Clinica Chimica Acta 436 (2014) 78–92

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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim

Invited critical review

Targeted anticancer therapy: Overexpressed receptors and nanotechnology Mohd Javed Akhtar a,⁎, Maqusood Ahamed a, Hisham A. Alhadlaq a,b, Salman A. Alrokayan c, Sudhir Kumar d a

King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia Department of Medical Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia Department of Biochemistry, King Saud University, Riyadh 11451, Saudi Arabia d Department of Zoology, University of Lucknow, Lucknow 226007, India b c

a r t i c l e

i n f o

Article history: Received 12 February 2014 Received in revised form 7 May 2014 Accepted 10 May 2014 Available online 15 May 2014 Keywords: Nanoparticles Overexpressed receptors Ligands Anticancer drugs Tumor microenvironment

a b s t r a c t Targeted delivery of anticancer drugs to cancer cells and tissues is a promising field due to its potential to spare unaffected cells and tissues, but it has been a major challenge to achieve success in these therapeutic approaches. Several innovative approaches to targeted drug delivery have been devised based on available knowledge in cancer biology and on technological advancements. To achieve the desired selectivity of drug delivery, nanotechnology has enabled researchers to design nanoparticles (NPs) to incorporate anticancer drugs and act as nanocarriers. Recently, many receptor molecules known to be overexpressed in cancer have been explored as docking sites for the targeting of anticancer drugs. In principle, anticancer drugs can be concentrated specifically in cancer cells and tissues by conjugating drug-containing nanocarriers with ligands against these receptors. Several mechanisms can be employed to induce triggered drug release in response to either endogenous trigger or exogenous trigger so that the anticancer drug is only released upon reaching and preferentially accumulating in the tumor tissue. This review focuses on overexpressed receptors exploited in targeting drugs to cancerous tissues and the tumor microenvironment. We briefly evaluate the structure and function of these receptor molecules, emphasizing the elegant mechanisms by which certain characteristics of cancer can be exploited in cancer treatment. After this discussion of receptors, we review their respective ligands and then the anticancer drugs delivered by nanotechnology in preclinical models of cancer. Ligand-functionalized nanocarriers have delivered significantly higher amounts of anticancer drugs in many in vitro and in vivo models of cancer compared to cancer models lacking such receptors or drug carrying nanocarriers devoid of ligand. This increased concentration of anticancer drug in the tumor site enabled by nanotechnology could have a major impact on the efficiency of cancer treatment while reducing systemic side effects. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overexpressed receptors, their ligands, and drug delivery to cancer using nanotechnology 2.1. G protein-coupled receptors . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Bombesin receptors . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Somatostatin receptors . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Endothelin receptors . . . . . . . . . . . . . . . . . . . . . . . 2.2. Integrin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Integrin αvβ3 . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Integrin α-3 . . . . . . . . . . . . . . . . . . . . . . . . . . .

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79 79 80 82 83 83 83 84 84

Abbreviations: Bn, bombesin; BnR, bombesin receptor; BR, biotin receptor; c(RGD-K), cyclic {Arginine-Glycine-Aspartic acid (RGD)} containing peptide; CA, cholic acid; DOX, doxorubicin; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ETRB, endothelin receptor B; FA, folic acid; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; FR, folate receptor; FSH, follicle stimulating hormone; FSHR, follicle stimulating hormone receptor; NPs, nanoparticles; Nrp-1, neuropilin receptor-1; PEG, poly(ethylene glycol); PLA, poly(lactic acid); PTMC, poly(trimethylene carbonate); PTX, paclitaxel; QDs, quantum dots; S1R, sigma receptor 1; S2R, sigma receptor 2; SRs, sigma receptors; SSTRs, somatostatin receptors; Tf, transferrin; TfR, transferrin receptor. ⁎ Corresponding author at: King Abdullah Institute for Nanotechnology (KAIN), King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. Tel.: +966 559981310 (mobile). E-mail addresses: [email protected], [email protected] (M.J. Akhtar).

http://dx.doi.org/10.1016/j.cca.2014.05.004 0009-8981/© 2014 Elsevier B.V. All rights reserved.

M.J. Akhtar et al. / Clinica Chimica Acta 436 (2014) 78–92

2.3. Folate receptors . . . . . . . . . 2.4. Transferrin receptors . . . . . . 2.5. Epidermal growth factor receptors 2.6. Fibroblast growth factor receptors 2.7. Sigma receptors . . . . . . . . . 2.8. Other overexpressed receptors . . 3. Conclusion . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .

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1. Introduction More than 10 million patients are diagnosed with new cases of cancer every year, and approximately 27 million new cases of cancer will have been recorded by 2030 [1,2]. While cytotoxic chemotherapeutic agents such as paclitaxel (PTX) and doxorubicin (DOX) effectively kill cancer cells, they cannot distinguish cancer cells from normal cells. This lack of selectivity leads to undesirable systemic toxicity when patients are exposed to the high dosages of cytotoxic agents required to eradicate the tumor. Improving the selectivity of anticancer drug delivery to cancer cells and the tumor microenvironment while sparing normal cells and tissues is a major challenge in the effective treatment of cancers of various tissues and organs. Marked differences are found in cancer cells and tissues in terms of biochemical, molecular and physiological features when compared with normal cells and tissues such as differences in redox status, pH levels, expression of certain cell membrane receptors, the leakiness of tumor tissues and the tumor vasculature. Therefore, cancer stands out as a disease likely to benefit from targeted drug delivery approaches exploiting these differences. These differences between healthy and cancerous cells and tissues, or hallmarks of cancer, have recently been reviewed [3]. Differences in normal and cancer biology are found at the level of anatomy, biochemistry and molecular biology. The characteristic anatomic features of tumor biology include the leakiness of blood vessels and poor lymphatic drainage in tumor tissues. The morphology and shape of blood endothelial cells are different in cancer vasculature due to the presence of fenestrae between adjacent cells, and thus the lack of contact inhibition. Most solid tumors, upon reaching a certain level of growth, exhibit enhanced vascular permeability, ensuring a sufficient supply of nutrients and oxygen to tumor tissues and outpacing the growth of surrounding tissues. Blood vessels in tumors are often dilated and convoluted, and compared with normal tissues, exhibit branching patterns that feature excessive loops and arteriolar–venous shunts [4]. All these features enhance the permeability of blood vessels in tumors compared with the vasculature in normal tissues, enabling the delivery and accumulation of molecules and other substances in tumor tissues that are generally not able to enter the vasculature in normal tissues. This is called the enhanced permeability and retention (EPR) effect, a phenomenon associated with the tumor vasculature. The net result of the EPR effect for circulating molecules depends on their properties including size, shape, charge, and polarity. The principal difference between EPR and passive localization lies in the characteristics of retention and tissue clearance rather than uptake. Small drug molecules rapidly penetrate into the tumor interstitial space, but in the absence of specific binding to cellular proteins, drug is not retained and may be free to diffuse out of the tissue back into the blood pool or the lymphatic system. In contrast, macromolecules have smaller diffusion constants, reducing the initial rate of tumor uptake but also tending to increase the half-life of blood-pool circulation by enhancing tissue retention and decreasing the rate of clearance [5]. Therefore, receptor–ligand interactions are the most important aspect of active targeting of nanocarriers (or nanoconjugates or macromolecules) and could be further potentiated by EPR (Fig. 1). The biochemical conditions of acidity (lower pH value) and hypoxia (lower concentration of oxygen) generally prevail in cancer cells and tumor tissues. The extracellular pH in tumor tissue is

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84 85 86 86 87 87 88 89 89

slightly lower than that in normal tissue, and this difference has been exploited to achieve pH-triggered drug release in tumor tissue [6]. A diverse group of functional materials has been designed to accomplish triggered drug release in response to other stimuli such as temperature [7,8], redox potential [9], ultrasound [10], and light [11]. These mechanisms of endogenously or exogenously triggered drug release [12] can only be applied once the anticancer drug reaches and accumulates at the desired site, i.e., tumor tissues. Molecular markers of cancer include the differential expression of proteins residing in the cytosol, organelles or membrane. The group of differentially expressed proteins includes receptors that are specifically expressed or overexpressed in cancer cells compared to normal cells. These overexpressed receptors have provided important endogenous tools for exploitation in the active targeting of drugs to cancer cells. Targeting nanocarriers to a particular organ or tissue through the blood or lymph circulation is referred to as primary targeting, while the accumulation around a cancer cell is named secondary targeting and manipulating the uptake of nanocarriers/drugs by cells and cellular compartments is known as tertiary targeting [13]. This review focuses on overexpressed receptors exploited for targeting drugs to cancer and the tumor microenvironment (Table 1). We briefly evaluate the structure and function of these receptor molecules, emphasizing the elegance of exploiting characteristics of cancer in cancer treatment. After discussing these receptors, their respective ligands are mentioned, followed by a review of the anticancer drugs that have been delivered by nanotechnology in preclinical models of cancer (Table 2). 2. Overexpressed receptors, their ligands, and drug delivery to cancer using nanotechnology Receptors overexpressed or specifically expressed in cancers of various tissues and cells are listed in Table 1. These receptors provide unique opportunities to understand cancer biology and its treatment. In attempts to treat cancer, overexpressed receptors are directly modulated/inhibited by agents such as antibodies or antibody fragments, and also by other small chemicals that directly bind these receptors and block their activities. These treatment strategies thus block the consistent unwanted stimulus for uncontrolled cell division, thus blocking cancer progression. Other approaches to cancer therapy do not intentionally interfere with receptor function, but rather exploit receptor overexpression for the targeted delivery of effective anticancer drugs that do not discriminate between cancer and normal cells. These drugs can be guided by linking them to suitable ligands against such overexpressed receptors. The field of nanotechnology has developed the ability to synthesize an enormous variety of NPs, providing a platform for guiding and carrying drugs to cancer cells and tumor tissues. These NPs can be loaded with anticancer drugs and conjugated with ligands. Nanosized liposomes, dendrimers, micelles, metals, alloys, mixtures of inorganics and organics, and other materials have been synthesized by nanotechnology for use in diverse applications such as catalysis, sensing and medicine. NPs loaded with drug molecules and conjugated with receptor ligands have been variously termed as nanoconjugates, nano-formulations, nano-carriers, etc. Table 2 summarizes some recent outcomes of the nanotechnology-based interventions in the targeting of anticancer drugs in preclinical in vitro and in vivo

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Endothelial cells

Normal vasculature should allow very little transport, if any, of nanoconjugatesfrom circulation to tissues.

Leaky tumour vasculature should allow nanoconjugate transportation in either direction with higher rate due to EPR effect.

Ligand-receptor interaction causing accumulation and retention of drug within tumor microenvironment

No ligand -receptor interaction

Unaffected normal cell

Normal cell

Demise of cancer cell

Cancer cell

Fig. 1. Different biological responses mediated by receptor ligands and the resulting cellular fate in cancer vs. normal cells. Ligand decorated nanoconjugates would in principle gradually concentrate within tumor tissues due to the higher leakage mediated by the EPR effect and the consequent entrapment caused by ligand binding with overexpressed receptors in cancer cells.

models of cancer exploring overexpressed receptors and their respective ligands. 2.1. G protein-coupled receptors Many tumor cells specifically express or overexpress G proteincoupled receptors (GPCRs) compared with the original tissue they are

derived from. Researchers have recently begun to exploit this difference in expression in the targeting of chemotherapeutics, radiodiagnostics and radiotherapeutics to tumors [14]. A variety of external stimuli including neurotransmitters, hormones, and phospholipids can activate G-protein coupled receptors (GPCRs), transducing signals from the extracellular environment to the cytoplasm. GPCRs share the common structure of a single polypeptide with seven membrane-spanning

Table 1 Description of overexpressed receptors widely used for selective drug delivery using receptor–ligand pairs in preclinical models of cancer. Major receptor type

Specific receptor(s)

Overexpression in cancer cell types

G protein coupled receptors (GPCRs)

Bombesin receptor (BnR) This family consists of three closely related proteins, based on their amino acid sequence homology — i) the gastrin-releasing peptide receptor (GRP receptor); ii) the neuromedin B receptor (NMB receptor) and iii) the orphan receptor, BRS-3 with unknown ligands. Somatostatins receptors (SSTRs) Five subtypes of SSTRs have been described so far termed as SSTR 1–5. SSTR-2 is being widely used. Endothelin receptors (ETRs) Two highly homologous receptors — i) ETRA and ii) ETRB bind, but with different affinities, to three closely related ligands, endothelins (ET; ET-1, ET-2 and ET-3) each consisted of 21 amino acids. Among several subtypes formed by various combinations of α and β subunits, ανβ3 is of particular interest in selective drug targeting. FRα, FRβ and FRγ. The best studied, and widely used in selective drug targeting, of these receptors is folate receptor-α (FRα), a cell surface glycosyl phosphatidylinositol-anchored glycoprotein that can internalize bound folates and folate-conjugated compounds via receptormediated endocytosis. Two types of receptors (ubiquitously expressed TfR1 and TfR2 restricted to hepatocytes) only have been described so far. The transferrin receptor 2 (TfR2) shares a 45% identity and 66% similarity in its extracellular domain with TfR1. EGFR family consists of four members: EGFR (or ErbB1, HER1), ErbB2 (HER2, neu in rodents), ErbB3 (HER3) and ErbB4 (HER4). A hallmark of FGFRs is the presence of an acidic, serine-rich sequence in the linker between D1 and D2, termed the acid box. The two sigma receptors – S1R and S2R – were distinguished classically on the basis of their binding affinity for pentazocine and guanidineDTG. Both bind pentazocine whereas only the latter binds with DTG. Follicle stimulating hormone receptors (FSHRs) Biotin receptors (BRs) C-type lectin receptors (CLRs). Asialoglycoprotein receptor (ASGPR) NRP-1

Lung, prostate, breast, pancreatic, head/neck, colon, uterine, ovarian, renal cell, glioblastomas, neuroblastomas, gastrointestinal carcinoids, intestinal carcinoids, and bronchial carcinoids.

Integrins Folate receptors (FRs)

Transferrin receptors (TfRs)

Epidermal growth factor receptor (EGFR) Fibroblast growth factors (FGFRs) Sigma receptors (SRs)

Others

Small cell lung, neuroendocrine tumor, prostate cancer, breast cancer, colorectal carcinoma, gastric cancer, hepatocellular carcinoma Melanoma tissues

Activated endothelial cells and tumor cells (such as U87MG glioblastoma cells), ovarian cancer cells. Most tissues including breast cancer cells.

Breast, ovary, and brain cancers such as glioma and glioblastomas.

Lung, breast, bladder, and ovarian cancers. Breast, prostate, bladder, and gastric cancer Non-small cell lung carcinoma, prostate cancer, melanoma, and breast cancer. Ovarian surface epithelium Leukemia Hepatocytes, dendritic cells, macrophages Human vascular cells

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81

Table 2 Some recent outcomes of nanotechnology in the delivery of anticancer drugs in preclinical models of cancer exploiting overexpressed receptors and their respective ligands. Note that specific names of nanocarriers, for example liposomes, may consist of the same or different building blocks/monomers. Similarly, micelles and other carriers might also differ in size, shape, charges, composition, and other properties. Specific Ligands/ligands analogues receptor(s)

Nanoparticles/nanocarriers

Anticancer drugs/agents

Major outcome

References

BnR

Peptide fragment called BN (7–14)

Liposome

DOX

[27]

BnR

BN (6–14)

No nanocarrier

Mitochondria-disrupting peptide, B28

SSTR

OCT

Liposome

DOX

SSTR

OCT

Liposome

DOX

SSTR

OCT

Micelles

DOX

ETRB

mAB against N-terminal tail of ETRB

Antibody–drug conjugate (ADC)

Monomethylauristatin E (MMAE)

αvβ3

c(RGDfK)

Liposome

DOX

αvβ3

c(RGDyK)

Iron oxide NPs coated with PEG Internalization potential testing

αvβ3

c(RGDyK)

Micelles

PTX

αvβ3

c(RGDyK)

Micelles

PTX and taxol

α-3

Peptide OA02

Micelles

PTX

FR

FA

Liposome

DOX

FR

FA

Copolymeric NPs

PTX

FR

FA

Surface functionalized QDs

–

FR

FA

Liposome

DOX

FR

FA

Liposome

DOX

FR

FA

Hybrid polymeric NPs

PTX

FR

FA

Liposome

DOX

TfR

Transferrin

Iron oxide NPs

MRI

TfR

Single-chain antibody fragment (TfRscFv)

Liposome

p53-gemcitabine

EGFR

Peptide ligand (D4: Leu-Ala- Liposome Arg-Leu-Leu-Thr)

Comparatively greater amount of DOX was delivered to PC-3 cancer cells and xenografts expressing higher BnR. Greater cytotoxicity to various human cancer cells (DU145, PC-3, MCF-7, HSF, MRC-5, PrSC)-expressing BnR. OCT enhanced DOX uptake in SMMC-7721 cells, and tumor of pancreas and melanoma of B16 tumor bearing BALB/c mice. Sterically stabilized liposomes (SSL) increased intracellular delivery of DOX in SSTR2-positive cells through a mechanism of receptor-mediated endocytosis. OCT caused higher cytotoxicity to MCF-7 cells with high expression of SSTR. Nude mice bearing MCF-7 cancer xenografts exhibited enhanced anti-tumor efficacy and decreased systemic toxicity. ADC demonstrated remarkable efficacy against human melanoma cell lines and xenograft tumor models in correlation with the levels of receptor expression. This delivery method resulted in a 15-fold improvement in tumor and anti-metastatic activity in human umbilical vein endothelial cells (HUVECs) expressing high levels of αvβ3 when compared with free drug. Integrin-specific association between the ligand c(RGDyK)-iron oxide nanoparticle adducts and U87MG glioblastoma cells. Presence of c(RGDyK) enhanced cytotoxicity by 2.5 folds in U87MG glioblastoma cells. Median survival time was prolonged in mice bearing intracranial U87MG tumor xenografts. Cellular uptake and, thus, cytotoxicity of c(RGDyK)NP/PTX was found to be significantly higher than that of NPs/PTX due to the integrin protein-mediated endocytosis effect. Better bioavailability of PTX than Taxol. Dramatically enhanced the uptake efficiency of these OA02-PEG-CA NPs in SKOV-3 and ES-2 ovarian cancer cells via receptor-mediated endocytosis, but not in α-3 integrin-negative K562 leukemia cells. Folate receptor targeted-liposomes loaded with doxorubicin (FA-L-DOX) inhibited proliferation of nonfunctioning pituitary adenoma (NFPA) cells NPs formulation has great advantages over the pristine drug, PTX, in achieving better therapeutic effect in MCF-7 breast cancer cells. Established folate targeting of QDs in FR expressing MCF-7 cells. FR mediated endocytosis induced higher cytotoxicity against the 4T1 mouse mammary carcinoma cell line. Higher cytotoxicity in KB human carcinoma cells; greater tumor growth inhibition and almost a 50% increase in life span of mice compared with mice taking drug in absence of targeting. Folate significantly promoted drug-loaded NP's cellular uptake through folate receptor-mediated endocytosis in FR overexpressing HeLa and glioma C6 cells as compared to no significant difference in FRmediated drug uptake in NIH 3T3 cells with normally expressed folate receptors. More effective drug uptake in the KB and KB-V xenograft models, and in the J6456 model irrespective of administered routes. Retention of Tf-SPIONs in cytoplasm of tumor glioma cells achieved The combination treatment prolonged median survival when compared with single drug treatment. Also decreased tumor burden. D4 peptide conjugated liposomes efficiently entered in EGFR expressing cancer cells (H1299) and specifically accumulated in EGFR-expressing xenografts tumor tissues.

Tested EGFR targeting potential

[31]

[45]

[46]

[47]

[56]

[67]

[68]

[69]

[70]

[72]

[91]

[92]

[93] [94] [95]

[96]

[97]

[116] [118]

[128]

(continued on next page)

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Table 2 (continued) Specific Ligands/ligands analogues receptor(s)

Nanoparticles/nanocarriers

Anticancer drugs/agents

Major outcome

References

HER2

mAB trastuzumab (TMAB)

Human serum albumin (HSA) NPs

Methotrexate (MTX)

[129]

EGFR

Heptapeptide

Liposome

DOX

EGFR

Pentapeptide

Lipid nanocarriers

Tested EGFR targeting potential

FGFR2

Truncated (t) fibroblast growth factor (tbFGF) peptide

Liposome

DOX

FGFR

tbFGF

Liposome

PTX

FGFR

tbFGF

Liposome

PTX

FGFR

tbFGF

Liposome

PTX

FGFR1

FGF1

Gold nanoparticles

Irradiation

S2R

SV119

Liposome

DOX

S2R

SV119

Gold nano-cages

FSHR

FSH33–53 peptide

Liposome

Tested targeting potential of SV119 PTX

BR

Biotin

Liposome

PTX

BR

Biotin

BR

Biotin

Gold NNPs

PTX

CLR

GalNAc residue

Liposome

Targeting asialoglycoprotein receptor.

CLRs

Lectin

Magnetic NPs (MNPs)

PTX

NRP-1

CRGDK peptide

Gold NPs

p-12 (a peptide)

The cytotoxicity of TMAB–MTX–HSA NPs on HER2 positive cells was found to be significantly higher than that for non-targeted MTX–HSA NPs and free MTX. Peptide-conjugated PLGA-PEG NPs loaded with DOX showed three-fold higher uptake than by peptidefree PLGA-PEG NPs in a SKOV3 cell line with high expression of epidermal growth factor receptor Higher internalization of pentapeptide ligand conjugated NPs in high EGFR expressing H1299 and K562 cells. An enhanced antiproliferative activity was achieved by co-delivering DOX and plasmid DNA with important a mutant gene simultaneously to the Lewis lung carcinoma cells (LLC). tbFGF-LPs-PTX significantly accumulated in tumor and prolonged the retention time in tumor-bearing mice. tbFGF, significantly enhanced cytotoxicity of PTX in FGFR expressing LL/2 cancer cells. Higher accumulation of PTX loaded with PEGylatedtFGF nanoconjugates in tumor tissues expressing FGFRs. FGF1 variant-AuNP conjugates was specifically internalized only in cells expressing FGFRs. Cell viability reduced significantly after irradiation with nearinfrared light whereas the proliferation potential of cells lacking FGFRs is not affected. DOX-loaded SV119 liposomes showed significantly higher cytotoxicity to DU-145 cells compared to the DOX-liposomes without SV119. SV119-ligand might be a promising tool in targeting certain tumor. FSH33–NP–PTX displayed stronger anti-proliferation and antitumor effects compared with free PTX or NP-PTX both in vitro and in vivo. Results showed that the biotin conjugated NPs could improve the selective delivery of PTX into cancer cells with overexpressed biotin receptors. Excellent drug uptake and cytotoxicity in L1210FR leukemia cells. Gold NPs surface-functionalized with PTX and biotin played a significant role in the diagnosis and therapy of the cancer cells. Glycolipid conjugated liposome internalized significantly into hepatocytes by ASGPR-mediated endocytosis. Five times higher uptake of PTX-MNPs conjugated with lectins in K562 leukemia cells as compared with lectin receptors negative HEK 293 cell line. CRGDK peptide increased intracellular uptake of gold NPs carrying therapeutic P12 peptide in NRP-1 receptor overexpressing MDA-MB-321 cells.

Taxoid (SB-T-1214)

domains. Binding of specific agonists (or ligands) to these receptors leads to the subsequent activation of heterotrimeric G-proteins (Gs, Gi/Go, Gq/G11 and G12/G13), which in turn regulate the activity of one or several effectors such as second-messenger-producing enzymes or ion channels [15,16]. Here, we focus only on those GPCRs reported to be overexpressed in various tumor cells, making them of interest to the field of cancer therapy. A diverse array of ligands have been developed for drug targeting against GPCRs. Bombesin and octreotide are among the major ligands for a certain class of GPCRs used in the targeted delivery of anticancer drugs by nanotechnology. 2.1.1. Bombesin receptors Bombesin receptors (BnRs), also known as gastrin-releasing peptide (GRP) receptors belonging to the GPCR superfamily, have been found to be overexpressed in cell lines derived from several human tumor types, including lung cancer, prostate cancer, breast cancer, pancreatic cancer,

[131]

[132]

[141]

[142]

[143] [144]

[145]

[175]

[176] [181]

[182]

[183] [184]

[194]

[195]

[198]

head/neck cancer, colon cancer, uterine cancer, ovarian cancer, renal cell cancers, glioblastomas, neuroblastomas, gastrointestinal carcinoids, intestinal carcinoids, and bronchial carcinoids. Thus, there is a great deal of interest in developing BnR-mediated agents to treat these tumors [17, 18]. The mammalian BnR family consists of three closely related G protein coupled receptors (GPCRs): the gastrin-releasing peptide receptor (GRP receptor), whose native ligand is the 27 amino acid peptide GRP; the neuromedin B receptor (NMB receptor), which mediates the action of the 10 amino acid peptide NMB, and the orphan receptor BRS-3, which shares 47–51% amino acid sequence homology with the GRP/ NMB receptors but still has an unknown ligand. Each of these receptors and their ligands are widely distributed in both the central nervous system and the peripheral tissues. Moreover, studies in animals suggest that these receptors are involved in a broad range of physiological and pathophysiological processes [19–22]. Numerous radiolabeled bombesin ligands (Bn) or their analogues are currently undergoing investigation for applications in tumor

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imaging and radiotherapy. A few non-radiolabeled ligand–drug complexes constructed by the conjugation of Bn with chemotherapeutic agents such as camptothecin, DOX, and PTX have successfully increased the selectivity and efficacy of these drugs in preclinical studies [23–25]. Recently, several studies have used nanotechnology to exploit the specific overexpression of BnR to target anticancer drugs loaded with NPs conjugated with Bn and/or Bn analogues. Accardo A et al. [26] have successfully delivered more of the anticancer drug DOX to PC-3 cancer cells using drug-containing liposomes conjugated with the BnR ligand (7–14) peptide fragment called BN(7–14). The same study reported significant cytotoxicity in PC-3 cells expressing high BnR as well as greater inhibition of tumor growth in PC-3 xenograft-bearing mice compared with liposome–DOX complexes without ligand. Previously, radiolabeled liposomes containing the BN(7–14) peptide fragment demonstrated that the peptide exposed on the liposome surface maintains the ability to selectively target aggregates of GRPR-expressing xenografts [27]. Many studies have demonstrated that the BN(7–14) fragment modified on its N-terminus with radiometal complexes for diagnostic or therapeutic nuclear medicine applications preserves its affinity for these receptors [28–30]. Yang et al. [31] again proved the ability of BnR to facilitate targeted drug delivery to several tumor cells such as human prostate cancer cells (DU145 and PC-3), human breast cancer cells (MCF-7), human skin fibroblast cells (HSF), human lung fibroblast cells (MRC-5), and human prostate stromal cells (PrSC) expressing BnR. They have used the mitochondria-disrupting peptide B28 as an antitumor agent together with the bombesin analogue BN(6–14), which contains a bombesin receptor-binding motif for the targeting of B28 to these tumor cells. B28 conjugated to BN(6–14) was far more cytotoxic to tumor cells than unconjugated B28. 2.1.2. Somatostatin receptors Somatostatin receptors (SSTRs), members of the superfamily of Gprotein coupled receptors (GPCRs), are widely expressed in a variety of tumors and cancer cell lines, including small cell lung cancer [32], neuroendocrine tumors [33], prostate cancer [34], breast cancer [35], colorectal carcinoma [36], gastric cancer [37] and hepatocellular carcinoma [38]. Not surprisingly, therefore, SSTRs have recently gained attention due to the potential for targeting anticancer drugs to cancer cells overexpressing SSTRs via suitable ligands against these receptors. Physiologically, these receptors are involved in mediating signaling to regulate the basic processes of secretion, cell division, proliferation and apoptosis. The ligand for these receptors is the neuropeptide hormone somatostatin (SST) [39,40]. Five subtypes of SSTRs have been described so far, termed SSTR 1–5 [41]. SSTRs, especially SSTR subtype 2, are found to be expressed at higher levels in many tumor cells and in tumoral blood vessels relative to normal tissues. The binding of somatostatin (SST) to endogenous SSTRs is followed by the internalization of SST, and several reports have shown that most hormone-secreting tissue tumors have a high density of SSTRs [42]. Almost a decade ago, Huang C.M. et al. [43] reported the use of SSTRs to target anticancer drugs to tumor cells by conjugating the anticancer drug taxol with the SSTR ligand octreotide (OCT). SSTR-mediated delivery of OCT-taxol conjugates triggered apoptosis and was exclusively toxic to SSTR-expressing human breast carcinoma (MCF-7) cells. OCT-conjugated taxol was less toxic to cells expressing low levels of SSTR compared with free taxol. These results suggested that OCTconjugated taxol demonstrated cell selectivity and might be used as a targeting agent for cancer therapy. Thereafter, Shen et al. [44] evaluated the anti-tumor effects of the anticancer drug PTX conjugated with the SSTR ligand OCT in human lung epithelial adenocarcinoma (A549) cells and human non-small-cell lung cancer (NSCLC) cells xenografted into nude mice. The PTX–OCT conjugate, made up of two molecules of PTX with one molecule of OCT, could enhance tumor growth inhibition and reduce toxicity when compared with unconjugated PTX. The two reports mentioned above did not use NPs to carry ligand–drug conjugates, but nanotechnology has since been applied to SSTR-mediated

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anticancer drug delivery. Liposomes containing the anticancer drug DOX with OCT ligand noticeably increased the uptake of DOX in human hepatocellular carcinoma (SMMC-7721) cells and led to greater cytotoxicity than liposomes without targeting ligands [45]. In contrast, no significant difference in cytotoxicity occurred between drugs encapsulated in liposomes with or without OCT in CHO cells not expressing SSTRs. Moreover, the study of the ex vivo fluorescence imaging of BALB/c mouse tissues and the in vivo tissue distribution of B16 tumorbearing mice indicated that OCT caused a remarkable accumulation of DOX in SSTRs expressing melanoma and pancreatic tumors compared with the DOX-liposomal formulation without ligand [45]. Using the same drug and ligand conjugated with novel sterically stabilized liposomes (SSLs) composed of polyethylene glycol (OCT-SSL-DOX) led to increased intracellular delivery of DOX in SSTR2-positive cells through a mechanism of receptor-mediated endocytosis [46]. Compared to SSL, OCT modification of SSL exhibited little effect on the physicochemical properties of SSL. In summary, OCT-modified SSL might be a promising system for the targeting of anticancer drugs in the treatment of SSTR2overexpressing cancers. Another study described efficient DOX targeting and thus significantly higher cytotoxicity in MCF-7 human breast cancer cells expressing SSTRs compared with normal human lung fetal (WI-38) cells with no significant SSTR expression. In this study, investigators constructed micelles of N-deoxycholic acid-O, Nhydroxyethyl chitosan (DAHC) with good DOX loading capacity (named DAHC-DOX). The in vivo administration of micelles to nude mice bearing MCF-7 cancer xenografts confirmed that OPD (OCT– PEG–deoxycholic acid)-DAHC micelles possessed a much higher tumor-targeting capacity than the DAHC control and exhibited enhanced anti-tumor efficacy and decreased systemic toxicity. These results suggest that OPD-DAHC micelles might be a promising anticancer drug delivery carrier for targeted cancer therapy [47]. Therapy with valproic acid and the SSTR2-targeting cytotoxic conjugate CPT–SST led to greater suppression of cervical cancer compared with each agent alone. These findings provide a promising clinical opportunity for enhanced cancer therapy using combinations of Notch1activating agents (valproic acid) and SSTR2-targeting agents [48]. We should expect more research in this area. 2.1.3. Endothelin receptors The endothelin (ET) family of molecules (acting as ligands) is composed of three polypeptides, ET-1, ET-2 and ET-3, each consisting of 21 amino acids that bind to two highly homologous G-coupled protein receptors (GCPRs), endothelin receptor A (ETRA) and endothelin receptor B (ETRB or EDNRB), triggering a variety of signals according to cell type. The three ETs are potent vasoconstricting peptides involved in the pathophysiology of various malignancies. ET-1, ET-2 and ET-3 are characterized by a single α-helix and two disulfide bridges, although the three peptides are encoded by distinct genes. ETRB binds with all three ETs with equal affinity, while ETRA binds ET-1 and ET-2 with a two-fold higher affinity than the ET-3 [49–52]. ETRB has been reported to be overexpressed in the vast majority of human melanoma tissue specimens examined [53,54]. Differential endothelin receptor expression and function has also been reported in rat myometrial and leiomyoma ELT3 cells [55]. An antibody–drug conjugate demonstrated preclinical efficacy, even in a model with a low level of ETRB expression [56]. The receptor ETRB is particularly attractive as a target for antibody/ligand–drug conjugate therapy due to its minimal expression on normal tissue, its cell surface localization and its rapid endocytosis. However, we are not familiar with any reports exploiting endothelin receptors in drug targeting nanocarriers. 2.2. Integrin receptors Among various integrins, αv (or αvβn) integrin receptors are found to be highly expressed in activated endothelial cells and tumor cells

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(such as U87MG glioblastoma cells), but not in resting endothelial cells or most normal organ systems. Therefore, these integrins represent potential targets for tumor imaging and therapy [57]. Researchers are currently investigating which integrins act as neoplastic markers. Upregulation of ανβ3 has been found to be tightly associated with a wide range of cancer types, making it a broad-spectrum tumor marker [58]. Similarly, another integrin, α-3, is also overexpressed in several types of cancers, especially in ovarian cancer, breast cancer, and melanoma [59]. Before discussing the roles of ανβ3- and α-3 integrin receptors in the targeted delivery of various anticancer drugs to cancer cells and exploring their respective ligands, it is imperative to briefly review the structure and function of integrins in normal biology. Integrins are transmembrane receptors that bind extracellular matrix proteins or other adhesion receptors on neighboring cells. Heterodimeric pairing of integrin α and β subunits confers specificity of binding to one or more substrates [60]. In particular, the αv subunit pairs with β1, β3, β5, β6, and β8. While some pairings preferentially bind a single ligand (αvβ5 for vitronectin), others recognize a number of ligands (αvβ3 binds vitronectin, fibronectin, vWF, tenascin, osteopontin, fibrillin, fibrinogen, and thrombospondin). At least 24 distinct integrin heterodimers are formed by the combination of 18 α subunits and 8 β subunits. Because the integrins expressed on the surface of a cell will determine whether it can adhere to and survive in a particular microenvironment, the matching of integrins and ligands plays a key role in the regulation of the sprouting ability of endothelial cells during angiogenesis, the recruitment of inflammatory cells to sites in need of repair, and the invasive potential of tumor cells [61,62]. The role of integrins in cell migration and invasion is one of their functions that has received the most study in tumor biology, and this has been reviewed elsewhere [63,64]. Antagonists to some integrins such as cilengitide have shown promising results in various phases of clinical trials, including for cancer therapy [65]. Our focus is to review the utility of these well studied integrin receptors for targeted anticancer drug delivery in nanomedicine. Integrins expressed by epithelial cells (including α6β4, α6β1, αvβ5, α2β1 and α3β1) are generally retained in the tumor but at different levels. Integrin expression also varies considerably between normal and tumor tissues. Most notably, integrins αvβ3, α5β1 and αvβ6 are usually expressed at low or undetectable levels in most adult epithelia, but can be highly upregulated in some tumors. However, the expression levels of some integrins decrease in tumor cells, potentially increasing tumor cell dissemination. In fact, re-expression of α2β1 in breast cancer cells reversed some of the malignant properties of those cells, suggesting that α2β1 could function as a tumor suppressor. Several integrins that might have important roles in cancer progression have now emerged with great potential in targeted drug delivery. As expression of the integrins αvβ3, αvβ5, α5β1, α6β4, α4β1 and αvβ6 is correlated with disease progression in various tumor types, these integrins are the most frequently studied in cancer [66]. 2.2.1. Integrin αvβ3 Recent studies showed that the delivery of targeted NPs loaded with DOX to the integrin αvβ3-positive tumor vasculature inhibited the growth of metastases while eliminating the toxicity and weight loss associated with systemic administration of this drug [67]. This delivery method resulted in a 15-fold improvement in tumor and antimetastatic activity when compared with administration of the free drug. The preferential activity of these NPs on metastases suggests that growing metastatic tumors may have a greater dependence on angiogenic vessels, making them more susceptible to integrin αvβ3targeted therapy. Chen et al. [68] coated iron oxide NPs with a PEGylated amphiphilic triblock copolymer and then conjugated the near-infrared fluorescent (NIRF) dye IRDye800 and a cyclic ArginineGlycine-Aspartic acid (RGD) containing peptide c(RGDyK) for integrin αvβ3 targeting. In vitro binding assays confirmed the integrin-specific association between the ligand c(RGDyK)-iron oxide nanoparticle adducts and U87MG glioblastoma cells. Successful tumor homing in vivo

was observed in a subcutaneous U87MG glioblastoma xenograft model by both magnetic resonance imaging (MRI) and NIRF imaging. The investigators achieved excellent tumor integrin targeting efficiency and specificity. Another study also used the same ligand–receptor pair [i.e., c(RGDyK)–αvβ3] in U87MG glioblastoma cells to deliver the anticancer drug PTX, which was encapsulated in poly(ethylene glycol)block poly(lactic acid) micelles, resulting in a nanoscale formulation of (c(RGDyK)–PEG–PLA–PTX). In vitro cytotoxicity studies proved that the presence of c(RGDyK) led to a 2.5-fold enhancement in the antiglioblastoma cytotoxic efficacy. In an in vivo model, the c(RGDyK)– PEG–PLA micelle accumulated in the subcutaneous and intracranial tumor tissues, and the PTX-loaded micelle (c(RGDyK)–PEG–PLA–PTX) exhibited the strongest tumor growth inhibition among the PTX formulations studied here [69]. Other investigators prepared polymeric micellar-like NPs (MNP) based on PEGylated poly(trimethylene carbonate) (PEG-PTMC) conjugated with c(RGDyK) for active targeting to integrin-rich U87MG cancer cells. A pharmacokinetic study in rats demonstrated that the polymeric micellar NPs significantly enhanced the bioavailability of PTX compared with Taxol. In vivo multispectral fluorescent imaging indicated that c(RGDyK)-MNP/PTX exhibited high specificity and efficiency in active tumor targeting [70]. 2.2.2. Integrin α-3 Similarly, α-3 integrin is overexpressed in several types of cancers, especially in ovarian cancer, breast cancer, and melanoma [59]. The high affinity α-3 integrin-targeting peptide OA02 has been shown to bind strongly to α-3 integrin-overexpressing ovarian cancer cells and specifically target ovarian cancer xenografts (ES-2) in nude mice when conjugated to near-infrared fluorescence dyes [71]. The overexpression of α-3 integrin on these ovarian cancer cells has been exploited [72] as a promising pharmacologic target for selective drug delivery in the treatment of these cancers. Conjugation of micellar NPs (PEG5kCA8 NPs) composed of polyethylene glycol (PEG) block dendritic cholic acid (CA) copolymers with the OA02 peptide dramatically enhanced the uptake and efficiency of these nanocarriers in SKOV-3 and ES-2 ovarian cancer cells via receptor-mediated endocytosis, but uptake was not increased in α-3 integrin negative K562 leukemia cells. Furthermore, the in vivo biodistribution study reported that the OA02 peptide greatly facilitated tumor localization and intracellular uptake of PEG5k-CA8 NPs into ovarian cancer cells as validated in SKOV3-luc tumor-bearing mice [72]. 2.3. Folate receptors Among cellular surface targets potentially suitable for use in drug targeting, folate receptors (FRs) stand out as one of the most promising and most investigated epithelial cancer markers. The FR constitutes a useful target for tumor-specific drug delivery primarily because it is up-regulated in many human carcinomas, including malignancies of the ovary, brain, kidney, breast, colon, myeloid cells, and lung, while it is expressed at low levels in normal cells and tissues [73–76]. FRs (FRα, FRβ and FRγ) are cysteine-rich cell-surface glycoproteins that bind folate with high affinity to mediate the cellular uptake of folate. Although expressed at very low levels in most tissues, FRs, especially FR α, are expressed at high levels in numerous cancers to meet the folate demand of rapidly dividing cells under low folate conditions [77,78]. The folate dependency of many tumors has been therapeutically and diagnostically exploited by the administration of anti-FRα antibodies, high-affinity anti-folates [79,80], folate-based imaging agents and folate-conjugated drugs and toxins [81–83]. The best studied isoform of these receptors is FRα, a cell surface glycosyl phosphatidylinositolanchored glycoprotein that can internalize bound folates and folateconjugated compounds via receptor-mediated endocytosis [84]. Very recently, its structural basis for the molecular recognition of folic acid was reported [85]. FR α has a globular structure stabilized by eight disulfide bonds and contains a deep open folate-binding pocket

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comprised of residues that are conserved in all receptor subtypes. The folate pteroate moiety is buried inside the receptor, whereas its glutamate moiety is solvent-exposed and protrudes out of the pocket entrance, allowing for drug conjugation without adversely affecting FR α binding affinity. The extensive interactions between the receptor and ligand readily explain the high folate-binding affinity of folate receptors and provide a template for the design of more specific drugs targeting the folate receptor system [85]. Sarcomas, lymphomas, and cancers of the pancreas, testicles, bladder, prostate, and liver often do not show elevated levels of folate receptors. When expressed in normal tissue, folate receptors are restricted to the lungs, kidneys, placenta, and choroid plexus; in these tissues, the receptors are limited to the apical surface of polarized epithelia [86]. Folate or folic acid (FA), the FR ligand is a small molecule (44 Da) stable over a broad range of temperatures and pH values, inexpensive, and non-immunogenic, and it retains its ability to bind to the folate receptor after conjugation with drugs or diagnostic markers [87]. The overexpression of folate receptor in cancers of several histologies relative to normal tissues, the low cost of folic acid (FA), and the vast library of conjugation reactions available make it one of the most used ligands for tumor-targeted drug delivery and tumor imaging (reviewed in [88]). Folate is internalized into cells via a low-affinity reduced folate carrier protein or via high-affinity folate receptors. Due to the elevated FR α expression in cancer cells, FR α has become one of the most intensively investigated cellular surface antigens for the targeted delivery of a variety of molecules, including imaging agents, chemotherapeutic agents, oligodeoxynucleotides, and macromolecules. Various types of drug carriers have been conjugated to folate such as liposomes, lipid NPs, polymeric NPs, polymers, and micelles filled with the active molecule [89]. Thus, solid tumors that are currently among the most difficult to treat by classical therapeutic modalities may be readily targeted with FA-linked therapeutics [73,90]. Folate receptor targeted-liposomes loaded with DOX (FA-L-DOX) inhibited the proliferation of human non-functioning pituitary adenoma (NFPA) cells and promoted apoptosis through the activation of caspase 8, caspase 9, and caspase 3/7 more effectively than L-DOX (liposomes without FR targeted but with DOX). Furthermore, FA-L-DOX also exerted greater anti-invasive ability in NFPA cells than L-DOX through the suppression of the secretion of matrix metalloproteinase-2 and matrix metalloproteinase-9. Addition of 1 mM free folic acid (to block FRmediated endocytosis of liposomes) significantly reduced the pleotropic effects of FA-L-DOX in NFPA cells, suggesting that FRα plays a critical role in mediating the antitumor effect of FA-L-DOX [91]. Another study using nanotechnology showed that a twocomponent copolymeric NP formulation has great advantages over the pristine drug PTX, achieving a better therapeutic effect in MCF-7 breast cancer cells. These results further showed that the folate decoration could promote significantly higher delivery of the drug into the corresponding cancer cells, enhancing therapeutic effect [92]. Later, the same group prepared folate-decorated quantum dot NPs to improve the imaging effects and reduce their side effects with surface functionalized poly(lactide)–vitamin E–D-alpha-tocopheryl polyethylene glycol succinate (PLA-TPGS) copolymer and vitamin E TPGS–carboxyl (TPGSCOOH) copolymer designed to conjugate folate-NH2. This system presents the advantage of making the targeting effect adjustable. Much higher internalization of the folate-decorated QD-loaded PLA-TPGS/ TPGS-COOH NPs was achieved in MCF-7 cells overexpressing folate receptors than in mouse NIH 3T3 fibroblast cells with low levels of folate receptor expression. Compared with free QDs, the QDs conjugated with PLA-TPGS/TPGS-COOH NPs showed lower in vitro cytotoxicity in both MCF-7 and NIH 3T3 cells [93]. Prabaharan et al. [94] encapsulated DOX into folate-conjugated amphiphilic hyperbranched block copolymeric micelles {a hydrophobic poly(L-lactide) inner shell and a hydrophilic methoxy poly(ethylene glycol) outer surface}. The DOX-loaded micelles provided an initial burst release (up to 4 h) followed by a sustained release of the entrapped DOX over a period of approximately 40 h. Cellular uptake of the DOX-loaded micelles linked with FA was

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found to be greater than that of the DOX-loaded micelles without FA due to folate-receptor mediated endocytosis in the 4T1 mouse mammary carcinoma cell line. Further, in vitro degradation studies revealed that the H40–PLA-b-MPEG/PEG–FA block copolymer hydrolytically degraded into polymer fragments within six weeks. Another study tested the anticancer efficacy of free DOX and DOX bound with folate targeted liposome. DOX-liposome was superior to free DOX in two human tumor models (KB, KB-V) and in one mouse ascitic tumor model (FRexpressing J6456). The therapeutic effect was dose-dependent in the KB model receiving DOX-liposome through the i.p. intra-cavitary route and was schedule-dependent in the J6456 mouse model receiving DOX-liposome via the intra-cavitary route. In some experiments, however, toxic deaths aggravated by the folate-depleted diet were a major confounding factor. While promising, this study indicates that more sophistication is needed in selecting cancer type associated receptors and ligands [95]. Another in vitro study [96] demonstrated significant FR-mediated endocytosis of PTX-loaded micellar NPs in FRoverexpressing HeLa and glioma C6 cells, compared with no significant drug uptake in NIH 3T3 cells with lower folate receptor expression. In their study, Riviere et al. [97] investigated the antitumor activity of FA-linked liposome-DOX injected intravenously in mice bearing KB tumor xenografts. In conclusion, systemically administered FAconjugated DOX-liposomes have demonstrated the potential to enhance the delivery of anticancer drugs in in vivo models. 2.4. Transferrin receptors The efficient cellular uptake of transferrin (Tf) makes this pathway a potential route for the delivery of anticancer drugs, proteins, and therapeutic genes primarily into proliferating malignant cells that overexpress transferrin receptors (TfRs) [98–100]. Transferrin (Tf) is a singlechain glycoprotein containing approximately 700 amino acids and is one of the most widely used tumor targeting ligands in tumor cells expressing transferrin receptors (TfRs) more than other cells [101–103]. TfRs are divided into two subtypes, TfR1 and TfR2. TfR1, also known as CD71, is found to be ubiquitously expressed at low levels in most normal human tissues, while TfR2, which is homologous to TfR1, is largely restricted to hepatocytes [104,113]. Despite its ubiquitous expression, TfR1 is expressed on malignant cells at levels several fold higher than those in normal cells, and its expression can be correlated with tumor stage or cancer progression [105–109]. TfR1 is found to be overexpressed in several human carcinomas including breast, ovary, and brain cancers such as glioma and glioblastomas [110–112]. This high expression of the receptor on malignant cells, its ability to internalize, and the necessity of iron for cancer cell proliferation make this receptor a widely accessible portal for the delivery of drugs into malignant cells, and thus an attractive docking site for targeting anticancer drugs in cancer therapy. TfR1, a type-II transmembrane protein, is a transmembrane homodimer that can bind up to two molecules of Tf through its extracellular carboxyl end binding sites. Transferrin receptor 2 (TfR2) shares a 45% identity and 66% similarity in its extracellular domain with TfR1 [113]. These findings make the transferrin family of receptors potentially valuable cancer biomarkers as well. Moreover, transferrin receptor and the receptor for insulin are highly expressed by the endothelial cells forming the blood–brain barrier (BBB), providing a way for drugs to enter the nervous system [114,115]. As the expression of TfR2 is restricted to hepatocytes, TfR1 will be treated as equivalent to TfR in the remainder of this review for the sake of clarity. Jiang et al. [116] reported that transferrin-conjugated superparamagnetic iron oxide NPs (Tf-SPIONs) acted as promising contrast agents in targeted magnetic resonance imaging (MRI) for the detection of glioma in the brains of rats. MRI showed obvious contrast enhancement of brain glioma that could still be clearly observed even 48 h post injection due to the retention of Tf-SPIONs in the cytoplasm of tumor cells, as proven by Prussian blue staining. A second factor supporting TfR as an appropriate target in pancreatic cancer is that the

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TfR is recycled during internalization in rapidly dividing cancer cells, thus improving the uptake of Tf-conjugated vectors [117]. Camp et al. [118] have delivered human wild type p53 protein (SGT53) (in combination with anticancer gemcitabine, probably to enhance the anticancer activity) bound with liposomal NPs, targeted to the transferrin receptor by a single-chain antibody fragment (TfRscFv) in an in vivo metastatic pancreatic cancer model. Compared with untreated mice harboring metastatic tumors with a median survival of 20 days, SGT-53, gemcitabine and SGT-53–gemcitabine complex increased the median survival to 29, 30 and 37 days, respectively. The tumor targeting liposomal-based SGT-53 nanoparticle is capable of sensitizing pancreatic cancer to conventional chemotherapy in pancreatic cancer models. This approach has the potential to be translated into a new, more effective therapy for pancreatic cancer. Further optimization is ongoing moving towards a Phase 1B/2 clinical trial [118]. Using transferrin-conjugated NPs, however, Salvati et al. [119] have found that proteins in the media can shield transferrin from binding to both its receptors on cells and soluble transferrin receptors. Although NPs continue to enter cells, the targeting specificity of transferrin is lost. Their results suggest that when NPs are placed in a complex biological environment, interaction with other proteins in the medium and the formation of a protein corona can ‘screen’ the targeting molecules on the surface of NPs and cause a loss of targeting specificity [119]. 2.5. Epidermal growth factor receptors Epidermal growth factor receptors (EGFRs) are important targets for anticancer therapy. Special attention has been given to ligands that interact with EGFRs. EGFRs are overexpressed in a wide variety of human cancers including cancers of the lung, breast, bladder, and ovary. EGFR expression has also been found to associate with various features of advanced disease with poor prognosis [120]. Various EGFRtargeting vectors and conjugates have been reported as delivery agents of cytotoxic drugs, toxins, or radionuclides [121]. Moreover, it is an ‘internalizing’ receptor, indicating that following ligand–EGFR binding, the ligand–receptor complex is actively endocytosed. Thus, directing nanocarriers by linking them with ligands specific to EGFRs provides a promising way to achieve receptor-mediated intracellular delivery of the carrier with its anticancer cargo. The EGFR family consists of four members: EGFR (ErbB1, HER1), ErbB2 (HER2, neu in rodents), ErbB3 (HER3) and ErbB4 (HER4). These structurally related receptors are single chain transmembrane glycoproteins consisting of an extracellular ligand-binding ectodomain, a transmembrane domain, a short juxtamembrane section, a tyrosine kinase domain and a tyrosinecontaining C-terminal tail. Binding of soluble ligand to the ectodomain of the receptor promotes homo- and hetero-dimer formation between receptors. Receptor dimerization is essential for activation of the intracellular tyrosine kinase (TK) domain and for the phosphorylation of the C-terminal tail [122]. Phosphotyrosine residues then activate, either directly or through adaptor proteins, downstream components of signaling pathways including Ras/MAPK, PLCg1/PKC, PI(3)kinase/Akt, and STAT pathways [123]. EGFR and ErbB4 can be thought of as fully functional receptors with the ability to bind ligands as well as autophosphorylate C-terminal tails through functional intracellular tyrosine kinase domains. ErbB2, however, is unique in that it has no known ligand but is the preferred dimerization partner for other EGFRs [124,125]. ErbB3 is also unique as it has no intrinsic tyrosine kinase activity but can transduce signals through interactions with kinase active receptors, namely EGFR, ErbB2, and ErbB4. Although all ErbB receptors have been localized to the nucleus, ErbB4 is notable for its ability to directly transduce extracellular signals to the nucleus through liberation of the intracellular domain by a ligand-dependent dual protease cleavage of the receptor [126]. In essence, EGFRs bind ligands, resulting in receptor homo- or hetero-dimerization followed by receptor internalization (primarily via clathrin-mediated endocytosis) and activation of the cytoplasmic tyrosine kinase domain [127].

In an initial study using EGFR to deliver a potential anticancer drug in nanocarriers conjugated with a novel EGFR ligand called D4 peptide (D4: Leu-Ala-Arg-Leu-Leu-Thr), liposomes accumulated in EGFRexpressing xenograft tumor tissues up to 80 h after intravenous delivery, a marked contrast compared with the control group [128]. Another study combined the somewhat less frequently used anticancer drug methotrexate (MTX) with human serum albumin (HAS) NPs decorated with trastuzumab (TMAB) molecules to form a nanoconjugate of TMAB–MTX–HAS. TMAB–MTX–HAS achieved significantly higher uptake and cytotoxicity in HER2 positive cells than non-targeted MTX– HSA NPs and free MTX [129]. TMAB is a monoclonal antibody working as ligand against HER2 receptors reported to be overexpressed in 20–30% of human breast cancers [130]. Other investigators prepared a functional liposome of poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) with an amino-active group for conjugation with a peptide ligand (a heptapeptide). The peptide-conjugated PLGA-PEG NPs loaded with DOX showed three-fold higher uptake than peptidefree PLGA-PEG NPs in human ovarian carcinoma (SKOV3) cells with high levels of EGFR expression [131]. Han et al. [132] found that conjugation of a pentapeptide (AEYLR or Ala-Glu-Tyr-Leu-Arg) with nanostructured lipid carriers caused increased cellular uptake of nanocarriers in tumor cells expressing EGFR. Analyses with flow cytometry and an internalization assay using human non-small-cell lung carcinoma (NCI-H1299) and human leukemia (K562) cells, with high EGFR and no EGFR expression, respectively, indicated that FITC-AEYLR had high EGFR targeting activity. Biotin-AEYLR specifically bound to human EGFR, demonstrating high affinity for human non-small-cell lung tumors. In a model of multidrug resistance (MTDR), EGFR binding peptide (EBP) linked DOX was found to accumulate equally in DOXresistant (SW480/DOX) and non-resistant (SW480) cell lines of human colon cancer, at higher levels than free, unconjugated DOX in DOX-resistant cells [133]. 2.6. Fibroblast growth factor receptors Overexpression of fibroblast growth factor receptors (FGFRs) has been reported in tumors of the breast, prostate, bladder, and gastric cancer, and has been associated with tumor progression and poor patient prognosis [134,135]. There are four FGFR genes (FGFR1–FGFR4) that encode receptors consisting of three extracellular immunoglobulin domains (D1–D3), a single-pass transmembrane domain and a cytoplasmic tyrosine kinase (TK) domain [136]. A hallmark of FGFRs is the presence of an acidic, serine-rich sequence in the linker between D1 and D2, termed the acid box. The D2–D3 fragment of the FGFR ectodomain is necessary and sufficient for ligand binding and specificity, whereas the D1 domain and the acid box are proposed to have roles in receptor autoinhibition [137]. Depending on the localization and cancer type, different types of FGFRs are overexpressed. For example, elevated levels of FGFR1, FGFR2, and FGFR4 are commonly found in cancers of the breast and prostate, whereas only FGFR2 overexpression is observed in gastric cancers, and papillary thyroid carcinoma is limited to the overexpression of FGFR1 and FGFR3 only [138]. Moreover, a low level of FGFR expression is found on the surface of non-cancerous cells in the vicinity of the tumor. Due to their specific expression in various cancer types, FGFRs represent potential therapeutic targets [139]. Fibroblast growth factors (FGFs), ligands of FGFRs, exert their physiological roles through FGFRs, regulating developmental pathways such as mesoderm patterning in the early embryo. The mammalian FGF family is composed of 18 ligands that elicit their actions through four (i.e., FGFR1, FGFR2, FGFR3, and FGFR4) highly conserved transmembrane tyrosine kinase receptors [140]. Xiao et al. [141] developed a novel cationic liposomal nanocarrier for DOX which was further conjugated with truncated human basic fibroblast growth factor (tbFGF) peptide, a modified peptide containing binding sites for the FGF2 receptor and part of heparin. This peptide could effectively bind FGFR2 on the cell surface without stimulating

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cell proliferation. As a result, conjugation of tbFGF led to an enhanced anti-proliferative activity in mouse Lewis lung carcinoma (LLC) cells by the synergistic actions of DOX and plasmid DNA (encoding the phosphorylation-defective mouse survivin threonine 34 → alanine mutant gene known as Msurvivin T34A plasmid) targeted in the same cells by the same cationic liposome. The concentration of DOX in the codelivery system causing 50% cell killing was nearly 3-fold lower than the concentration of free DOX responsible for the same cytotoxicity. Furthermore, the co-delivery system suppressed tumor growth more efficiently than either DOX or the Msurvivin T34A plasmid alone in the Lewis lung carcinoma-bearing C57BL/6 mice. The co-delivery system also caused a 15 day delay of tumor growth, longer than the other treatment groups. In conclusion, this novel cationic liposome is an efficient vector for the simultaneous delivery of drugs and DNA to the same cells in vitro and in vivo. Another study compared the pharmacokinetics and tissue distribution of a novel truncated basic fibroblast growth factor peptide-mediated cationic liposomal PTX (tbFGF-LPsPTX) with free PTX and cationic liposomal PTX (LPs-PTX) in tumorbearing mice. The data indicated that the concentration of tbFGF-LPsPTX significantly increased in the tumor and that the retention time was prolonged [142]. Another group prepared biocompatible and biodegradable nanocarriers of cholesterol-block-poly(ethylene glycol) (Chol-PEG2000-COOH) polymer with PTX linked with truncated bFGF fragments (tbFGF). This nanocarrier significantly enhanced the cytotoxicity caused by targeted PTX in FGFR expressing LL/2 (a modified Lewis lung carcinoma) cells as measured by an MTT {3-(4,5-dimethyl thiazol2-yl)-2,5-diphenyl tetrazolium bromide} assay. Flow cytometry revealed that the cellular uptake of rhodamine B, encapsulated in the tbFGF conjugated micelles, was increased by 6.6-fold for HepG2 (human liver carcinoma), 6.2-fold for A549 (human lung adenocarcinoma), 2.9-fold for C26 (mouse colon adenocarcinoma) and 2.7-fold for LL/2 (mouse Lewis lung carcinoma) cells, respectively, compared with micelles without tbFGF. The fluorescence spectroscopy further demonstrated that the tbFGF-conjugated micelles specifically bound to tumor cells over-expressing FGFRs and then released rhodamine B into the cytoplasm [143]. Biodistribution studies in C57BL/6J mice bearing B16 melanoma revealed a higher accumulation of tFGF targeted PTX delivered by PEGylated liposomes in tumor tissue and organs containing the mononuclear phagocyte system (liver and spleen), but a considerable decrease in other organs (heart, lung, and kidney) as compared with PEGylated-liposome-PTX without tFGF and free PTX [144]. Szlachcic et al. [145] developed novel FGF1 variant-AuNP conjugates found to be specifically internalized only in cells expressing FGFRs. FGF1 coated AuNPs significantly reduced viability in cells expressing FGFRs after irradiation with near-infrared light (down to 40% of control cell viability), whereas the proliferation potential of cells lacking FGFRs was not affected. 2.7. Sigma receptors Researchers have recently become interested in exploring sigma receptor ligands for tumor imaging and targeted therapy. Sigma receptors are overexpressed in a variety of human tumors including cancers of non-small cell lung carcinoma (NSCLC), prostate, melanoma, and breast [146–148]. Sigma receptor was initially proposed to represent a subtype of opioid receptors [149]. These subtypes display different tissue distributions and distinct physiologic and pharmacologic profiles in both the central and peripheral nervous systems. Sigma-1 and -2 receptors were classically distinguished on the basis of their binding affinity for [3H](+)-pentazocine and [3H]-1,3-di(2-tolyl)guanidine ([3H]-DTG). Both sigma receptors bind pentazocine, while only the sigma-2 receptor binds guanidine-DTG [150–152]. Although the sigma-1 receptor is a well-characterized protein, the sigma-2 receptor protein has largely remained elusive [153]. However, the novel sigma-2 receptor ligand SW43 was reported to stabilize the progression of pancreatic cancer in combination with gemcitabine [154]. The sigma-1 receptor (S1R), a

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ligand regulated membrane protein chaperone of 25 kDa possessing one putative transmembrane domain and an endoplasmic reticulum retention signal, is involved in the ER stress response and inter-organelle communication [155–157]. S1R is expressed primarily in the cerebral cortex, hippocampus, and cerebellar Purkinje cells [158,159], and has been proposed as a target for the treatment of central nervous system diseases including amnesia, pain, schizophrenia, clinical depression, Alzheimer's, stroke, and addiction [160,148]. S1R is primarily localized to mitochondria-associated ER membranes [161,162], which are the sites for mitochondrial bioenergetic regulation through the release of ER calcium [163]. S1R activity is modulated by a number of endogenous molecules such as N,N dimethyltryptamine, progesterone and sphingosine, and exogenous molecules including opiates, antipsychotics, antidepressants, antihistamines, phencyclidine-like compounds, βadrenergic receptor ligands, and cocaine [164–166]. Upon binding to these molecules, activated S1R causes the dissociation of ankyrin from the inositol triphosphate receptor (IP3R) [167], resulting in calcium release at the ER–mitochondrial interface. This released calcium is efficiently taken up by mitochondria to increase energy production and other stress responses [168]. Although the natural ligand for the sigma-2 receptor (S2R) and the detailed structure of S2R itself remain unknown, this receptor has been shown to be overexpressed in a variety of human tumors including breast, pancreas, neuroblastoma, bladder, and lung. As such, various S2R ligands have been extensively studied for their effectiveness in the treatment of solid tumors due to their preferential uptake in proliferating cells [169]. Equally important is the notion that S2R ligands are rapidly internalized into cancer cells [170,171], including pancreatic cancer which expresses the receptor at low levels [172]. Synthetic ligands to this receptor could play an important role in cancer diagnosis, imaging, and targeted drug delivery [173]. SV119, an S2R ligand, binds to pancreatic cancer cells and induces target cell death in vitro and in vivo. When conjugated to SV119, small molecular modulators known to interfere with intracellular prosurvival pathways retained their ability to induce cell death, and their efficiency was enhanced by the targeting effect of SV119. These findings indicate the utility of S2R ligands to increase the tumor-specific delivery of small molecules [174]. SV119 targeting of DOX-loaded liposomes to S2R-overexpressing cells led to significantly higher uptake of SV119conjugated liposomes in cancer cells of human breast (MCF-7), human prostate (PC-3, DU-145) and human lung (A549, 201T) lineages compared to normal human bronchial (Beas-2B) cells. Thus, the SV119 ligand caused significantly higher cytotoxicity in DU-145 cells when compared with the cytotoxicity induced by liposomal DOX without ligand [175]. Similarly, gold nano-cages functionalized with SV119 again demonstrated the promising ability of this ligand–receptor system for cancer cell targeting [176]. These results suggest that the SV119–S2R system might be an effective tool for targeting certain tumors. 2.8. Other overexpressed receptors Some less common receptors recently used for targeting drugs to cancer cells are follicle-stimulating hormone receptors, biotin receptors, C-type lectin receptors and neuropilin receptors. Overexpression of follicle-stimulating hormone receptors (FSHRs) during ovarian cancer makes FSHR a possible target against ovarian cancer. Follicle stimulating hormone (FSH) is a glycoprotein consisting of α and β chains. Some FSHR-binding domains in FSH have been identified including amino acids 1 to 15, 33 to 53, 51 to 65, and 81 to 95 of the FSH β chain [177, 178]. The ovary is the target organ of FSH, which binds to the FSH receptor (FSHR), a G protein-linked receptor. Overexpression of FSHR was found on the ovarian surface epithelium and in some ovarian cancer cell lines [179,180]. Zhang et al. [181] developed a ligand of FSHR called FSH33–53 that was derived from 33 to 53 amino acids of the FSH β chain. Later,

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FSH33–53 was conjugated with NPs constructed from PEG-PLA [polyethylene glycol-poly(lactic acid)] forming FSH33–NP complexes. The anticancer drug PTX was chosen to test the targeting potential of FSH33–NPs against ovarian cancer. The FSH33–NP–PTX nanoconjugates displayed stronger anti-proliferation and antitumor effects compared with free PTX or PTX-loaded NPs without FSH33 both in vitro and in vivo. In summary, this novel FSH33–NP delivery system showed very high selectivity and efficacy for FSHR-expressing tumor tissues. Therefore, it has a strong potential to become a new therapeutic approach for patients with ovarian cancer. Kim et al. [182] reported that biotin-conjugated poly(ethylene oxide)/poly(ε-caprolactone) (PEG/PCL) amphiphilic block NPs carrying PTX induced higher cytotoxicity in cancer cells than NPs carrying PTX but not conjugated with biotin. These results showed that biotinconjugated NPs could improve the delivery of PTX into cancer cells via interactions with overexpressed biotin receptors. Chen et al. [183] have shown a good correlation between drug uptake and biotin receptor expression. These investigators have conjugated secondgeneration taxoid (SB-T-1214) as the cytotoxic agent with biotin and reported excellent drug uptake and cytotoxicity in L1210FR leukemia cells overexpressing biotin receptors compared with two biotin receptor negative cell lines, L1210 leukemia cells and WI38 normal human lung fibroblast cells. Other investigators have shown that gold NP surface-functionalized with PTX and biotin are promising for applications in cancer diagnosis and therapy [184]. C-type lectin receptors (CLRs) have emerged with great potential in cell-specific drug and gene delivery [185–187]. CLRs belong to a large family of receptors that share a structurally homologous carbohydrate-recognition domain and often bind to glycan structures in a Ca2+-dependent manner [188], including the collectins, selectins, lymphocyte lectins, and proteoglycans. Because of their endocytic properties, CLRs are suitable targets for cell-specific drug delivery [189]. In addition to cell-specific drug delivery, CLRs may also be exploited to modulate the functions of CLR-expressing cells such as endocytosis or cell activation. In innate immunity, CLRs are mainly expressed by antigen presenting cells such as dendritic cells and macrophages. They serve as pattern-recognition receptors and bind to pathogen-associated molecular patterns (PAMPs) [190], but may also sense self-antigens released by damaged tissue or dead cells [191]. Hepatic lectin receptors including asialoglycoprotein receptor (ASGPR) are capable of mediating the endocytosis of bound ligands. Following recognition, bound ligands are internalized via coated pits and the complex is then released into endosomal compartments while the lectin receptor is recycled to the cell surface for another round of endocytosis [192]. Thus, hepatic lectin receptors represent promising targets to be exploited in liver-specific drug and gene delivery. The human ASGPR, a type II transmembrane protein, consists of two subunits, H1 and H2, with the H1 subunit mediating

Ca2+-dependent galactose/GalNAc recognition [193]. In a more recent study, liposomes incorporated with synthetic glycolipids containing a terminal GalNAc residue (acting as a ligand for ASGPR) were shown to be internalized into hepatocytes by ASGPR-mediated endocytosis [194]. Singh et al. [195] reported five times higher uptake of PTX-loaded magnetic NPs (PTX-MNPs) conjugated with lectins in K562 leukemia cells compared to lectin receptor-negative human embryonic kidney (HEK 293) cells. Neuropilin receptors (NRPs) were initially considered to be expressed in neuronal cells but were later found to be expressed in many cell types such as myofibroblasts, endothelial cells and tumors. The two NRPs (NRP-1 and NRP-2) expressed in vertebrates are transmembrane glycoproteins that show 44% homology at the amino acid level. These receptors contain four distinct extracellular domains that mediate ligand binding and a short cytoplasmic domain that lacks catalytic activity [196]. NRP-1 is highly expressed in diverse tumor cell lines including human neoplasms, and has potential implications in tumor growth and vascularization [196,197]. CRGDK peptide, a ligand of the NRP-1 receptor, increased the intracellular uptake of gold NPs carrying the therapeutic P12 peptide (p12) in MDA-MB-321 human breast cancer cells overexpressing the NRP-1 receptor [198]. 3. Conclusion Targeting drugs to cancer cells and the tumor microenvironment is a major challenge in the field of anticancer therapy. This article has discussed strategies for achieving the specific delivery of anticancer drugs to cancer cells and tissues by exploiting specific or overexpressed receptors through the use of nanotechnology. Nanotechnology has enabled many options in manipulating nanocarriers based on proteins, lipids and metals, which can be loaded with anticancer drugs and directed mostly to cancer cells by the attachment of moieties that recognize cancer phenotypes. NPs ranging from lipids to metals are under exploration as vehicles for anticancer drug delivery and as moieties guiding drugs to tumor cells. Lipid-based NPs include polymers, micelles and liposomes. Note that lipid-based NPs are composed of the same blocks/ monomers, but their main structural differences lie in their threedimensional structures. Polymeric NPs are single, linear but folded polymers made of the same or different blocks, whereas dendrimers are composed of many macromolecules and are thus tree-like and hyperbranched. Liposomes are well known vesicular structures composed of lipid bilayers enclosing an aqueous core, whereas micelles are composed of lipid monolayers without internal aqueous compartments. All are colloidal structures with sizes ordered as polymeric NPs ( 10 nm), dendrimers (2–10 nm), micelles (10–100 nm) and liposomes (100–200 nm) [199]. Polymeric NPs may be homopolymers (composed of the same monomer) or heteropolymers (composed of more than two

Size and stability of nanoparticle-ligand complex determining its circulation time? Nanoparticle’s drug release efficiency at the desired site?

NP nanoparticle Anticancer drug molecules

Ligand immunogenicity? Nanoparticle’s loading efficiency with anticancer drug? Effect of bound ligand on its native affinity with receptor?

Receptor ligands

Receptor

Nanoparticle’s biocompatibility and systemic toxicity? Spatial and temporal variability in receptor overexpression? Fig. 2. Major issues that need to be addressed in multidisciplinary research involving material sciences and life sciences.

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different monomers). Liposomal NPs are mainly composed of PEG, whereas micellar NPs are primarily based on PEG with another block of cholic acid (CA) [72], poly(lactic acid) [69] and poly(trimethylene carbonate) (PEG-PTMC) [70]. Instances of metal-based NPs include iron oxide [116] and gold NPs [145,176,184,198]. Human serum albumin (HSA) NPs [129] and quantum dots are other nanoscale drug carriers [93]. Lipid-based NPs have been frequently used in anticancer therapy as carriers of anticancer drugs, especially in targeted delivery facilitated by overexpressed receptors. Physicochemical factors such as the dimensions and stability of NPs, the ease of ligand conjugation, the drug loading ability, and retention times all are likely to affect the suitability of individual nanocarriers to varying degrees. While nanotechnology is a highly promising technique for targeted drug delivery to cancer cells and the tissue microenvironment, this approach will not achieve its potential success unless some technological and toxicological challenges are addressed (Fig. 2). Several mechanisms of triggered drug release can only be applied once the anticancer drug reaches and accumulates at the tumor site. Major issues in nanocarrier design include nanoparticle–drug loading efficiency, the overall stability of nanoconjugates with attached ligands, optimal receptor–ligand interactions, and the duration of expression of the targeted receptor. Nanoparticle toxicity and ligand immunogenicity are additional critical factors that require proper attention in the engineering of clinically effective nanocarriers for the targeting of anticancer drugs. Acknowledgment This work was supported by the Research Chair of King Saud University on Drug Targeting and Treatment of Cancer using Nanoparticles. References [1] Boyle P, Levin B. World Cancer Report. Lyon, France: International Agency for Research on Cancer Press; 2008. [2] Sutcliffe SB. Cancer control: life and death in an unequal world. Curr Oncol 2012;19:12–5. [3] Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646–74. [4] Iyer AK, Greish K, Fang J, et al. Exploiting the enhanced permeability and retention effect for tumour targeting. Drug Discov Today 2006;11:812–8. [5] Maeda H. Tumour-selective delivery of macromolecular drugs via the EPR effect: background and future prospects. Bioconjug Chem 2010;21:797–802. [6] Lee ES, Bae YHJ. Recent progress in tumour pH targeting nanotechnology. J Control Release 2008;132:164–70. [7] Wei H, Zhang XZ, Chen WQ, et al. Self-assembled thermo sensitive micelles based on poly(L-lactide-star block-N-isopropyl acrylamide) for drug delivery. J Biomed Mater Res 2007;83:980–9. [8] Wei H, Zhang X, Cheng C, et al. Self-assembled, thermosensitive micelles of a star block copolymer based on PMMA and PNIPAAm for controlled drug delivery. Biomaterials 2007;28:99–107. [9] Saito G, Swanson JA, Lee KD. Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities. Adv Drug Deliv Rev 2003;55:199–215. [10] Rapoport NY, Christensen DA, Fain HD, et al. Ultrasound-triggered drug targeting of tumours in vitro and in vivo. Ultrasonics 2004;42:943–50. [11] Han G, You CC, Kim BJ, et al. Light regulated release of DNA and its delivery to nuclei by means of photolabile gold nanoparticles. Angew Chem Int Ed Engl 2006;45:3165–9. [12] Mura S, Nicolas J, Couvreur P. Stimuli-responsive nanocarriers for drug delivery. Nat Mater 2013;12:991–1003. [13] Schroeder A, Heller DA, Winslow MM, et al. Treating metastatic cancer with nanotechnology. Nat Rev Cancer 2012;12:39–50. [14] Zwanziger D, Beck-Sickinger AG. Radiometal targeted tumour diagnosis and therapy with peptide hormones. Curr Pharm Des 2008;14:2385–400. [15] Wettschureck N, Offermanns S. Mammalian G proteins and their cell type specific functions. Physiol Rev 2005;85:1159–204. [16] Oldham WM, Hamm HE. How do receptors activate G proteins? Adv Protein Chem 2007;74:67–93. [17] Jensen RT, Battey JF, Spindel ER, et al. International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states. Pharmacol Rev 2008;60:1–42. [18] Sancho V, Di Florio A, Moody TW, et al. Bombesin receptor mediated imaging and cytotoxicity: review and current status. Curr Drug Deliv 2011;8:79–134. [19] Fathi Z, Corjay MH, Shapira H, et al. BRS-3: novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells. J Biol Chem 1993;268:5979–84.

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