Nanoparticles targeting mechanisms in cancer therapy

Review

Nanoparticles targeting mechanisms in cancer therapy; great efforts, less implementations

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It has been more than one century since Paul Ehrlich spoke about the idea of targeting specific molecules in the cell when he coined the ‘Magic Bullet’ principle. In most occasions, we seek new pharmacodynamic models for therapy, but nanoparticles provide a chance to modify the already existing pharmacokinetics of drugs to meet needed pharmacodynamic models. In the scope of ‘nanoscale’, every entity has different characters, no general rules control pharmacokinetics of nanoparticulate drugs as new physical and physicochemical properties are added to equations. However, such remarkable drug models are still quite far from achieving their potential in clinical application. Among the major obstacles is that most available results in nanoparticles targeting rely upon in vitro and animal models that do not match the tumor environment characteristics in humans. This Review discusses the concept of targeting tumor cells with nanoparticles, the limitations that lead to its incomplete application in clinical practice along with some of the promising solutions to such limitations. Indeed, only a few advances in nanomedicine have translated into clinical practice. To date, there are few clinically approved nanoparticle chemotherapeutics for cancer, for example, doxil, abraxane and oncaspar, and the rest are under clinical investigation [6,7] . We will discuss the concept of targeting and some of the methods used to fabricate new NP drugs such as functionalization, paramagnetism and magnetic targeting.

10.4155/TDE.13.75 © 2013 Future Science Ltd

Ther. Deliv. (2013) 4(9), 1–13

Kyrillus S Shohdy*1 & Ahmad Samir Alfaar2 1 Faculty of Medicine, Cairo University, Cairo University Hospitals, AlSaray Street, Al-Maniel, 11451, Cairo, Egypt 2 Research Department, Children’s Cancer Hospital, CCHE 57357, Egypt. *Author for correspondence: Tel.: +20 122 996 1016 [email protected]

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Many nanoparticles (NPs) are developed on the basis they will be nanocarriers for assemblies of functional molecules to overcome problems of bioavailability, biologic barriers and specific targeting of tumor cells [1] . Nanoparticles are colloidal particles approximately 10–1000 nm in size, with a size range of 1–100 nm in at least one dimension, generally made of polymers but a few exceptions are found, the used polymers may be either artificial or biopolymers [2] . In most cases, NPs serve as carriers for other therapeutic molecules, recently it has been elucidated that some NPs have a therapeutic effect themselves, a striking example of this are HDLs, which are basically dynamic natural NPs and play a major role in cholesterol transport, and as cancer cells need more cholesterol due to their high rate of proliferation, HDL-NPs can function to efflux cholesterol from such cells thereby depriving them of cholesterol and, hence making them starve to death [3] . NPs are preferable carrier systems for chemotherapeutic drugs, because they specifically target cancer cells, enhance efficacy and reduce systemic toxicity [4] , in regard to other colloidal drug systems, NPs are more stable and keep considerable stability in body fluids after different modes of administration [2] . In addition, they have unique physicochemical characteristics that can be modified accordingly [5] , but this Review excludes discussion of those characteristics, rather we focus on limitations produced from their modification.

Targeting Nowadays, laboratory research in the field of cancer tries to discover specific and well-defined targets or biologic pathways, and finding appropriate drug models that can act on them to cause shutdown of malignant process [8] . Simply, targeting is how to use the drugdelivery system (DDS) to fulfill the following four criteria [9,10] : n Retain: efficient drug loading, allowing sufficient time of drug in circulation to reach its specific site; Evade: minimal immunogenicity of the DDS;

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Target: accurate drug delivery up to cellular and subcellular levels;

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Release: timely, controlled unloading of DDS cargo. Many modalities are nowadays developed to get full use of targeting in cancer therapy. However, optimization of nano-DDS should consider the previous four criteria equally because

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ISSN 2041-5990

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Review | Shohdy & Alfaar

polymeric drug-delivery system with a size range between 10 and 100 nm, they have high loading capacity and specific targeting capabilities.

Targeting: Using a therapeutic agent that can specifically reach a tissue to cellular and subcellular levels, overcoming the various biological barriers.

Functionalization: The

ability to modify the surface of a nanocarrier with a tissuerecognition ligand.

Enhanced permeation and retention effect:

enhanced permeation and retention effect

(EPR). We will discuss the latter in more detail. Enhanced

permeation & retention effect EPR was described first by Matsumura and Maeda [12] . Rapid growth of tumor increases its demand for blood supply. A tumor volume of over 2 mm 3 limits the diffusion of nutrients into the tumor. The tumor overcomes that through angiogenesis [13] , leading to forming neovasculature with leaky reduced structure and a discontinuous endothelium with large fenestrations of 200–780 nm [14] . Conventional chemotherapeutic agents are of small size, so they accumulate in both normal and cancerous tissues via free diffusion-dependant equilibrium [15] . But they also immediately disappear from the tumor by back-diffusion. Moreover, the small size of the conventional agents reduces their half-life in mouse models to about 20 min, violating an essential requirement of enhanced permeation. On the other hand, large-sized NPs facilitate targeting tumors rather than normal tissues as most normal tissues have tight intracellular junctions less than 10 nm (Table 1) [16] . Moreover, large molecular size NPs will evade renal

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Sequestration of nanoparticles in tumor tissue due to its vasculture leakiness and impairment of lymphatic drainage.

Passive targeting Cellular uptake of NPs occurs through nonspecific endocytosis, it depends mainly on physicochemical properties of NPs e.g. surface, functionalization and charge [11] . Mechanisms of passive targeting are simple diffusion and

filtration and clearance, hence prolonging their plasma half-life and achieving more accumulation in the tumor, helping EPR take place [6] . Additional retention occurs as the clearance of NPs is delayed due to the compromised lymphatic system in tumors. which gives sufficient time for NPs to disintegrate and release their cargo [1] . Maeda applied this concept on SMANCS, which is poly(styrene-co-maleic acid/half-nbutyl-ester [SMA]) conjugated with neocarzinostatin, as the anticancer neocarzinostatin, size of 16 kDa, but it did not show an EPR effect. When it was conjugated to synthetic polymer SMA and bound to albumin it reached a molecular size of nearly 80 kDa [15] and EPR was observed. SMANCS was approved in Japan in 1993 for hepatocellular carcinoma [17] . It is obvious that what matters for the EPR effect is the size. The core principle in achieving that is to find a suitable polymer or any carrier that could be coupled to the drug either via a degradable linker or with certain release mechanisms depending on the tumor microenvironment [18] . The physicochemical nature of NPs implies the occurrence of the EPR effect whether it is intended or not. Further studies should be conducted to compare between endothelium of tumor vasculature and healthy vasculature under radiation or heat to evaluate the impact of traumatization and the benefit of inducing an artificial EPR effect in healthy tissue, as the vasculature in early neoplastic lesions may be more restrictive with properties similar to healthy tissue [19] .

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Nanoparticles: Colloidal

any limitations in one criterion will cause the inevitable failure of DDS. Another failure facing the DDS is that tumor progression depends on widescale transduction pathways; hence, targeting a single molecular target or antigen will not achieve complete remission, moreover, cancer is genetically unstable and various mechanisms of resistance are continuously evolving [8] .

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Key Terms

Table 1. Fenestrations size in different tissues indicating the basis for enhanced permeation and retention effect. Tissue type Tumor Others organ

Inflamed organs

Fenestration size Implanted tumor in mice Intravenously inoculated brain tumor in rat Kidney (in guinea pig, rabbit, rat) Liver in mice Spleen in mice Lung in dog In hamster

200–780 nm 100–380 nm 20–30 nm 150 nm 150 nm 1–40 nm 80 nm–1.6 µm

Adapted from [14].

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Limitations

of passive targeting Although tumors’ defective vasculature allows enhanced permeation, the very reason causing the formation of such defective vasculature is that tumors suffer ischemia and low perfusion in the first place [20] . Such defective perfusion reduces delivery of blood-borne compounds to the tumor environment. Thick fibrosis and low blood vessel density are among the reasons for such phenomenon in some tumor tissues [21] . Moreover, the dysfunctional lymphatics responsible for retention also leads to high interstitial pressure, which counteracts the diffusion of drugs from blood vessels into the tumor [13] . Another limiting factor is the size of NPs, the optimal size ranges mainly between 10 and 200 nm, those lesser are cleared by the kidneys and those larger often accumulate in extracellular space and fail to reach the tumor site [22] . The future science group


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targeting (pathotropic, synaphic) NPs are grafted with ligands that can be recognized by receptors on the cell surface, this ensues receptor-mediated endocytosis [1] . Common ligands used nowadays include transferrin [25] , folate [26] , peptides (e.g., arginine–glycine– aspartic acid) [27] , antibodies or antibody fragments [28] . Receptors here are either uniquely expressed or overexpressed in tumor cells. For instance, the highly dividing tumor cells need more transferrin and folate, so overexpression of these receptors occurs specifically on tumor cells rather than normal cells [29] . Another approach to active targeting is to modify the drug pharmacokinetics and release profile. Sahoo and Labhasetwar found that paclitaxelloaded NPs show higher efficacy in breast cancer cell lines when they are conjugated to transferrin (Tf ) ligand, due to increase of cellular uptake and reduced exocytosis as opposed to the case with unconjugated NPs [30] . The uptake of Tf-conjugated NPs is done via Tf receptors while uptake of nonconjugated NPs is through a nonspecific endocytic pathway. Tf receptors (TfRs), as they deliver iron into cells, cycle into acidic endosomes and are recycled back to cell surface [25] . Thus, TfRs can be targeted in two ways: for delivery of therapeutic agents (enhance agonism) or by blocking their function causing cell death (enhance antagonism). Moreover, TfRs expression is proportional to tumor progression [25] . According to Sahoo, Ma and Labhasetwar animal studies in which mice received a single-dose intratumoral injection of NPs-Tx-Tf (Taxol® [Tx] dose = 4 mg/kg) demonstrated complete tumor regression and greater survival rate than those mice that received either NPs-Tx or paclitaxelcremophor EL formulation (conventionally used paclitaxel formulation) [31] .

Heterogeneity of cancer cells

The use of NPs conjugated to one type of ligand is limited by heterogeneous expression of the receptor itself from one cancer cell surface to another in the same tumor, which implies the risk of residual disease [37] . Also, the microenvironment of tumors modulates such heterogeneity, especially with hypoxia [38] . One recent interesting study by Bae et al. succeeded in preparing a human serum albumin-based nanoparticle system (HAS-NP) with three cytotoxic mechanisms added [39] : doxorubicin, which is easily loaded in HSA-NPs; tumor-related apoptosis-inducing ligand (TRAIL) – one of the TNF receptor superfamily, it was anchored to surface amines on HSA-NPs; and Tf, which was added to overcome multidrug-resistant tumor cells efflux, via its receptor-mediated endocytosis system in addition to it being highly expressed in MCF-7 cells. They reported that molecular imaging detected a markedly enhanced vasculature in most targeted tumor tissue, in HCT 116-xenografted hairless nu/nu mouse. More proof-ofconcept studies are needed to assess in vivo antitumor efficacy. The only remarkable results are the cytotoxic effect of the TR AIL/Tf/Dox/HSANPs on the MCF-7/ADR cell lines (IC50: 5.2 µg/ml), which are characterized by doxorubicin resistance and low folate receptor

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Active

availability and their affinity. When receptors reach saturation, any excess targeting molecules will be cleared from the body like any nontargeted molecules [20] . On the other side, when targeted nanomedicines have a very high affinity for the tumor cells, they are getting trapped once they extravasate in the part of the tumor surrounding the capillaries, consequently they do not diffuse to the entirety of the tumor, especially large solid tumors, so recurrence of cancer is more likely even with considerable tumor volume reduction with the treatment [32] . This phenomenon was first pointed out by Weinstein and Fujimori and called the ‘binding site barrier’ hypothesis [33,34] . Interesting experimental data proving occurrence of this phenomenon have been introduced based on antitumor medicines such as monoclonal antibodies and other biological ligands [35,36] . The field of nanotechnology needs such experiments to be conducted after conjugation of antitumor drugs with NPs, and the differences compared.

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vascular fenestrations vary from each patient to another, each tumor type to another, even over time during treatment of the same tumor, so developing size-specific targeting modalities will be difficult [23] . Most approved nanotherapeutics depend mainly on passive targeting, yet the EPR effect does not achieve a high level of drug concentration in the tumor site [24] .

| Review

Limitations

of active targeting

Capacity of targeted receptors

The number of targeting molecules, which bind specifically at the tumor site, is determined by the number of cell surface receptors, their future science group

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Review | Shohdy & Alfaar Therapeutic Delivery © Future Science Group (2013)

Limitations Mechanisms

TRAIL Tumor capsule

HCT 116

Pericyte

TRAIL-induced apoptosis

NPs

TRAIL

at str e fen

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TfR Transferrinmediated endocytosis

EPR effect

CAPAN-1

Both TfR & TRAIL

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Heterogeneity of tumor cells

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High cellular density

TfR

Normal cells

Fibroblasts

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TRAIL

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MCF-7/ADR

Normal-sized fenestrations

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Blood vessel

Low cellular density

High IFP

Normal IFP

Tumor cells

Figure 1. Nanoparticles targeting mechanisms and some of their limitations, also indicating heterogeneity of three tumor cell lines. EPR: Enhanced permeation and retention effect; IFP: Interstitial fluid pressure; NP: Nanoparticle; TfR: Transferrin receptor; TRAIL: Tumor related apoptosis-inducing ligand. Based on results from [39] .

Key Term Controlled release: Spatial

and temporal control over the drug-release profile in tumor tissue.

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expression (Figure 1) . Another internalization mechanism besides Tf is the human epidermal growth factor receptor-2 (HER2), a monoclonal antibody (mAb) and a well-known tumortargeting marker, which is overexpressed in the MCF7 cell line. Humanized anti-HER2specific antibody trastuzumab was conjugated to HSA-NPs by Wartlick et al., which resulted in effective internalization into these cells via receptor-mediated endocytosis [40,41] . The possibility of coupling the two mechanisms together on HSA-NPs needs to be addressed. In that regard, in vivo studies in HER2 + breast tumor have shown that multimodal NPs conjugated to HER2 antibodies preferentially accumulate in this tumor [42] . Furthermore, Bae et al.’s model Ther. Deliv. (2013) 4(9)

(TRAIL/Tf/Dox/HSA-NPs) provides inspiration for studies on fabrication of Tf/folate gold NPs to get full use of the photothermal effect of AuNP and endocytosis pathway of TfR on such drug-resistant tumor cell lines [39] . Overexpression in tumor tissue versus whole body

Some literature question the futility of targeting the overexpressed receptors in tumor cells on the basis that most of the studies conducted on animal tumors disregard the fact that even though the number of receptors per nontarget cell is relatively low, the total mass of the nontarget receptors is still far bigger than their tumor’s mass, which limits the accessibility of the carrier future science group


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release The jewel in the crown of targeting mechanisms is the efficient release of therapeutic agents. It is either through maintained release over time or through triggering the release by stimulus unique to delivery site. The therapeutic agents are encapsulated in NPs to improve their solubility and stability. These encapsulated molecules can be released over time to achieve concentration within therapeutic window [44] . Triggering the release of drugs from NPs (also known as smart drug delivery system) is either based on internal stimuli governed by naturally occurring biological events or is tumor induced. Examples include pH, enzymes, redox potentials and external stimuli including light, magnetic fields and ultrasound, which provide spatio-temporal control over the release [45,46] . Further information on the subject can be found in the Advanced Drug Delivery Reviews journal’s themed issue on ‘Stimuli-Responsive Drug Delivery Systems’ [47,48] . The concept of controlled release will not be fully exploited unless we can control the location

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Controlled

and timing of drug release simultaneously (spatiotemporal availability of the anticancer drug). Gullotti and Yeo indicate that difference between location and timed of drug release really matters [49] . They conjugate poly(lactic-co-glycolic acid [PLGA]) NPs to a cell-penetrating peptide, TAT, to increase intracellular delivery of paclitaxel to non-drug-resistant cells. Drug-release kinetics were further investigated in phosphate-buffered saline containing Tween 80. Efficient uptake of paclitaxel/PLGA-TAT NPs was observed, yet no increase in killing of multidrug-resistant cells occurred. Table 2 summarizes the results of the study. The kinetics of paclitaxel release from the NPs varied with the release medium. Further studies are needed to put proper distinction between in vitro and in vivo discrepancies. Figure 2 indicates results of two studies assessing the in vitro release profiles of 9-nitrocamptothecin (9-NC)-loaded PLGA NPs [50] and PLGA-PEG-NPs loading 9-NC [51] , respectively. Both studies use phosphate-buffered saline, the point to be investigated is that the release profiles are varying totally and simple buffered saline as phosphate-buffered saline does not reflect the complexity of biological environments. The study may also apply some of the approaches proposed by Adams to improve anticancer development [52] :

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to receptors on target cells [9] . This is reflected in the fact that the volume of distribution of the carrier to nontarget cells is higher than that with respect to the tumor tissues. However, some molecules have the ability to induce more overexpression in tumor cells for certain receptors. For instance, overexpression of FOLR2 (a-FRs), one of the folate receptors and a promising target in myeloid leukemia, can be induced by the all-trans retinoic acid ATRA effectively via its differentiation-promoting effects [26] . It is noteworthy that FOLR1 is expressed more abundantly on cancer cells than FOLR2 and that the latter prefers to interact with the reduced form of folic acid [43] .

| Review

Employ preclinical cell-culture models at physiological tumor pH (6.5–7.0) and oxygen (0–5%) instead of pH 7.4 and ambient O2 (21%);

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Screen for antitumor activity against tumor stem cells instead of tumor bulk;

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Measure tumor pharmacokinetics directly for drug or drug carrier; apply miniaturized, implantable imaging technology as plasma

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Table 2. Summary of Gullotti and Yeo study indicating importance of location and timing of drug release. Drug system

SKOV-3 cells (paclitaxel sensitive) NCI/ADR-RES cells (multidrug resistant)

Overall cytotoxicity are the same Free PTX PTX/PLGA NPs PTX/PLGA-TAT NPs Explanation

Location of drug source (extra vs intracellular) makes little difference

Most of the encapsulated PTX had been released extracellularly before the majority of PLGA–TAT NPs were taken up by the cells

NP: Nanoparticle; PLGA: Poly(lactic-co-glycolic) acid; PTX: Paclitaxel; RES: Reticulendothelial system; TAT: trans-activating transcriptor. Data taken from [49] future science group


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Review | Shohdy & Alfaar High interstitial fluid pressure within tumor stroma due to lack of functional lymphatics and high permeability of vessels, whilst in normal tissues interstitial fluid pressure is around 0 mmHg [54] ;

n

80 70 60 50 40

PLGA-PEG-NP PLGA-NP

10 0

Various cell layers between endothelium and tumor for example, pericyte, smooth mucle cell and fibroblast.

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30 20

0

20

40

60

80

100

120

140

Time (h)

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Figure 2. 9-NC release curve from optimum formulation of poly(lactic-coglycolic acid-PEG and poly(lactic-co-glycolic acid nanoparticles. The results from studies in BPS medium need to be correlated with others performed in biological medium similar to the cancer microennvironment. Reproduced with permission from Derakhshandeh. 9-NC; 9-nitrocamptothecin; NP: Nanoparticle; PLGA: Poly(lactic-co-glycolic acid.

It is evident that the cancer-associated fibroblast is the most prominent cell type within the tumor stroma of many cancers, for example, breast and pancreatic carcinoma [55] . It is also noteworthy that these cells can be actively targeted, for instance, pericytes lack expression of a-smooth muscle actin; however, tumor pericytes express this marker [55] . A new trend is investigated nowadays for using tumor stroma itself as a target, so-called cancer stromal targeting therapy [56] .

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Release of 9-NC (%)

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Treat patients based on their unique tumor physiology (e.g., level of hypoxia) and pharmacogenomics; utilize therapeutic drug monitoring for pharmacokinetically guided dose adjustment.

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of targeting in brief Active and passive targeting are not two different modalities occurring separately, but they occur quite sequentially, as the EPR effect brings the drug into tumor tissue as a whole and the ligand–receptor interactions bring it into the cells. Moreover, a third modality also occurs when the drug reaches intracellular targeting. The activation mechanisms either use a default pathway for delivery to lysosomes or they avoid that [9] . But this sequence faces some challenges between the step of passive targeting (i.e., EPR) and the step of active targeting (i.e., binding site on the tumor cell). It is not an easy way between the leaky vasculture that mediates passive targeting and the tumor cell wall that mediates active targeting. There are many anatomical and physiological barriers inbetween and recent studies largely neglect them [53] . Herein, some of these barriers that impede the proper coupling of passive and active targeting are enumerated:

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High cellular densit y within solid malignancies;

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In vivo

tumor models The success of development of nanomedicines preclinically depends on availability of in vivo tumor models that can mimic the real human tumor environment [57] . A study was conducted by Zhang et al. to prove that pericyte coverage of human tumor vasculture is an important factor in determining the accumulation of NPs in the tumor [58] . They found remarkable differences between animal models and human histopathology specimens taken from various tumors, especially pancreatic carcinoma. Therefore, experimental models that are more relevant to human tumors need to be established on the basis of human tumor vascular characteristics, as illustrated in Figure 1. The preclinical experiments use two types of animal xenograft models – ectopic and orthotopic tumors. In the first, tumor cells are introduced as subcutaneous injections, regardless of their native tissue type, but in the second the cells are injected into an environment relative to their native tissue type, a common example for this is breast cancer cells injected into mammary tissue of animals; thus, the cancer cells are subject to the as much as possible the same environment as the actual human tumor is subject to. Both models face many challenges, for example, their histology show many discrepancies with the human tumors, in particular the stroma, which governs important tumor microenvironment features such as high interstitial pressure. Moreover, murine tumor models lack tumor heterogeneity (see ‘Heterogeneity

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pharmacokinetics produce some artefacts, and mainly pass unnoticed, except in a few cases like in results of study summarized in Table 2 ;

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future science group

Nanoparticles targeting mechanisms in cancer therapy; great efforts, less implementations Ectopic xenograft

Advantages –Simplicity of implantation –Obvious tumor volume measurement

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Orthotopic xenograft

Disadvantages –Slower rate of development –Unlikely to simulate the in situ tumor phenotype

Advantages –Microenvrionment more closely simulate the in situ tumor –Allows for rapid screening –Greater permeability (needed for EPR effect)

Disadvantages –Difficult implantation –Lower perfusion –Lack of tumor cell heterogeneity –Resemblance of cellular not stromal components

Figure 3. Advantages and disadvantages of both ectopic and orthotopic models. Data taken from [60,61,90] .

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of such DDS in vitro, then they examined it in more clinically relevant models with addition of fetal bovine serum at one time and human serum at another time, there was a decrease in overall uptake of the NPs [63] . They postulated that protein corona is formed from the added serum around the NPs and that this shields the Tf and prevents it from binding either to its targeted receptors on cells or to soluble Tf receptors.

Which way to go? Many opposite approaches are arising in the field of nano-oncology. For example, some suggest preserving the leaky vasculature of tumors for EPR effect while others recommend to ameliorate the defects to enhance the perfusion, hence extravasation of polymeric NPS to tumors [64] . Analogously, promoting angiogenesis helps obtain full use of the EPR effect, while developing anti-angiogenesis approaches enhances drug perfusion and release profile. Examples of the latter approach include NPs targeting the VEGF receptors (VEGFRs), avb3 integrins and matrix metalloproteinase (MMP) receptors [13] . The moieties added to NPs – to achieve better targeting to tumor site – increase difficulty in mass transport across biological barriers. That is, the increased cross-section of the NPs decreases the benefit of the EPR effect. It is simply a compromise between active or passive targeting [23] . Currently, most approved nanomaterials for oncology rely mainly on passive targeting. Furthermore, targeting is not only used for therapy, but also for diagnostic imaging, socalled ‘theranostics’, an approach that would not have been achieved if not for the versatile nature of NPs. Many schemes are emerging for developing various targeting systems for better drug delivery. Table 3 outlines those schemes with some examples based on experimental data. Some of the

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of cancer cells’) [59] ; Figure 3 summarizes the disadvantages of both models. Ho et al., conducted an interesting study to compare between two xenograft models of breast cancer, where the tumor cells were injected to non-obese diabetic/severe combined immuno deficiency gamma mice either orthotopically (mammary fat pad [MFP]) or ectopically (subcutaneous [sc.]) [60] . When high molecular weight dextran was injected as a model nanocarrier, it showed higher accumulation in MFP tumors 3 weeks after injection, this proves that the MFP has a greater permeability to macromolecules – the same feature contributed to the EPR effect. In addition, greater vascular density and size are detected by immunostaining in MFP tumors collected 3 weeks after implantation, but in comparison to the liver as a positive control in this study, both models showed poor dextran accumulation, this poor tumor perfusion can be attributed to pathological features of tumors such as low vascular density and small blood vessel size. Impact

of serum proteins on NPs During their transport in circulation, NPs have to interact with a multitude of plasma proteins, amongst those are albumin and transferrin [61] . These serum proteins are employed themselves in a drug-delivery system, but recently it has been detected that they induce some constraints against some nanoparticulate delivery systems. It is found that the targeting ability of some functionalized NPs in vitro may totally change in vivo due to interaction with proteins in biological media, where proteins bind to the surface of NPs and form the so-called protein corona [62] . A recent study discussing this limitation was conducted by Salvati et al., where PEGylated fluorescent silica NPs were conjugated to human Tf with a thiol–PEG linker (SiO2–PEG8–Tf). The researchers examined the targeting efficiency future science group


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Review | Shohdy & Alfaar Table 3. Scheme for developing nanoparticulate targeting systems. Scheme

Example

Drug model

Remarks

1. Targeted molecules on targeted cells 2. Ligand

Transferrin receptors

NPs-Tx-Tf

–

[30]

Trastuzumab Anti-HER2 receptors Albumin-based NPs

Trastuzumab/HAS-NPs

mAb for receptor-mediated endocytosis

[91]

3. Appropriate NPs carrier 4. Synergistic molecules

5. Limitations

TRAIL and Dox

Ref.

[92]

Dox/folate conjugated albumin NPs TRAIL/Tf/Dox/HAS-NPs

A combination of Dox and TRAIL at concentrations of only 1 ng/ml and 1 µg/ml reduced CAPAN-1 cell viability to below 40%; comparable to dox control solution

[39]

[93]

HAS-NPs proliferative Dox-loaded HAS-NPs effect

was uptake of drug by RES [69] . This limitation was overcome through a hydrophilic polymer PEG, as PEGylation of the nanoparticle surface prevents opsonization (proteins adsorbed to surface of NPs), hence the uptake by RES [70] . Also, PEG improves colloidal stability as it forms a hydrophilic layer on the surface of NPs [1] . Moreover, the PEG steric hydrophobic barrier impedes action of enzymes on NPs, delaying their digestion [40] . This phenomenon was termed ‘Stealth®’ liposomes by Frank Martin of LTI, which means unseen or unrecognized by the RES. It was the basis for stealth cisplatin. Cisplatin failed to get approval as it did not enable drug bioavailability to the tumor cells. On the other hand, Doxil overcomes that through collapse or partial collapse of the ammonium sulfate gradient and/ or phospholipases that hydrolyze the liposomal phospholipids and cause destabilization [69] . PEG makes NPs stealth from the RES, yet it also makes them stealth from tumor cells, a pitfall that could be addressed by adding active targeting systems to PEGylated NPs [23] . Interestingly, the converse of the PEGylation stealth phenomenon has recently been experimented with using the phenomenon of uptake of NPs by macrophages of the RES as DDS. Fidler et al. report that the macrophages originating from blood monocytes, despite an intact blood– brain barrier, can actually infiltrate experimental brain metastasis [71] . The bottom line is that modification of NPs surface enhances passive targeting, on one hand through PEG stealth phenomenon, and on the other hand because ligands for active targeting are often grafted at NPs surface via a linkage on PEG chains [72] .

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common approaches used for efficient targeting are surface modification and paramagnetism, which will be discussed in detail.

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Dox: Doxorubicin; HAS: Human serum albumin; mAb: Monoclonal antibody; NPs: Nanoparticles; Tf: Transferrin; TRAIL: Tumor-related apoptosis-inducing ligand.

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Modification of surface properties of NPs (functionalization) A surface-modifying ligand is conjugated to the NPs, either via covalent bond formation between that ligand and a functional group on NPs surface, for example, a carboxylic and an amino group on albumin-base nanoparticle, or via surface coating of NPs with certain polymers such as PEG, poloxamines and polysaccharides [40] . The surface modification by organic molecules serves many tasks [65] : improving physicochemical properties such as stabilizing the NPs in a biological suspension with a pH around 7.4 and a high salt concentration preventing formation of aggregation, controlling chemical reactions, and supporting well-dispersed size and its uniformity [66] ; providing functional groups at the surface increasing the possibilities of conjugation with more ligands (derivatization); and finally, avoiding immediate uptake by the reticulendothelial system (RES) mainly by means of repulsion of plasma proteins through the formation of a dynamic cloud of hydrophilic and neutral chains at the NPs surface [67] . Liposomes were the first controlled-release DDS to be modified with PEG and used later to develop doxil, which is an anionic vesicle delivery system encapsulating doxorubicin, and the first liposomal nanodrug to be approved by the US FDA (in 1995) [68] . Two failures led to doxil success; the first generation of liposomal doxorubicin was dependent on medium-size oligolamellar liposomes composed of two low (gel to fluid transition temperature) Tm (fluid) phospholipids and cholesterol. The main limitation

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Superparamagnetism & magnetic targeting Association of drugs to superparamagnetic NPs, essentially iron oxide, is a promising technology for drug targeting through guidance by means of an external magnetic field [74] . The relation between magnetic field strength, H (which is equivalent to the external magnetic field) and magnetization, M, is linear as shown by the following equation [75] :

For instance, gold-coated silica nanoshells are used to target human breast cancer-derived SKBR3 cells. Illuminating the nanoshell-treated cells with a diode laser can induce thermal destruction of the targeted cells [79] . Besides, in a single-arm study conducted by Maier-Hauff et al. on patients with recurrent glioblastoma multiforme, the study concluded that thermotherapy using magnetic NPs in conjunction with a reduced radiation dose is safe and effective and leads to longer overall survival following diagnosis of first tumor recurrence compared with conventional therapies in the treatment of recurrent glioblastoma [80] . Magnetic targeting depends on creating magnetic field gradients that attract the NPs to localized sites. However, this gradient undergoes fast decay away from the magnet, which, in turn, limits magnetic targeting to superficial tissues [81,82] . To overcome this, Fu et al. implemented a novel approach using a magnetic micromesh and biocompatible fluorescent magnetic NPs to enhance magnetic targeting of systemically administered individual fluorescent magnetic NPs containing a single 8 nm superparamagnetic iron oxide core in a human glioblastoma mouse model [83] . The magnetizable embedded micromesh produces very strong magnetic field gradients attracting individual magnetic NPs to multiple locations of the mesh. The importance of this study lies in using two magnetic entities to overcome the spatial magnetic distribution barrier, something we need to seriously consider to address the limitations of magnetic targeting.

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modification dictates change in multiple physicochemical parameters Direct modification of the NP system to take advantage of a specific physicochemical parameter often results in changes in other parameters hindering the efficacy of the system. One of the studies that overcomes this limitation was conducted by Choi et al. where the chitosanconjugated nano-carrier (chito-NC[PF 68]) was prepared as a vehicle for iron oxide NP-enhanced MRI contrast agent, and the loading contents of IONP could reach 40% wt without affecting the size and zeta potential of the nano-carrier at 37°C [73] . But only the mechanical properties of the nano-carrier were varied by loading different amounts of IONPs. In fact, the term surface modification gives an impression of changing multiple parameters in NPs, while most research could merely succeed to modify the charge on the NPs surface.

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M = cm H

Equation 1

Where the constant cm is the magnetic susceptibility of the material. However, some ferromagnetic materials do not obey this linear relationship. They have a magnetic memory left after removal of the external magnetic field, known as remanence. Therefore, such materials solve thrombotic risk related to magnetic aggregation of injectable delivery system as they retain all or some of their induced magnetism when the external magnetic field is removed [74] . The common model used is SPION (superparamagnetic iron oxide NPs), which is nontoxic and enhances contrast in MRI used for monitoring drug delivery [76] . Moreover, the production of thermal energy, when NPs are subjected to alternating magnetic fields (rotation of particles on the field – in that, brownian relaxation, could be utilized in cancer treatment [77] .They induce thermal ablation of tumors, especially surgically inaccessible ones [78] . future science group

Pitfalls in mainstream NP research Clinical studies are based on interpreting statistics of multiple experiments. A cornerstone to such studies is the validity of the statistical conclusions, which are largely based on the statistical significance of the results. An example of a famous study where such a pitfall was pointed out was Lui et al. [84] . Florence [85] pointed out the lack of the statistical significance of the results reported by Lui et al. on the fate of carbon nanotubes in mice, in which tumor accumulation did not exceed 6% of the dose, and the study simply declared that carbon nanotubes effectively target tumor. Moreover, some studies about NP targeting ignored the importance of satisfying all of the criteria for a successful DDS (discussed in ‘Targeting’), declaring the success of some targeting mechanism based on it satisfying only a subset of those criteria. Choi et al. fabricated gold nanoshells, isolated monocytes from buffy


9

Review | Shohdy & Alfaar The new studies lack characterization of tumor biology with more focus on NPs physicochemical parameters alone. Discouraging clinical results in the field of nano-oncology dictate that cancer biology comes first [89] .

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Conclusion & future perspective In vitro and animal models studies show encouraging results, however, the results are often far away from reality in vivo. This can be explained on the basis of great diversity of cancer biology. Likehood parameters of in vitro media and models have to be investigated to provide the best similarity between them and what actually happens in the human cancer environment. The efforts paid in designing new drug-loaded NPs have to go hand in hand with understanding of the nature of cancer’s biological environment and its interaction with NPs in order to narrow the gap between promising in vitro results and discouraging results in humans and clinical trials. The link between bench and bedside is still missing in the field of nano-oncology.

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coat of human whole blood, and to test their hypothesis they systemically injected a mouse model of breast cancer metastatized to brain with nanoparticle-laden macrophages, and tracked macrophage location with another NP, for instance, fluorescently labeled microspheres [86] . Their results indicate that macrophages did cross the blood–brain barrier, delivered loaded NPs to less than a cell width away from the nearest metastatic cell, giving such a paradigm to a Trojan horse delivery method. They consider it “the first successful demonstration of the active delivery” [86] . However, the timely controlled release criterion is not discussed. More studies are needed to know how macrophages will unload their cargo before we consider that a triumph. In spite of our great knowledge of cancer biology, the development of cancer nanoparticulate therapeutics is still only moving along slowly. The late-stage attrition rate for oncology drugs is as high as 70% in Phase II and 59% in Phase III trials [87] . Many cancer biology characters have been elucidated, yet little preclinical models developed. The recent research focuses more on medicines and little on the cancer cell itself, more studies are needed to detect the behavior of cancer cells; we believe that many failures in the field of nanomedicine come from this point. For instance, results from a single randomized trial, BCA-3001 led the Oncologic Drugs Advisory Committee to state: “Please note that important disease characteristics that are currently used in the United States for selecting breast cancer therapies were unknown. 30% of patients had an unknown ER/PR status and 50% of patients enrolled in the trial had unknown HER-2/neu status” [88] .

Acknowledgement: The author acknowledges the great help from CCHE 57357 Foundation staff in preparing the article and the writing assistance of Osama Khalil and Radwa Nour.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary

Targeting capabilities of nanoparticles

Nanoparticles (NPs) can retain the drug efficiently, evade the immune system and deliver their cargo to specific cellular and subcellular levels with timely, controlled unloading.

Two types of targeting are identified – active and passive – and most approved nanomaterials for cancer therapy rely mainly on passive targeting. Limitations of targeting mechanisms

NPs face many biologic barriers on their way to their target cells.

Interesting examples of those limitations are heterogeneity of cancer cells, overexpression in tumor tissue versus whole body and capacity of targeted receptors. Recent solutions for nanoparticle limitations

Conjugation of NPs with various ligands results in a change of their physicochemical parameters, modifying their surface properties, termed functionalization.

Superparamagnetism and magnetic targeting have recently been investigated with promising results.

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