1987, 1989). The Her2dimerization is essential for an activation of signaling cascadeto promote cell survival through the Ras–Raf–mitogen-activated protein kinase–extracellular-signal-regulated kinase(ERK) kinase (MEK)/ERK pathway (Fig. 2) (Yarden andSliwkowski 2001). These findings led to the development oftrastuzumab (anti-Her2 Mab; Herceptin®, Genentech), thefirst genomic research-based, targeted anti-kinase therapyapproved by the Food and Drug Administration for thetreatment of patients with invasive breast cancers over-expressing Her2 (Fendly et al. 1990). Trastuzumab binds tothe extracellular membrane domain of Her2 and inhibits theproliferation and survival of Her2-dependent tumors byblocking the dimer formation. In a phase III comparison trialin which trastuzumab was added to first-line therapy with

anthracycline–cyclophosphamide or paclitaxel for patientswith Her2+ metastatic breast cancer, the addition of trastuzu-mab provided significantly better results (25.1 months mediansurvival) than standard therapy alone (20.3 months mediansurvival), with a 20% overall survival improvement(Cobleigh et al. 1999; Slamon et al. 2001).

2.3 Systemic chemotherapy

Large prospective clinical studies have clearly demonstrat-ed that the hormone and/or immune therapy greatly reducethe mortality of patients with ER+/HER2 or PR+/HER2breast cancer, a group that comprises 75% and 15–20% ofbreast cancer cases, respectively (Slamon et al. 1989;Konecny et al. 2003). However, the remaining 10–15% ofbreast cancers comprise a “receptor-negative’ or “triple-negative” category defined by the absence of expression of

Fig. 1 Estrogen receptor (ER) protein structures are subdivided intodistinct functional domains that are responsible for different functions:the N-terminal ligand independent transactivation domain AF-1, theDNA-binding domain, a flexible hinge region, the ligand-bindingdomain, and a ligand dependent transactivation domain, AF-2, locatedat the C-terminus within the ligand-binding domain. Estrogen (E)

binding to the ER induces a homodimerization followed by nucleartranslocation. Liganded ER binds to the estrogen receptor responseelement (ERE) as a homodimer and recruits co-activator complexes toactivate transcription. With estrogen, full activation of transcriptionthrough both the AF1 and AF2 is observed. In contrast, tamoxifen (T)bound ER only induces AF1 mediated transcription

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these three receptor proteins (Cleator et al. 2007). The triplenegative breast cancer is highly proliferative and aggressivewith poor prognosis due to a lack of specific treatmentguidelines, and therefore, triple-negative breast cancers aremanaged with standard chemotherapy (Carey et al. 2007).Unfortunately, such treatment is associated with high ratesof local and systemic recurrence (Carey et al. 2007). Manycytotoxic agents (such as cyclophosphamide, 5-fluorouracil,doxorubicin, taxanes, capecitabine), either as single agents orin combination regimens, have demonstrated activity againstadvanced breast cancer (Parkin 2001). The most commonlyused nonanthracycline-based regimens cyclophosphamide,methotrexate, and 5-fluorouracil (CMF) have objectiveresponse rates of 50–70%, with a median duration ofresponse of 10–12 months. More recently, single agentCapecitabine has shown activity in advanced disease andsuperiority to CMF regimens in patients with metastaticbreast cancer. Anthracycline-based regimens (such as fluo-rouracil, doxorubicin, and cyclophosphamide) have objectiveresponse rates of 50–80%, with fewer than 10% completeresponses. The median duration of response is usually 10–18 months, with median survival times of 18–26 months(1998). Substantial therapeutic effects have been observedamong patients who respond to this type of intervention,unfortunately, their remissions are usually short-lasting.Taxanes (e.g. paclitaxel and docetaxel) are among the mosteffective and currently used cytotoxic agents in breast cancer.

The combination docetaxel/capecitabine has shown survivaladvantages when compared to single agent docetaxelsuggesting that the combination regimen may show asuperior benefit. In spite of those results, the median survivalof patients with metastatic breast cancer is still approximate-ly 18 months. The limited efficacy of cytotoxic chemother-apy is partially due to the use of suboptimal dosages of thosetherapeutic agents in attempts to prevent both acute andchronic toxicities.

2.4 Anti-cancer therapy associated side effects

Most tumors, including breast cancer, are treated with acombination chemotherapy strategy with the commonaddition of biological agents that demonstrate synergisticor additive effects by multiple mechanisms. Even thoughchemo and adjuvant therapies have proven their efficacy asdiscussed above, side effects associated with these therapiesare serious and sometimes even life threatening. The knownside effects of chemotherapy are caused by the cell killingeffect of such agents. This derives from the fundamentalphenomenon that available cytotoxic agents are notselective in their activity, and therefore non-specificallydamage normal replicating cells in the bone marrow,gastrointestinal epithelia, and hair follicles. For example,acute toxicities associated with conventional doxorubicininclude myelosuppression, nausea, vomiting, mucositis, and

Fig. 2 A mechanism of therapeutic action of humanized monoclonalantibody against HER2 for breast cancer therapy: Aberrant expressionof HER2 on the surface of the cell membrane followed by thedimerization induces both cell proliferation and survival signaling in aligand independent manner. The phosphorylated tyrosine residues onthe intracellular domain of HER2 activate the phosphoinositide

3-kinase (PI3-K), which phosphorylates a phosphatidylinositol that inturn binds and phosphorylates the Akt, driving cell survival. In parallel,son of sevenless (SOS) activates the rat sarcoma Ras protein (RAS), inturn, activates raf protein (RAF) and then mitogen-activated proteinkinase (MAPK) and ERK kinase (MEK). Herceptin binds to HER2monomer and inhibits the dimer formation

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alopecia. The most serious, conventional doxorubicin-induced toxicity is irreversible congestive heart failure(Von Hoff et al. 1979). Tamoxifen is also associated withserious side effects and complications including anincreased risk for endometrial cancer by 2.4 times inwomen aged 50 years or older (Fisher et al. 2005) andthromboembolic disease by 1.9 times (Cuzick et al. 2003).Targeted therapies showed significantly positive effect asevidenced by multiple clinical studies, however, even thesetargeted therapies caused serious side effects. Trastuzumabalone or in combination with chemotherapy may causeserious heart problems including ventricular dysfunctionand congestive heart failure in addition to common flu-likesymptoms (Slamon et al. 2001). Therefore, the develop-ment of a novel treatment strategy including selectivedelivery of cytotoxic agents to tumor mass for the treatmentof advanced breast cancer is critical to improving thetherapeutic index and efficacy/toxicity balance.

3 Application of nanotechnology for anti-cancer therapy

Application of nanotechnology to medical science has beenemerging as a new field of interdisciplinary research amongmedicine, biology, toxicology, pharmacology, chemistry,material science, engineering, and mathematics, and isexpected to bring a major breakthrough to address unsolvedmedical issues. Nanotechnology was originally defined as“the creation of useful materials, devices, and systems usedto manipulate matter that are small scale ranging between 1and 100 nm” (http://nano.cancer.gov). As nanotechnolog-ical applications in the field of medical science haveexpanded rapidly towards multiple directions in the past10 years, the definition of nanotechnology has beenbroadened. Based on our definition, four ingredients arenecessary to identify a nanotechnology tool (Thei et al.2006): (1) the characteristic size of the device has to benano, (2) the device has to be man-made; (3) the device hasto exhibit properties that only arise because of the nano-scopic dimensions; and (4) the peculiar behavior of thedevice has to be predictable through the construction ofappropriate mathematical models.

Many different types of nano-delivery systems withdifferent materials and physio-chemical properties havebeen developed for application to different diseases. Mostwell studied among these are liposomes (Rivera 2003),polymer-based platforms (Duncan 2003; Green et al. 2007),dendrimers (Cloninger 2002) (Pan et al. 2007), goldnanoshells (Hirsch et al. 2003; Loo et al. 2005), nanocrystal(Yong et al. 2007), carbon-60 fullerenes (Kam et al. 2005),silicon- and silica-based nanoparticle (Yan and Kopelman2003; Martin et al. 2005; Peng et al. 2006), and superparamagnetic nanoparticulates (Oyewumi and Mumper

2002; Yan et al. 2004) among others. An excellent examplethat nanotechnology has already achieved in the field ofmedicine is liposomal drug delivery. Several differentformulations of liposomal doxorubicin have successfullybeen used in the clinic for the treatment of breast, ovarian,and Kaposi sarcoma (Di Paolo 2004).

The field of nanotechnology has rapidly evolved asevidenced by the fact that there are more than 150 ongoingclinical trials investigating the efficacy of nanotechnology-based drug delivery carriers targeting cancer (ClinicalTrial.gov). Various liposomal doxorubicin formulations weredeveloped in an effort to improve the therapeutic index ofthe conventional doxorubicin chemotherapy while maintain-ing its anti-tumor activity. For example, the efficacy of threeliposomal doxorubicins are currently being used: liposomaldaunorubicin (DaunoXome®), liposomal doxorubicin (D-99,Myocet™J), and pegylated liposomal doxorubicin (Doxil®marketed and distributed in the U.S. and Caelyx® distributedoutside the U.S.). Generally, these agents exhibit efficaciescomparable to those of conventional doxorubicin, except withbetter safety profiles and less cardiotoxicity (Von Hoff et al.1979; Hortobagyi 1997). The delivery strategy of thesevectors is based on enhanced permeation and retention(EPR) effect (Maeda 2001; Wu et al. 2001; Maeda et al.2003): the circulating vector accumulates in the tumor massover time because it is sufficiently small (<300 nm) toextravasate by crossing passively the fenestrations in thediseased vasculature (passive targeting). In addition toliposomal doxorubicin, albumin-bound paclitaxel (Abrax-ane®) is another example of an EPR based nanovectorapplication for breast cancer chemotherapy. Paclitaxel ishighly hydrophobic and dissolved in cremophor to preventpaclitaxel precipitation. However, cremophor-associated tox-icities are severe (hypersensitivity reaction and neurotoxicity)and challenge the application of paclitaxel (Liebmann et al.1993). Albumin-bound paclitaxel was developed to improvethe solubility of paclitaxel. This formulation improves thetoxicity profile of conventional paclitaxel therapy formulatedwith cremophor (Nyman et al. 2005). These vectors are notspecifically targeted against any molecule expressed on thetumor cells or the endothelium and have been classified as‘first generation’ vectors (Ferrari 2008a).

The ‘second generation’ of therapeutic nanovectorsevolved to be able to recognize and target specificbiological molecules on the surface of the cancer cells(active targeting). Such application will promise to improvetherapeutic window to delivery higher concentration todiseased lesion, while reducing life-threatening systemiccytotoxicity. This can be achieved by chemical coupling ofhigh affinity ligand, such as Arg–Gly–Asp (RGD) (Pasqua-lini et al. 1997), folate (Gabizon et al. 2004), prostatespecific membrane antigen (Farokhzad et al. 2006a), on thesurface of the nanoparticles, and it facilitates the interaction

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of nanoparticles and cancer cells, resulting in a dramaticimprovement of the biodistribution of nanoparticles com-pared to the non-targeted first generation nanovectors. Weare currently developing a ‘third generation’ of nanovectors(Tasciotti et al. 2008), which relies on a multi-stage strategyand is characterized as a carrier for nanoparticles and ahigher level of multi-functional integration. Biodegradablemesoporous silicon microparticles (1st stage) can be loadedwith one or multiple types of nanoparticles (2nd stage)containing different types of payloads, both for therapy andimaging (Fig. 3). The 1st stage particle is designed tonavigate within the circulatory system and to recognizespecifically the diseased endothelium through a judicious

(mathematically driven) choice of its geometrical (size,shape) and surface physico-chemical properties. The 2ndstage nanoparticles within the pores of the 1st stage, arereleased towards the tumor mass from the site of vascularadhesion (tumor endothelium) as the 1st stage degradesover time. The 2nd stage nanoparticles are sufficientlysmall (<20 nm) to easily cross the inter-endothelialjunctions and diffuse within the extravascular compartment.The delivery strategy of the third generation vectors doesnot rely on the EPR effect, in that the 1st stage particles aredirected towards the vascular endothelium and the 2ndstage particles pass the fenestrations. The modularity of thethird generation vectors presents a powerful tool to address

Fig. 3 Illustration of the con-cept of angiogenic vessel tar-geting multistage nanovectordrug delivery to overcome bio-logical barriers: (a) Systemicinjection of drug delivery vectoror drug alone has a risk to beeliminated from the circulationdue to immune cell uptake.Drug delivery vector needed toextravasate from the vessels tothe tumor mass to exert theirtherapeutic action. Thus, thedelivery vector needs to besmall enough or flexible to passthrough interendothelial space.(b) A drug delivery vector isdesigned to spontaneously mar-ginate and lodge to the endo-thelial surface through theinteraction between the ligandgrafted on the vector and thesurface receptor on the endothe-lial cells will release nanopar-ticles or therapeutic agents. Thevector can be designed to avoidthe recognition from immunecells by conjugation of PEGaround the vector surface. Smallsize of nanoparticles loaded withpayload (2nd stage) can bereleased from 1st stage vector.Further, the use of vasoactiveagents may aid to open endo-thelial wall temporary to en-hance the rate of drugpenetration into the tumor mass

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multiple unmet medical issues, with a focus of developmentof multifunctional and multimodal therapies.

4 Obstacles of anti-cancer therapies

In general, breast cancer therapeutic agents are intrave-nously or orally administered and are required to penetratemultiple and sequential barriers to reach the tumor mass at aconcentration capable of inflicting lethal toxicity. Theseobstacles include physical barriers, e.g. absorption throughskin and gastrointestinal tract (e.g. luteinizing hormone–releasing hormone agonists and Fulvestrant, capecitabineand progestins), physiological barriers (e.g. the reticulo-endothelial system, epithelial/endothelial membranes, andcellular drug extrusion mechanisms), and biophysicalbarriers (e.g. tumor vascular architecture and interstitialpressure gradients, transport across the extracellular matrixand stromal impediments, specificity and density of tumorspecific surface receptors) (Ferrari 2005a, b). In this review,we discuss the biological nature of each barrier that smallforeign substances need to overcome to reach tumor massand further discuss possible solutions that nanotechnologycan offer to solve long standing medical issues that impedetherapeutic effect.

4.1 Clearance of therapeutics from the circulation

Intravenously administered therapeutic molecules mustcirculate in the system long enough to reach their biologicaltarget and exert their therapeutic effects. However, foreignsubstances including single drug molecules as well asnanoparticles can be cleared from the circulation bymultiple defense mechanisms, generally, identified withthe reticulo endothelial system. The circulation half-life ofsingle drug molecules is limited to few minutes, whereassimilar and higher dose of drugs can be administered atthe systemic level using particulate formulations with half-lifes of several hours. This evidently constitutes a greatadvantage of nanotechnology over conventional therapiesand it may remove and certainly reduce the need ofrepeated injections of scheduled chemotherapy. In additionto this, nanovectors can be designed to avoid and escapesequestration by their geometrical and physico-chemicalproperties. The average diameter of small capillary isapproximately 5–8 μm, and they generally occur in thelungs (Illum and Davis 1982; Rapp and Bivins 1983).Therefore, rigid particles of sizes larger than 5 μmwould cause vascular embolization (Martin et al. 2005),which would be nonetheless limited to the smallestcapillaries compared to the particle size. In contrast,particles smaller than 20–30 nm would tend to extravasatefrom the systemic circulation through the internedothelial

gap junctions (Kanan et al. 1975; Illum and Davis 1982;Simberg et al. 2007). The particles filtered from circulationinto tissue are most likely phagocytosed by tissue macro-phages. Fully differentiated tissue macrophages are highlyphagocytic and can be found tightly bound to the capillarybed of each organ. Therefore, it would be ideal if thedelivery carriers are able to circulate long enough to reachtheir targets, and their sizes are small enough to passthrough the capillaries but large enough not to slip throughfenestrea.

The lungs, liver and the spleen are the most criticalorgans in terms of particle trapping and sequestration. Theliver has the highest microvasculature number and density,with a size of 10–13 μm in diameter. The endothelial cellsof the sinusoid walls, where liver Kupffer cells attach, havenumerous small pores ranging in size from 100–300 nm(Wisse et al. 1996; Bibby et al. 2005). The Kupffer cellsconstitute approximately 30% of liver sinusoidal cells.Therefore, nanoparticles are likely to be sequestered in theliver sinusoid and phagocytosed by Kupffer cells. Finally,the spleen is most likely the site where intravenouslyinjected particulates are trapped because the microcircula-tion of the spleen is quite complex. The major role of thespleen is to remove damaged or old erythrocytes, patho-gens, and particulates from the circulation. Everyday,approximately 1011 erythrocytes are phagocytized bymacrophages in the red pulp cord. The venous sinuses(sinusoids) are enveloped by a framework of reticular fibersthat lie between the splenic cords. These venous sinuses are100–150 μm wide and are lined with discontinuousendothelium that allows blood cells to re-enter to thecirculation. There are small slits between the endothelialcells, referred to as interendothelial slits, that are approx-imately 4 μm wide, depending on the species (Fujita 1974).Normal erythrocytes, which are 7–12 μm in diameter, areable to squeeze through the interendothelial slits to re-enterthe circulation, while damaged rigid erythrocytes are unableto pass through these narrow slits because of their loss offlexibility (Murakami et al. 1973). Similarly, it is likely thatrigid particles larger than the slits size would be trapped inthe red pulp due to the limited size of the splenicinterendothelial slits and, as a result, would be phagocy-tized by splenic macrophages. Aside from the geometrictrap and phagocytosis mediated by tissue macrophages,intravenously administered particles may encounter addi-tional circulating phagocytic cells, such as monocytes. Forexample, the half-life of systemically circulating amino-modified small particles with sizes between 100 nm and1 μm is only 80–300 s because of monocyte particle uptake(Murakami et al. 1973). Thus, developing a drug deliverystrategy to minimize the contact and recognition of thedelivery carrier by phagocytes and to maximize the timeremaining in the circulation is critical. Polyethylenglycol

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(PEG) provides a shielding “STEALTH®” effect, bydelaying recognition and sequestration by circulatorymonocytes and tissue macrophages. This finding led to adevelopment of Pegylated liposomal doxorubicin that is themost-widely used liposomal doxorubicin formulation inpatients with breast cancer in the US and Europe (Rivera2003). Although therapeutic efficacy of liposomal doxoru-bicin and pegylated liposomal doxorubicin is almost thesame, a substantial difference between these two formula-tions is the half-life, which ranges from approximately 2–4 hto more than 55 h, respectively (Rivera 2003), significantlylowering a risk of cardiotoxicity relative to conventionaldoxorubicin.

4.2 Tumor vascular architecture

The alteration in hemodynamics and vasculature architec-ture has been recognized as essential characteristics ofmetastatic disease. For example, tumor vessels are discon-tinuous and organized in a chaotic fashion lacking thehierarchical branching pattern of normal vascular networks(Less et al. 1991, 1992b; Jain 2003). The vessel diametersare uneven, due in part to the compression of the immaturewall by proliferating tumor cells (Less et al. 1991). As aresult of this abnormal organization and structure of thetumor vessels, blood flow in tumor vessels is, in general,slower and is associated with a characteristic transcapillary“leaking” phenomenon. Most of the blood vessels in theinternal region of tumor are venules, while cells within theperiphery of the tumor are more viable (viable rim) andcontain arteries or arterioles. Therefore, the pressure differ-ences between arterioles and venules in the necrotic coreare extremely low, but are larger in viable rims of the tumor.This heterogeneity in blood flow within the tumor partiallyexplains the uneven drug distribution pattern observedwithin some tumors. Intratumoral injection of therapeuticsmay be one way of bypassing endothelial barriers, since itis associated with an increase in the levels and retention oftherapeutic molecules near the tumor mass while preventingsystemic side effects (Azemar et al. 2003). However,clinical application of intratumoral injections (e.g. genetherapy) has been restricted to cases where the exactlocation of the tumor is known and reachable; unfortunate-ly, many tumors do not fall into this category. Recentprogress in nanotechnology offers significant advancementto tackle this problem.

During disease progression, the expression of receptorson the surface of endothelial cells can be significantlyaltered. This is evidenced by the presence of specificendothelial markers [such as integrins, vascular endothelialgrowth factor receptor 2 (VEGFR2), Delta like 4, andtumor endothelial marker, cellular adhesion molecules,E-selectin] on the surface of tumor vasculature endothelium

(Neri and Bicknell 2005). The differences in the expressionof surface receptor proteins on normal and tumor endothe-lium make the tumor vasculature an alternative drugdelivery target and provide an excellent rationale forvascular targeting strategy and therapeutic exploitation.The third generation vectors idea relies on the biologicalvascular diversity within normal and diseased tissues. Forexample, molecules (such as ligands (Buchler et al. 2007),antibody (Witte et al. 1998), aptamers (Farokhzad et al.2006b; Yang et al. 2006), and synthetic peptides (Pasqualiniet al. 1997; Infanger et al. 2005) that specifically interactwith surface receptors could be used as a tumor vasculatureselective targets, rather than targeting the tumor mass. Sucha targeting strategy can be directed by a surface modifica-tion of the nanoparticles or therapeutic agents to recognizethe surface receptors on the endothelial cells of the tumorvessels (active targeting). One of the most successfulexample of tumor vasculature selective targeting strategyis to target αvβ3 integrins, which are cell adhesionmolecules overexpressed on the actively proliferatingendothelium of the tumor vasculature but not on restingendothelial cells in normal vasculature (Pasqualini et al.1997). Peptides with Arg–Gly–Asp sequence in a cyclicframework were shown to selectively bind to the αvβ3integrin receptors (Pasqualini et al. 1997; Bibby et al. 2005;Infanger et al. 2005). These high-affinity RGD peptideshave been used for the active and selective delivery oftherapeutics, imaging agents, viruses, polymers, liposomesand other gene delivery vehicles to αvβ3-expressing tumorvasculature of multiple different cancer types (Bibby et al.2005; Infanger et al. 2005).

4.3 Tumor interstitial pressure

Interstitial fluid pressure (IFP) is increased in most solidtumors, including breast (Less et al. 1992a; Nathanson andNelson 1994), melanoma, head and neck carcinoma, andcolorectal carcinoma (Heldin et al. 2004). Increased IFPcontributes to decreased transcapillary transport in tumorsand drug retention time in the tumor. Therefore, it presentsan obstacle to treatment, as it leads to a decrease in theuptake of drugs or therapeutic molecules into a tumor. Thetumor IFP is uniform throughout the necrotic core, whereas,at the center of the tumor, it drops steeply toward theperiphery of the tumor mass (Boucher et al. 1990, 1991;Roh et al. 1991). High tumor IFP not only preventstherapeutic agent to reach tumor mass but also pushes suchagents back into the circulation, leading to a reduction ofretention time of therapeutic agents in the tumor. Manyfactors are suggested to involve elevated tumor interstitialpressure. These include blood vessel leakiness, the lack oflymphangiogenesis, interstitial fibrosis, and a contraction ofthe interstitial space mediated by stromal fibroblasts.

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Several studies have indicated that high IFP in the tumorcorrelates with poor prognosis (Nathanson and Nelson1994). In most normal tissues, the IFP is about 0 mmHg,whereas for different types of carcinoma in which it hasbeen measured to date, the mean IFPs vary from 14 to30 mmHg (Stohrer et al. 2000). In patients with invasiveductal carcinomas, the IFP was 29+/−3 (SE) mm Hg, anextremely high value compared to the −0.3+/−0.1 mm Hgin normal breast parenchyma, the 3.6+/−0.8 mm Hg inbenign tumors, the −0.3+/−0.2 mm Hg in noninvasivecarcinomas, and the 0.4+/−0.4 mm Hg in other benignbreast conditions (Nathanson and Nelson 1994). There is adirect correlation between IFP and tumor size (Nathansonand Nelson 1994). Interestingly, the administration ofpaclitaxel decreases the mean IFP by 36% and improvestumor oxygenation by almost 100%. In contrast, theadministration of doxorubicin did not significantly affecteither interstitial pressure or oxygenation (Taghian et al.2005). The use of metal based biocompatible nano-particles (i.e., iron oxide and gold nanoparticles) as atherapeutic modality for remotely controlled thermalablation will hold promise in the development of noveltherapy over conventional chemotherapy. Gold nanoshellswere developed for laser-induced thermal therapy as anew class of nanoparticles with tunable optical absorp-tivities, and systemic injection of gold nanoshell followedby near infrared treatments effectively inhibited tumorgrowth and prolonged tumor free survival in micebearing xenograft tumors (Hirsch et al. 2003; Gobinet al. 2007).

4.4 Endothelial cell barrier on the vessels

Blood vessels are lined with a single layer of endothelialcells that are surrounded by mural cells, pericytes, and acontinuous basement membrane composed of severalextracellular matrix molecules. The endothelial cell liningof the vasculature defines a semi-permeable barrier betweenthe blood and the interstitial spaces of all organs. Thisstructure may present a barrier for both injectable nano-particles as well as small therapeutic molecules such asantibody. During tumorigenesis, blood vessels aroundtumor undergo dramatic morphological changes and theendothelial cells create a large number of fenestrations, withsizes about 200–300 nm and sometimes up to 1,200 nm(Hashizume et al. 2000). For anti-cancer therapy, non-targeted therapeutic molecules and small nanoparticlesreach tumor mass by extravasation through the fenestrationpores (passive targeting of the first and second generationvectors). The vast majority of drug delivery particulatesrely on this morphological change, however, this pore sizeand location of fenestrea change overtime; permeability andpore size of the tumor is affected by the host microenvi-

ronment; permeability of orthotopic tumor is higher thansubcutaneous tumor. Therefore, the development of a drugdelivery strategy that is independent of vessel leakiness iscrucial and this is precisely the aim of the third generationvectors. The permeability of the endothelium lining of thevessels can be enhanced; several vasoactive compounds,such as vascular permeability factor (VPF)/VEGF, havebeen tested pre-clinically and clinically for their abilities toenhance vascular permeability. They include bradykinin,nitric oxide (NO), peroxynitrite (ONOO−), histamine,prostaglandins, collagenases or matrix metalloproteinases,tumor necrosis factor (TNF)α, interferon alpha, and others(Maeda et al. 2003). These compounds can be loaded on a1st stage particle and released simultaneously or individu-ally to enhance locally the transient formation of smallendothelial openings, through which 2nd stage particles canpass and easily reach the extravascular compartment. Forexample, an immunoconjugate with a permeation enhancerwas taken up by tumor cells but was not in normal tissue(Takeda et al. 1999). The pre-treatment of sarcoma andmelanoma patients with the pro-inflammatory cytokineTNFα results in increased perfusion of chemotherapeuticagents into tumors, an effect that is associated withimprovement in overall tumor response rates (Khawli etal. 1994). Thus, incorporating a drug delivery strategy thatenhances vascular permeability only at sites proximal to atumor mass could circumvent this problem. Examples ofhow nanotechnologies can overcome endothelial cellbarriers is a multi-functional delivery, the co-delivery oftherapeutic agents with a penetration enhancer, and the pre-treatment (i.e. prior to the administration of the therapeuticagent) of the endothelium with VPF to open temporarilyintracellular tight junctions to facilitate the therapeuticagents’ reaching the tumor mass.

4.5 Cellular uptake of therapeutic agent

Most of the biologically active compounds and therapeuticagents currently in use for chemo and adjuvant therapysetting are required to act at either the surface receptor ofthe tumor cell, within the cytoplasm or locations within thenuclear component. Contrary to the compounds that exerttheir therapeutic effect through the cell surface or extracel-lular component, the majority of standard chemotherapyagents such as doxorubicin, paclitaxel, and etoposide needto gain entry into the cells to exert their therapeutic effectsthrough an inhibition of macromolecular biosynthesis(Karon et al. 1965), inhibition of microtubule function(Kumar 1981) or induction of DNA damage (Fornari et al.1994). The cell membrane acts as a regulator and defensiveunit to protect the cell from the outside environment bycontrolling the influx and outflow of chemicals, proteins,and other biologically significant compounds permitting the

Biomed Microdevices (2009) 11:49–63 57

cell’s functionality and survival. However, membranescould be additional barriers for drug delivery. Many typesof cells including endothelial cells, fibroblasts, osteoclasts,and pericytes have some phagocytic or pinocytic activity(Henneke and Golenbock 2004). Pinocytosis refers to theuptake of fluids and solutes and is closely related toreceptor-mediated endocytosis. For example, one of theroles of endothelial cells is to transport nutrients from theblood to adjacent tissue, and therefore, possesses highphagocytic nature. Pinocytosis and receptor-mediated en-docytosis share a clathrin-based mechanism and usuallyoccur independently of actin polymerization. By contrast,phagocytosis, the uptake of large particles into cells, occursby an actin-dependent mechanism and is usually indepen-dent of clathrin. Both non-specific binding and surfacereceptor binding events could trigger further receptorrecruitment and surface migration events, to possiblystrengthen the binding (Fig. 4). Up to date, there are noclearly defined and readily available design criteria thatwould provide a method of delivery through the cellmembrane with a high degree of both selectivity andefficiency. Dependent on the host cell’s endocytic pathway,nanoparticles can follow different multistep entry routes.Conjugation of a thiolated trastuzumab antibody against theher2 receptor to nanoparticles comprised of human serumalbumin has been described as a successful way to increaseintracellular uptake by cells overexpressing her2 viareceptor-mediated endocytosis (Steinhauser et al. 2006).Another possible delivery system proposed by nanotech-nology approach takes advantage of a lipid raft-dependentinternalization process called macropinocytosis. By fusing

protein transduction domains (PTDs), such as the HIV-1transactivator protein, it has been shown that it is possibleto deliver a number of different types of cargo andbiologically active, transducible nanoparticles in cell cul-ture and to treat preclinical models of cancer (Snyder et al.2005).

4.6 Tumor heterogeneity

One of the central problems of breast cancer treatment istumor heterogeneity, which includes aberrant expressionand mutation of oncogenes and tumor suppressor genes,which leads to alterations in multiple cellular mechanismssuch as apoptosis, cell cycle control, repair mechanisms,drug resistance, local invasion, and metastasis. There arenumerous studies that point out the heterogeneity of thebreast tumor, and this includes spatial heterogeneity withinthe tumor (Sharifi-Salamatian et al. 2004) and betweentumors from different individuals (Perou et al. 2000). Themost common somatic mutations found in breast tumorlesions are sporadic mutations in HER2 (Slamon et al.1989), p53 (Davidoff et al. 1991), checkpoint kinase 2(CHEK 2) (Bogdanova et al. 2007), phosphatase and tensinhomolog (PTEN) (Li et al. 1997), and germline mutation inbreast cancer (BRCA)1 and BRCA2 is another examples ofthose(Welcsh and King 2001). These mutations ultimatelylead to uncontrolled cell proliferation and also to differentresponses to therapy. Thus, current cancer therapies rely oncombined approaches that simultaneously modulate multi-ple pathways. Statistically, the use of adjuvant polychemo-therapy has been proven to reduce the breast cancer death

Fig. 4 Cellular uptake mecha-nisms: uptake of particulates byendocytosis can be divided intothe clathrin-mediated and cla-thrin independent endocytosis.Each of endocytic pathways isalso defined by a specific size ofthe engulfed soluble or particu-lates. (1) Particulates with thesize up to 200 nm are endocy-tosed through clathrin coatedpits in the membrane (classicreceptor mediated endocytosis).(2) Caveolae are flask shapedinvaginations of the plasmamembrane with a diameter of50–80 nm. (3) Endocytosis ofbacteria and large size of partic-ulates larger than 0.5 μm occurmainly via macropinocytosisand phagocytosis

58 Biomed Microdevices (2009) 11:49–63

rate by about 38% in women under the age of 50 (2005).For example, the recurrence of breast cancer in ER+patients treated with a combination of polychemotherapyand tamoxifen is lower (14%) than that in patients treatedwith tamoxifen alone (21.6%) (2005). Although combina-tion therapy has a demonstrated significant impact onpatient survival, this approach remains challenging inadvanced disease because increased toxicity associatedwith this modality. Therefore, the co-delivery of multipletherapeutic molecules and compounds to the same locationpromises to significantly improve current therapeuticeffects, evaluation of therapeutic responses, and patientquality of life. In order to reduce such toxicity, the use ofreduced combined doses of chronomodulated administra-tion has been investigated but continues to be challenging.As demands for personalized medicine are widely realizedto overcome tumor heterogeneity, nanotechnology baseddrug delivery will offer the perfect platform for personal-ized polychemotherapy and simultaneously modulate mul-tiple pathways which contribute tumor survival.

5 Discussion

Nanotechnology has already provided significant break-throughs and advantages in several areas of medicine asdiscussed, and both are non-targeted nanotechnology-basedtherapeutics for breast cancer treatment (Doxil® andAbraxane®). While basic and clinical science have revealedand identified multiple problems that cause a reduction oftherapeutic efficacy of systemic chemo and immunotherapyfor breast cancer, numerous new nanotechnology-baseddrug delivery platforms have been tested to address theseunmet clinical problems. Though nanomedicine holds greatpromise, there are still multiple challenges in order to bring

this novel technology to the clinic (Sanhai et al. 2008). Inparticular, controlling the biodistribution of nanoparticu-lates in vivo and the avoidance of biological barriers are twoof the most important challenges. We believe that the thirdgeneration of particulate systems can help in addressingthese challenges. The main advantage of these over theprevious generations relies on their modularity: each stageis dedicated to a specific function and can be rationallydesigned to execute that specific function with superiorperformances. For a multi-stage third generation particu-late, the 1st stage particulate is designed to navigate into thecirculatory system, avoid or limit the recognition from thecells of the immune system and accumulate with higherpercentage in the organs of interest; whereas the 2nd stageparticulates, loaded within the 1st stage, are designed todiffuse within the organ of interest, interact specificallywith the target cells and release their payload. Clearly thefunctions of the two particulates are different and theirgeometrical and physico-chemical properties should bedifferent so that the 1st stage could be optimally designedfor vascular targeting, whereas the 2nd stage would beoptimally designed for extravascular targeting. Obviouslythe whole delivery process can be broken down into moresteps (specific functions), meaning more stages, leading tofully multiple stage particulate systems. The work ofDecuzzi and Ferrari over the past years has shown howthe behavior of particulate systems can be fine tuned notonly by tailoring their surface physico-chemical properties(decoration with ligand molecules; polymeric coating withPEG) but also controlling their geometrical properties, assize and shape. These three engineering parameters (size,shape and physico-chemistry) play a crucial role inparticulate (i) transport within the circulation and in thetissue; (ii) recognition of vascular and extravascular targets;(iii) interaction with target cells and cells of the immune

Fig. 5 Rational design of nano-vector: A design map will aid toidentify the ideal nanoparticlessize and density of surfaceligands from three parameters;margination, specific adhesion,and endocytosis

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system; and can be tailored during the fabrication andsynthesis process with great accuracy. Particles with non-spherical shapes have been shown to drift laterally towardsthe vessel walls in capillary flows, mimicking the behaviorof platelets (Decuzzi et al. 2005; Gentile et al. 2008), andby doing so the likelihood of recognition of specificbiological targets in the vasculature can be significantlyincreased. Non-spherical particles have been shown toadhere more strongly to the vessel walls under flow, andin particular for oblate spheroidal particles it has beenestimated an increased of about 50 times in the deliverablepayload compared to classical spherical particles with thesame strength of adhesion (Decuzzi and Ferrari 2006). Non-spherical particles have been also shown to resist moreinternalization (Decuzzi et al. 2008), so that can adhere tocells of the vessel wall without being internalized whilereleasing their payloads. A fine tuning between size, shapeand surface physico-chemical properties can lead to aprecise control of the particulate behavior in terms ofmargination dynamics, vascular adhesion and internaliza-tion (Decuzzi and Ferrari 2008), and mathematical model-ing can lead to define Design Maps, as that given in Fig. 5,which can help predict particle behavior and drive particledevelopment. These study clearly suggests that the geom-etry of the delivery carriers is one of critical determinant fortheir behavior in the circulation (Ferrari 2008b).

In conclusion, it is evident that anti-cancer therapycertainly needs a breakthrough to eradicate cancer relateddeath. Nanotechnology is one of the growing fields inmedical science with a promise to address long standingclinical issues. There are an overwhelming number ofdistinct nanoparticles that have been developed which varywith respect to many properties, such as particle size,shape, charge, surface modification, and drug payload/therapeutic effect. The future challenges in the successfulclinical applications of nanotechnology based drug deliveryare not the lack of novel technologies, it is rather the needto identify favorable physio-chemical properties that willallow injectable nanovectors to overcome multiple barriers.

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