Nanoparticles and targeted drug delivery in cancer ...

Accepted Manuscript Title: Nanoparticles and targeted drug delivery in cancer therapy Authors: Behdokht Bahrami, Mohammad Hojjat-Farsangi, Hamed Mohammadi, Enayat Anvari, Ghasem Ghalamfarsa, Mehdi Yousefi, Farhad Jadidi-Niaragh PII: DOI: Reference:

S0165-2478(17)30176-1 http://dx.doi.org/doi:10.1016/j.imlet.2017.07.015 IMLET 6077

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Immunology Letters

Received date: Revised date: Accepted date:

14-4-2017 4-7-2017 26-7-2017

Please cite this article as: Bahrami Behdokht, Hojjat-Farsangi Mohammad, Mohammadi Hamed, Anvari Enayat, Ghalamfarsa Ghasem, Yousefi Mehdi, Jadidi-Niaragh Farhad.Nanoparticles and targeted drug delivery in cancer therapy.Immunology Letters http://dx.doi.org/10.1016/j.imlet.2017.07.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nanoparticles and targeted drug delivery in cancer therapy Behdokht Bahrami1,2, Mohammad Hojjat-Farsangi3,4, Hamed Mohammadi5,6, Enayat Anvari7, Ghasem Ghalamfarsa8, Mehdi Yousefi6,9, and Farhad Jadidi-Niaragh5,6,10*

1. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 2. Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. 3. Department of Oncology-Pathology, Immune and Gene Therapy Lab, Cancer Center Karolinska (CCK), Karolinska University Hospital Solna and Karolinska Institute, Stockholm, Sweden. 4. Department of Immunology, School of Medicine, Bushehr University of Medical Sciences, Bushehr, Iran. 5. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 6. Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. 7. Department of Physiology, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, Iran. 8. Medicinal Plants Research Center, Yasuj University of Medical Sciences, Yasuj, Iran. 9. Stem Cell and Regenerative Medicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran. 10. Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

*Corresponding author: Farhad Jadidi-Niaragh, Ph.D. Assistant Professor of Immunology, Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Tel: +98 411 336 46 65 Fax: +98 411 336 46 65 Email: [email protected]

Running title: Targeted cancer therapy by nanoparticles

Highlights   

Nanomedicine improved the current immunotherapeutic approaches. Nanomaterials can reduce the adverse effects of current anti-cancer drugs. Targeted drug delivery by nanomaterials is an effective anti-cancer therapeutic approach.

Abstract Surgery, chemotherapy, radiotherapy, and hormone therapy are the main common anti-tumor therapeutic approaches. However, the non-specific targeting of cancer cells has made these approaches non-effective in the significant number of patients. Non-specific targeting of malignant cells also makes indispensable the application of the higher doses of drugs to reach the tumor region. Therefore, there are two main barriers in the way to reach the tumor area with maximum efficacy. The first, inhibition of drug delivery to healthy noncancer cells and the second, the direct conduction of drugs into tumor site. Nanoparticles (NPs) are the new identified tools by which we can deliver drugs into tumor cells with minimum drug leakage into normal cells. Conjugation of NPs with ligands of cancer specific tumor biomarkers is a potent therapeutic approach to treat cancer diseases with the high efficacy. It has been shown that conjugation of nanocarriers with molecules such as antibodies and their variable fragments, peptides, nucleic aptamers, vitamins, and carbohydrates can lead to effective targeted drug delivery to cancer cells and thereby cancer attenuation. In this review, we will discuss on the efficacy of the different targeting approaches used for targeted drug delivery to malignant cells by NPs. Keywords: Nanoparticles, cancer immunotherapy, targeted drug delivery

1. Introduction Targeted cancer therapy can discriminate the small differences between normal and cancer cells. Targeted therapies are usually more effective than other conventional treatments and exhibit lesser unwanted adverse effects. Since the non-specific and systemic drug delivery leads to rapid elimination of drug, administration of the highest tolerable dose of the drug is needed which is not economical and usually exhibits high toxicity. In recent years, accumulating studies have been shown the efficacy of nanosized materials for tumor targeting, diagnosis (imaging) and therapy (1). Nanoparticles (NPs) are nanosized materials that can embed drugs, imaging agents, and genes (2). NPs can deliver the high doses of therapeutic factors into tumor cells while bypass normal cells. While the scaffold structure of NPs enables the attachment of drugs and contrast agents, their surface facilitates biodistribution and specific delivery through conjugation with ligands that bind to tumor biomarkers (3). NPs have solved the problems of conventional chemotherapy, including non-specific biodistribution, drug resistance, and unwanted adverse effects. Interesting features of NPs have been led to the entrance of several NP-based therapeutics into the clinical trial stage during the last two decades (4). Possibility of modulation of various features of NPs has made them as potent therapeutic vectors for cancer therapy. Nanocarriers increase the circulation half-life of therapeutics in body and enhance their accumulation in tumors site, which is in part related to the small size of NPs and deregulated vascular structure and enhanced permeability and retention (EPR) effects (5). The physicochemical features of nanocarriers significantly affect the half-life and biodistribution of NPs (6, 7). Size of NPs is an important factor in the fate of particles. While NPs smaller than 7 nm in hydrodynamic diameter fall into renal filtration and urinary excretion (8, 9), nanomaterials larger than 100 nm are usually cleared from the circulation by phagocytic cells (10, 11). Moreover, the surface positive charge of particles facilitates internalization into the cancer cells. Surface addition of some polymers such as polyethylene glycol (PEGylation) to NPs can also enhance the circulation half-life of particles in part through prevention of clearing by reticuloendothelial system and enhancing the accumulation of particles in tumor site (12). Although, surface modification of NPs may improve their circulation time, however, it can affect their internalization into cancer cells. Therefore, addition of some tumor-specific ligands to the surface of NPs (active targeting) which facilitates internalization of NPs into cancer cells may lead to overcome this problem (13). 2. Therapeutic NPs Several NPs have been used in the wide variety of pathologic conditions during the last two decades (14). Liposomes with a lipid scaffold structure were discovered 40 years ago by Bangham (15). Liposomes are composed of self-assembled phospholipids into bilayers with spherical shape (16). Size of these nanomaterials is varied from the 30 nm to microns (17). Liposomes can encapsulate both the hydrophilic and hydrophobic therapeutic factors within the vesicles and lipid bilayer, respectively. These nanocarriers are highly biocompatible and can easily modified for exhibition of better properties, such as increased circulation time and active targeting (18). Currently, multiple liposome-based anti-cancer therapeutic compounds such as DaunoXome®, Myocet®, VincaXome®, DepoCyt®, Doxil®, Caelyx® are available in the market for clinical use (19). Nanostructured lipid carriers (NLC), which were identified in the late 1990s are composed of a mixture of a solid and liquid lipid (20). These nanocarriers can potently internalized by tumor cells and exhibit several advantages, including high drug loading potential, controlled drug release, increasing drug stability, and the ease of large-scale generation (21, 22). Solid Lipid NPs (SLNs) are non-toxic nanocarriers generated with natural lipids or synthetic lipids (23). Production of SLNs does not need to use of toxic organic solvents, which help to intact maintenance of the drug composition. These nanomaterials can carry both the lipophilic or hydrophilic drugs. SLNs are versatile

nanocarriers since they are capable to controlled release and protection of drugs which lead to possibility of administration through both the parenteral and non-parenteral routes (24). Poly (lactic-co-glycolic acid) (PLGA) is a biodegradable polymeric NP composed of co-polymerization of the glycolic acid and lactic acid, and approved by the Food and Drug Administration (FDA) for drug delivery (25). Due to hydrolysis of PLGA in the body to its original components, it is considered as a very useful nanovector. Lupron Depot ® which is PLGA-based commercial nanocarrier is used for the attenuation of advanced prostate cancer. Dendrimers are composed of the repeatedly highly branched polymeric star-like molecules with a 3D geometric shape. Dendrimers exhibit three different parts including a central core, the branches, and an exterior surface with various surface functional groups (26). There are two main strategies for production of these dendrimers including divergent (outward from the core) and convergent (inward towards the core) strategies (27). Presence of tertiary amines in the structure of dendrimers let us to add various molecules for active targeting (28). Dendrimers are characterized through the generation of monomers (G) added to a main core. Dendrimers are the smallest nanocarriers generated with size of 1.9 nm for G1 and 4.4 nm for G4 which facilitates their application in the some specific conditions (29). They are used for both the diagnostic (imaging) and therapeutic purposes (30). Vivagel® is the first dendrimer-based compound which is considered as the Fast Track Status by the FDA (31). Iron oxide NPs are of important types of inorganic NPs with size of 1 to 100 nm in diameter. Since these particles can be visualized by Magnetic Resonance imaging (MRI), they have been used for imaging purposes in various tumors (32). Regarding the magnetic feature of these nanomaterials, they can be used for therapeutic goals via hypethermia through conduction by external magnetic field into tumor site (33). These NPs can also be used for in vivo investigations, because they are biodegradable and degraded iron can be absorbed by hemoglobin in body (34). A superparamagnetic iron oxide NPs (SPIONs) are potent useful nanomaterials that can be applied for both the imaging and therapeutic applications (35). There are multiple iron oxide based NPs in market which can be used for therapeutic or imaging applications such as Ferridex I.V.® , Ferumoxytol® , and Combidex® (35). Gold NPs were identified by Michael Faraday for the first time (36). The surface of gold NPs can be easily modified by amine and thiol groups for tumor specific targeting. Moreover, gold NPs show surface plasmon resonance (37). Regarding the small size of these nanocarriers, they can enter to tumor cells through EPR effect. Gold NPs-based therapeutics have also experienced the early-phase clinical trials which were associated with hopeful outcome (38). Due to the high atomic number of gold NPs, they can also be used as imaging vectors and tumor-selective photothermal therapy (39).

3. The mechanisms of nanoparticle internalization in cancer cells Endocytosis which is the main mechanism of NP internalization in target cells can be categorized into phagocytosis and pinocytosis. The phagocytosis is the main mechanism of capture by phagocytic cells including neutrophils, dendritic cells, and macrophages, whereas the pinocytosis is observed in all cells and may be classified into clathrin- or caveolae-mediated endocytosis, clathrin/caveolae-independent endocytosis, and micropinocytosis (40-42). Large particles are usually captured by phagocytosis pathway. For phagocytosis, NPs should be covered with the opsonins which facilitate their adherence to phagocytic cells through opsonin receptors such as mannose and scavenger receptors. The interaction of receptor–ligand results in actin rearrangement and phagosome formation, leading to the induction of cup-like membrane extension, which capture and internalize the NPs (41).

Pinocytosis is usually effective in the enclosing fluids and suspensions containing small particles. Based on the type of proteins involved, it can be divided to clathrin-dependent and caveolae-dependent endocytosis, macropinocytosis and clathrin- and caveolae-independent endocytosis. Clathrin-dependent endocytosis is observed in all mammalian cells and is involved in the capture of essential nutrients such as cholesterol (LDL) through the LDL receptor, or iron through the Tf receptor (43). Ligated receptors bind to cytoplasmic adaptor proteins to form a clathrin lattice (44). The GTPase activity of dynamin detaches the vesicle from the plasma membrane which leads to generation of a clathrin coated vesicles (45). Poly(ethylene glycol)-polylactide (46), Poly(lactic-co-glycolic acid) (PLGA) (47), Silica-based nanomaterials (48), chitosan (49), surface-modified NPs that target Clathrin-dependent endocytosis (modified with Tf) (50) are some examples of NPs which use Clathrin-dependent endocytosis mechanism for the cellular entry. Caveolae are a type of lipid rafts at cholesterol-rich area of membrane enriched with caveolin-1 (51). As this internalization mechanism bypass lysosomes, several pathogens use caveolae-mediated transport to enter target cells (51). The caveosome has a neutral pH and use actin to move within the cell (52). NPs which use this internalization mechanism bypass a degradation and increase the drug delivery to an ER or nucleus. It has been shown that the anionic NPs usually apply caveolae-dependent endocytosis (53). Macropinocytosis is a growth factor-induced, actin-promoted endocytosis that encloses a large content of fluid-phase (54) and is observed in almost all cells. This route is usually started following stimulation with growth factors such as a colony-stimulating factor-1 (CSF-1), epidermal growth factor (EGF) and plateletderived growth factor or tumor-promoting factor. Activation of receptor tyrosine kinases enhances the formation of membrane ruffles (55, 56). The close fluid and particles can be then taken up into the macropinosomes. There is no general agreement regarding the final fate of macropinosomes (57, 58). Cationic NPs such as chitosan can also use this internalization pathway (59).

4. Drug delivery systems In a traditional oral or intravascular drug delivery approaches, therapeutic factors are distributed throughout the body and only a small part of the drug reaches to tumor site. Tumor specific targeted drug delivery leads to accumulation of drug in the tumor region and decreases the drug leakage into other healthy organs. This approach increases treatment efficacy, while decreasing adverse effects (60). There are two general drug delivery approaches including passive and active (which is also known as ligandbased) targeting (61). NPs can usually be concentrated in the tumor area due to abnormal leaky vasculature of tumor tissue which is also known as enhanced permeation and retention (EPR) effect. EPR effect facilitates transition of nanovectors with size of 400 nm in diameter into the surrounding tumor tissue (62, 63). Therefore, passive targeting depends on some pathophysiological features of tumor tissue including the abnormal vasculature, temperature, pH, and surface charge of tumor cells (61). It is evident that some physicochemical properties of nanocarriers such as size, surface charge, molecular weight, and hydrophobic or hydrophilic feature are crucial for passive targeting. Although passive targeting is interesting approach, however, it suffers from the serious limitations such as inefficient drug diffusion into tumor cells, the random nature of targeting, and the lack of EPR effect in some tumors (64) (Figure 1). One of the best ways to solve the problems of passive targeting is conjugation of ligands of tumor specific biomarkers with nanocarriers which is referred as active targeting (65). There are several targeting moieties such as monoclonal antibodies and their variable fragments, peptides, aptamers, vitamins, and carbohydrates (66). It is evident that tumor specific biomarkers should be overexpressed on tumor cells to reach high specificity (1). Following interaction of ligands with receptors, they can internalize by tumor cells through receptor-mediated endocytosis and their cargo can be released by acidic pH or enzymes (1). Regarding the higher chance of endocytosis by tumor cells, active targeting is more preferred compared to passive approach (67). Among the various targeting ligands, folate, transferrin, epidermal growth factor receptors (EGFRs), and

glycoproteins are the most investigated molecules for active targeting (61). In here, we will discuss on the various active targeting strategies using different targeting molecules.

4.1. Transferrin receptors Iron homeostasis in body which is an important factor in several biochemical processes is regulated by transferrin. Regarding the non-immunogenic, biodegradable, and nontoxic features of transferrin, it is interesting molecule for active targeting (Table 1). Transferrin (with molecular mass 80 kDa) is mainly produced by hepatocytes (68). The human transferrin receptor1 (TfR1) is a homodimer molecule expressed in all nucleated cells (with different expression levels) in the body. While the TfR1 is overexpressed on fast dividing cancer cells, it is low or undetectable in non-proliferating normal cells (69). TfR2 exhibits a 66% homology in its extracellular domain with TfR1. It has been shown that the affinity of TfR1 for iron-loaded Tf is 25-fold higher than TfR1 (70). TfRs are overexpressed in 90% of tumors which is in part due to an augmented need to iron. It is estimated that both the expression of TfR (up to 100-fold) and its binding affinity (10–100 times) are greater on tumor than normal cells (71). Following high affinity attachment of TfRs with diferric Tf (holo-Tf) internalization will be occurred through receptor-mediated endocytosis process. Upon endocytosis, iron will be released in early endosomes and apoTfR recycled to the apical plasma membrane. Regarding the overexpression of TfR on tumor cells and its correlation with cancer progression, Tf has been applied as a ligand for tumor targeting (72). Accordingly, conjugation of Bortezomib-loaded PLGA NPs with holo-transferrin (hTf) led to the increased targeted delivery of bortezomib-loaded NPs to pancreatic cancer cells, in vitro. Interestingly, it has been shown that conjugation of Tf with doxorubicin (DOX) led to exhibition of higher cytotoxicity in DOX-sensitive tumor cell lines such as HL60, Hep2, and particularly in resistant cell lines compared to free drug (73). Surface modification of poly(γ-glutamic acid-maleimide-co-llactide)-1,2-dipalmitoylsn-glycero-3-phosphoethanolamine (-PGA-MAL-PLA-DPPE) copolymer with Tf also resulted in a better cellular uptake of NPs by nasopharyngeal carcinoma (C666-1) and human cervical carcinoma (Hela) cells (74). Ligation of Tf with the PEG-PLA biotin (Tf–PEG-PLA) micelles was also associated with specific targeting of nanocarrier into rat C6 glioma cells in vitro and in vivo (75). In another study, Ding and colleagues developed a multifunctional DOX loaded Fe3O4@SiO2-Tf based drug delivery system which displayed high cellular uptake by TfR-expressing cancer cells and potent cytotoxicity (76). Dual delivery of both DOX and sorafenib to Hepatocellular carcinoma (HCC) cells by Tf modified core-shell nanocarrier enhanced cellular uptake and cytotoxicity in tumor cells, examined in both 2D and 3D cultures (77). Conjugation of Tf with DOX-loaded lipid coated NPs and their coculture with A549 lung cancer cells inhibited the proliferation of malignant cells with more efficacy compared to non-targeted NPs (78). Moreover, Bao et al. showed that specific targeting of Daunorubicin-PLGA-PLL-PEG NPs by Tf increased concentration of drug in K562 cells in vitro and arrested tumor growth in the xenograft models, in vivo (79). In another investigation, conjugation of plasmid DNA-loaded dendrimers of diaminobutiric poly(propylene imine) (DAB) G-3 with Tf increased the intracellular expression of plasmid DNA in tumor cells. Moreover, application of these nanovectors in A431 tumor bearing mice was associated with higher expression of plasmid DNA in tumor samples compared to other tissues (80). Targeting of TfR was also performed by conjugation of PAMAM-PEG G-5 dendrimers with peptide HAIYPRH (T7), which binds to TfR, in order to deliver ADR to TfR overexpressing tumor cells (81).

The presence of the blood-brain barrier (BBB) in the central nervous system (CNS) is the main limiting factor for specific drug delivery (82). High expression of TfR on the BBB makes it possible

to develop Tf conjugated SPIONs that act as potential and specific MRI targeting contrast agent for brain glioma (83). Consistently, Porru and colleagues have been shown that administration of Tf conjugated NPs into mice intramuscularly bearing U373MG-LUC xenografts exhibited higher antitumor efficacy compared to one treated by NPs-ZOL (84). In another study, Kopecka and colleagues used zoledronic acid complexes with calcium phosphate NPs (CaPZ NPs) and cationic liposomes and doxorubicin to overcome chemo- and immune-resistance in breast cancer. They showed that zoledronic acid decreased IC50 of doxorubicin in chemoresistant breast cancer cells and enhance its activity in the chemo- and immune-resistant tumor bearing mice. This ameliorating effect was associated with downregulation of Ras/ERK1/2/HIF-1α axis, apoptosis induction, increased dendritic cells and downregulation of Treg cells (85, 86). It has also been demonstrated that zoledronic acid loaded NPs can effectively activate anti-cancer function of Tγδ cells in a monocyte/macrophage-mediated manner (87). It is confirmed that these zoledronic acid loaded NPs can overcome multidrug resistance in cancer cells (88). It should be noted that self-assembly CaPZ NPs showed higher physicochemical and biologic features compared to liposomal carriers (89). Similarly, another study showed that Tf-modified AuNPs demonstrated high therapeutic potential for brain tumor therapies and/or imaging (90). Similar approach was applied using human serum albumin NPs conjugated with Tf for brain imaging (91, 92). Treatment of CNS related malignancies such as glioblastoma multiform with DOX loaded Tf-coupled PEG-liposomes was also associated with higher cellular uptake of NPs compared to non-targeted particles (93). Administration of Tfconjugated solid lipid NPs into rats led to higher accumulation of quinine dihydrochloride in brain of rats compared to the free drug (94). Administration of Tf-decorated liposomes loaded with p53 encoding DNA into the DU145 prostate cancer model was associated with ameliorating effects (95). A similar nanocarrier loaded with Bcl-2 directed antisense ODN G3139 arrested the growth of K562 cancer cells in vitro and in vivo (96). Oxalate stabilizes the iron atoms in Tf and decreases iron release in the endosome. Accordingly, oxalate-Tf conjugated with diphtheria toxin demonstrated high toxicity in ovarian cancer and glioblastoma cell lines, in vitro. Furthermore, intratumoral administration of these therapeutics into xenografted glioma bearing mice led to tumor regression (97-99). Lactoferrin (Lf), which is a member of the transferrin family, can also be used as a promising targeting ligand. Accordingly, it is reported that Lf-PEG-PLA NPs loaded with fluorescent 6-coumarin could efficiently reach to the brain parenchyma (100). Regarding the successful outcome of initial investigations on the efficacy of Tf-coupled NPs in cancer therapy, various Tf decorated NPs undergone clinical trials. Accordingly, a Tf-coupled cyclodextrin polymerbased NP, CALAA-01, was the first targeted delivery of siRNA in a Phase I clinical trial (101). However, further investigations are required to bring Tf surface modified NPs to market. 4.2. Cell-penetrating peptides (CPPs) Cell-penetrating peptides (CPPs) are short peptides (5–30 amino acids) with a positive charge, which facilitates their penetration into cells across the cell membrane (102). CPPs can enhance the delivery of their cargo into cells through endocytosis process (Table 2). The trans-activating transcriptional activator (Tat) derived from Human Immunodeficiency Virus 1 (HIV-1) was the first identified CPP in 1988 (103, 104). CPPs are usually classified into three groups including proteins-derived peptides, chimeric peptides that are formed by the fusion of two natural sequences, and synthetic CPPs which are rationally designed sequences usually based on structure–activity studies (105). Based on another classification, there are cationic, amphipathic and hydrophobic CPPs (106). It has been suggested that CPP mediated uptake of cargo by cells is mediated by mechanisms such as endocytosis (energy dependent) and direct penetration (energy independent) (106).

Tat (103, 107), the herpes simplex virus type 1 protein VP22 (102), and the homeodomain transcription factor Antennapedia (107, 108) are the most extensively studied CPPs. It has been shown that NPs can be conjugated with CPPs for both the therapeutic and imaging applications (109). Since the first study related to the conjugation of CPPs with NPs in 1999 (110), several papers are published which applied various CPPs for targeted drug delivery and imaging (111). Many studies have demonstrated the effectiveness of surface modified superparamagnetic contrast agents with Tat peptide for intracellular noninvasive tracking of various cell types with MRI (112, 113). Similar strategy was applied to target the brain via magnetic conduction using the Tat conjugated magnetic poly (PLGA)/lipid NPs (114). CPPs are also used for targeted delivery of siRNA-loaded lipid NPs into cells (115). Moreover, surface modification of DNA loaded liposomes with Tat peptide increased transfection efficacy and decreased cytotoxicity in mouse fibroblast NIH 3T3 cells and cardiac myocytes H9C2 cells, in vitro (116). Transfection of DCs with DNA-loaded liposomes exhibited higher efficiency when NPs were modified by Tat peptide (117). In an interesting strategy, Tat-conjugated paclitaxel (PTX)-loaded liposomes were shielded by PEG layer which increased blood circulation time. PEG layer was detached at the tumor region by the exogenous reducing factor [glutathione (GSH)] and exposed Tat peptide facilitated cell internalization (118). In another similar investigation, PTX-loaded and CPP (R6H4) conjugated liposomes which were masked with hyaluronic acid could significantly arrest tumor growth in the Heps xenograft tumor mouse model (119). PTX-loaded nanocapsules conjugated with a glioblastoma-specific CPP (NFL-TBS.40-63) also decreased the tumor progression in the GL261 glioma brain tumor bearing mice (120). Delivery of PTX into tumor bearing mice was more effective when nanostructured lipid carrier (NLC) was functionalized with photon-sensitive cell penetrating peptides (psCPP) and Asn-Gly-Arg (NGR) (121). Surface modification of liposomes with octaarginine led also to better drug delivery to airway epithelial cells (122). Interestingly, it has been shown that conjugation of nanolipid crystal NPs with Tat could also increase skin permeation of NPs up to a depth of 120 µm (123). Tat CPPs were also used to functionalization of a micelles for targeting tumors (124). The targeting potential of Tat as active drug delivery molecule has also been shown in other investigations on several cell lines in vitro or in vivo (125). Targeting of CNS using the BBB permeability (penetratin)-deferasirox micelles also enhanced hydrophobic drug delivery (101). Surface modification of SLNs with Tat CPPs enhanced gene-transfer efficiency (126). Specific drug delivery was also done using multifunctional tandem peptide R8 conjugated liposomes for treatment of glioma (127). In an interesting strategy, PTX and DOX loaded liposomes were functionalized using two different targeting molecules including Tf and Tat peptide that was associated with enhanced targeting and therapeutic efficacies (128). Another study confirmed the efficacy of this drug delivery system for brain drug delivery in glioma (129). Combination of CPPs and quantum dots (QDs) is another approach by which several investigators demonstrated the hopeful results for both the diagnostic and therapeutic applications (130-134). It has been shown that conjugation of gold NPs with CPPs such as Tat can also be used for treatment of lysosomal storage diseases (135). Moreover, it is reported that Tat modified gold NPs can pass through nucleus pores and target the nucleus (136). Tat functionalization can also enhance the efficacy of plasmonic photothermal therapy agents in tumor therapy (137). Surface modification of cisplatin-hydrogel NPs with F3 peptide led to tumor regression via anti-angiogenesis effects (138). Conjugation of biodegradable polymer based NPs such as chitosan and PLGA with CPPs such as Tat enhanced drug delivery in various cancers (139-142). Qin et al showed tha Tat-modified liposomes could significantly accumulate in the brain (143). Similarly, Tatfunctionalized chitosan NPs improved gene delivery to the brain (140). Tat-conjugated gold NPs could also deliver Gd3+ and doxorubicin to glioma with higher efficiency compared to Gd3+ and free doxorubicin (144).

As a new strategy, combination of intracellular delivery by CPPs and mobile transposition via PiggyBac (PB) transposase may provide promising potential for efficient gene therapy (145). Although there are several in vitro studies regarding the efficacy of CPP targeted NPs for intracellular drug delivery, limited studies have been shown their efficacy in vivo (146-150). It seems that combinatorial approaches using two targeting moieties including CPPs (for enhancing cell permeation) and ligands of tumor cell biomarkers (for specific delivery) may be the potent therapeutic approach for cancer therapy (146).

4.3. LDL-LDL Receptor Based on densities, there are five types of lipoproteins including chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low-density, lipoproteins (LDL), and high-density lipoproteins (HDL). Lipoproteins can transport cholesterol and other lipids in the blood. Apolipoprotein B-100 which covers about half of the LDL surface area targets the LDL to LDL receptors (LDLRs) in some tissues, such as the liver, adrenal glands, and ovaries (151). Regarding the endogenous nature of lipoproteins, phagocytic system cannot recognize them. Since LDLRs are overexpressed in various cancers including acute myelogenous leukemia, adrenal adenoma, colon, pancreatic, lung, brain, and prostate cancers (152), they can be considered as the appropriate targeting molecule with high biocompatibility (Table 3). Accordingly, LDL modified NPs have been used for specific drug delivery into different tumor cells (153-156). Based on hydrophobic features of drug, they can incorporate into LDL particles through protein loading, core loading, and surface loading (153). Due to the difficulties in isolation of LDL, synthetic LDL-like particles are made for targeting applications (157). Administration of LDL-DOX complex into mice led to significant accumulation of drug in the liver compared to free DOX (158). Hematoporphyrin (HP) can also bind to LDLRs on tumor cell membrane (159). HP is a potent photosensitizer for photodynamic therapy (PDT). Surface modification of DOX-loaded NPs with HP enhanced the ameliorating effect of PDT in treating liver cancer (160). Conjugation of Osthole-loaded chitosan NPs with LDL led to high targeting efficacy of nanocarrier for treatment of liver cancer both in vitro and in vivo (161, 162). Similarly, coupling siRNA-loaded N-succinyl chitosan NPs with LDL was also associated with higher cellular uptake of siRNA by tumor cells (163). In another attempt, Kim and coworkers generated LDL similar SLNs loaded with PTX which exerted high anti-tumor function compared to Taxol and Genexol-PM (164). Regarding the high expression of LDLRs on BBB, it seems that LDL can be considered as the effective targeting molecule for drug delivery into the CNS (165, 166). Statins, which are effective therapeutics against CNS cancer cells, can increase the expression of LDLRs on the both BBB and cancer cells such as human glioblastoma (167). Accordingly, coadministration of statins with DOX-loaded LDL conjugated NPs enhanced drug delivery into the CNS and induced tumor growth arrest (168). Moreover, surface modification of NPs with apolipoprotein E (ApoE) can also enhance the crossing the BBB and effective drug delivery through LDLRs mediated endocytosis (169). Several anti-tumor drugs are delivered into the CNS through this drug delivery system (169-171). In an another interesting strategy, PTX-loaded NPs were decorated with peptide-22 which has a high affinity for LDLRs on the BBB and glioma cells (172). 5FU loaded LDL NPs could also significantly accumulate in liver tumor site compared to free drug (173). Similar results were observed when LDL NPs were loaded with the natural omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), which has anticancer properties (174). It has been shown that photosensitizer-reconstituted LDL NPs can effectively be uptaken by HepG2 tumor cells (175). The monitoring potential of LDLRs as a targeting ligand has also been demonstrated in A549 cancer cells (176).

4.4. Integrins and integrin ligands There are 24 different integrins, which are composed of heterodimers of α and β chains (177). It has been demonstrated that integrins are overexpressed in cancers and can enhance disease progression in part through angiogenesis and metastasis (178). For example, it is reported that integrins αvβ3, αvβ5, and α5β1 are involved in angiogenesis process of cancer (179) and integrins αvβ6 and α6β4 are overexpressed on tumor cells (180). Increased expression of integrins in cancer and their important role in tumor progression have made them as worthy molecules for targeted drug delivery into tumor tissues (Table 4). The tripeptide ArgGly-Asp (RGD) is usually used as the ligand for targeting αvβ3/αvβ5 integrins, overexpressed in tumors. Moreover, αV integrins, two β1 integrins (α5, α8) and αIIbβ3 can recognize RGD sequence (181). Several RGD containing peptides such as cRGDfX, cRGDeV, and cRGDyV are developed for selective targeting of tumor site (182). Also, RGD peptides can be conjugated with nanocarriers to deliver their cargo to tumor site. Accordingly, DOX loaded poly-glutamic acid polymer was conjugated with the high affinity αvβ6-specific 20-mer peptide H2009.1 and could successfully target αvβ6-expressing cells, in vitro (183). Administration of cisplatin loaded cRGDyk-conjugated liposomes into prostate cancer models led to the potent cellular uptake and higher cytotoxicity of encapsulatedd cisplatin compared to free drug or non-targeted NPs (184). Similar results were achieved when Gemcitabine-loaded c(RGDyK) conjugated albumin NPs were administered into BxPC-3 pancreatic cancer bearing mice (185). Targeting potential of cRGD for targeted drug delivery into cancerous cells using albumin NPs has also reported by other investigators in both the in vitro and in vivo (186, 187). In another study, DOX-loaded cRGD-conjugated unimolecular micelles exhibited better uptake by U87MG human glioblastoma cells and higher accumulation in tumor site compared to non-targeted NPs (188). Efficient drug delivery into U87MG cells is also demonstrated using cRGD targeted NPs by other investigators (189-191). Treatment of U87MG glioma beating mice with a cRGD targeted micelle-type bioconjugated PLGA-4-arm-PEG branched polymeric NPs showed their higher cellular uptake and lower cytotoxicity compared to non-targeted NPs (192). In another investigation, it is demonstrated that polyglutamic acid-PTX-E-[c(RGDfK)(2)] NPs could potently inhibit angiogenesis process in part through the suppression of the growth of proliferating αvβ3 expressing endothelial cells and tumor cells (193). Direct conjugation of anti-tumor drugs such as PTX with a dimeric RGD peptide E[c(RGDyK)]2 (RGD2) was also associated with accumulation of drug in the orthotopic MDA-MB-435 tumor and tumor growth arrest (194). Similarly, direct conjugation of biologic drugs such as TRAIL and TNF-α with cRGD targeting moiety led to specific drug accumulation in tumor site and inhibition of tumor growth (195-197). Efficacy of cRGD moiety for specific drug delivery to αvβ3-positive PC-3 prostate cancer cells using gold NPs has demonstrated, in vitro and in vivo (198, 199). Targeting α5β1 integrin by a fibronectin mimetic α5β1 RGD-containing peptide (PRb) which was conjugated with 5-Fluorouracil-loaded NPs resulted in potent downregulation of tumor growth in a CT26 colon carcinoma mouse experimental model (200). This targeting approach has also been investigated for delivery of DOX (201) and siRNA (202) in other studies which was associated with significant inhibition of tumor growth. Conjugation of NPs with the H2009.1 peptide which specifically targets αvβ6-expressing cells led also to significant accumulation of cargo in the targeted cells (203, 204). A 20-mer peptide A20FMDV2 is another αvβ6-specific molecule which can be used for specific drug delivery (205). A siRNA transfection was done with higher efficacy in tumor cells when chitosan-PEG NPs were coupled with RGD peptide (206). Moreover, administration of VEGFR2-specific siRNA-loaded PEG-PEI NPs which were coupled with RGD into tumor bearing mice inhibited both tumor angiogenesis and growth rate (207).

Targeting integrin molecules can also be used for PDT. It has been reported that conjugation of Pc4 loaded iron oxide NPs with fibronectin-mimetic peptide exhibited significantly greater inhibition of tumor growth than non-targeted NPs (208). It is also reported that targeting potential of integrin ligand coupled NPs depends on the number of conjugated ligands (209). In addition to therapeutic applications, RGD containing NPs are extensively used for tumor imaging which helps to evaluation of tumor stage or treatment monitoring (210-215).

4.5. Carbohydrates (lectin ligands) Glycan groups on the cell surface play an important role in a various physiological actions, such as cell-cell and cell-substrate interactions, immunogenicity and protein targeting (216). Interestingly, their structure or expression may be modified during various pathologic conditions such as cancers which lead to generation of tumor-associated carbohydrate antigens (TACAs) (217). TACAs are also crucial molecules for cancer progression processes such as metastasis (218). It is suggested that binding of surface carbohydrates with their ligands (lectins) leads to accumulation of glycans inside the cells in part through endocytosis process (219). Intracellular lectins, which are mainly involved in secretion, trafficking, sorting and targeting of maturing glycoproteins, include calnexin family (Calnexin and Calreticulin), M type (mannosidases), L-type (ERGIC53) and P-type (phosphomannosyl receptors). Extracellular lectins are involved in various actions such as adhesion, signaling, glycoprotein clearance and pathogen recognition and include C selectins (mannose receptor), R proteins (macrophage mannose receptor family), and Siglecs (galectins) (220). The carbohydratebased targeted drug delivery approach, which is also known as “glycotargeting”, is the subject of investigations from the more than two decades ago (221) (Table 5). Majority of glycotargeting approaches have used the lectin-mediated endocytotic process (222). Regarding the high biocompatibility and specific recognition of carbohydrates by cell surface receptors, several carbohydrate-targeted NPs have been used for specific drug delivery in the past decades (220). There are several targeting molecules for binding to endogenous lectins such as galactose, mannose, fucose, sialic acid, GalNAc (all from monosaccharides), lactose, N-acetyllactosamine (disaccharides), low molecular-weight HA fragment, sialyl Lewisx (oligosaccharides), hyaluronan, pullulan, arabinogalactan, dextran, and chitosan (polysaccharides). The affinity of carbohydrates for their protein receptors and the expression level of receptor on the target cells are two main criteria for the choice of ligand in drug targeting (223). Carbohydrate ligands composed of multivalent glycoside clusters enhance binding affinity and recognition potential on target cells which this phenomenon is known as the “glycoside cluster effect" (224). As mentioned, the role of lectins in the growth and metastasis of tumor is largely anecdotal. So, in addition to use of lectins as targeting moieties, a reverse scenario is also possible in which carbohydrate can be conjugated with NPs to target lectins (225). This strategy was used in phase I/II clinical trials in patients with primary or metastatic liver cancers, in which N(2-Hydroxypropyl)methacrylamide ((HPMA)–DOX–Gal) copolymer (PK2, clinical candidate FCE28069) exhibited hopeful results (226). In another study, galatose based hepatocyte asialoglycoprotein receptor (ASGP-R) ligands (arabinogalactan and pullulan) showed high affinity to ASGP-R compared to glucosebased polymer (kappa carrageenan) in hepatic targeted NP mediated curcumin delivery (227). In a similar study, pullulan conjugated polyethylene sebacate-DOX NPs could successfully accumulate in the hepatocytes (228). Administration of PTX-loaded mannosylated SLNs into lung cancer bearing mice led to significant accumulation of drug in the alveolar cell site which implies the efficacy of carbohydrate-based targeting system for drug delivery into lung (229). Similar results were achieved when these mannosylated NPs were loaded with DOX (230). Interestingly, it is reported that quantum dots lectin conjugates can be applied to evaluate the glycoconjugate pattern in normal and tumor tissues (231). Moreover, (99m)Tc-labelled gold NPs targeted with mannose were used to assess their function as a radiopharmaceutical for sentinel lymph node

detection (SLND) in mannose-receptor positive tissue (232). Regarding the high expression of mannose receptors on macrophages, these cells were tracked through mannosylated PEG(2,000) (Man-PEG(2,000)) quantum dots (233). In vitro assessment of DOX-loaded glucose-conjugated chitosan NPs showed that these targeted NPs could potently uptake by glucose transporters (Gluts) over-expressing tumor cells (234). In another study, phthalocyanine surrounded by a carbohydrate shell of sixteen galactose units used for PDT against HT-1376 and UM-UC-3 bladder cancer cell lines. Galacto-dendritic units significantly enhanced the uptake of batch through interaction with GLUT1 and galectin-1(235). Galectin-1, which is mainly expressed on macrophages, acts as an adhesion molecule. Dual functionalized liposomes by the galectin-1-specific anginex (Anx) and the αvβ3 integrin-specific RGD could effectively target tumor cells in the B16F10 melanoma bearing mice (236). Selections which are expressed on the leukocytes and endothelial cells play an important role in the lymphocyte homing and inflammation (237). Conjugation of PLA NPs with a ligand of E- and P-selectin led to increased adhesive capacity of NPs to human umbilical cord vascular endothelial cells compared to nontargeted NPs (238). Active targeting of gold NPs stabilized with a hydrophobic zinc phthalocyanine photosensitiser (C11Pc) and PEG through jacalin, a lectin ligand for the cancer-associated Thomsen-Friedenreich (T) carbohydrate antigen, was associated with receptor-mediated endocytosis of NPs by the HT-29 colon cancer cells and the SK-BR-3 breast cancer cells, in vitro (239). Using lectin-Fe2O3@Au NPs, it has also been reported that lectin can be used as tumor targeting ligand for generating NP-based contrast agents for MR and CT imaging of colorectal cancer in vivo (240) CD44 and RHAMM (receptor for hyaluronan-mediated motility) are two cell-surface carbohydrate receptors which can be used for targeted drug delivery. These receptors bind to hyaluronan-binding proteins. CD44 is usually modified during cancer progression and is crucial factor for metastasis process (241). Treatment of B16F10-CD44+ cells with hyaluronan (HA-SLNs) NPs was associated with potent intracellular delivery of PTX and significant apoptosis of tumor cells, in vitro (242). Analysis of anti-tumor function of docetaxelloaded PLGA/PEI/HA NPs in the CD44+ (A549) and CD44− (Calu-3) lung cancer cells demonstrated that these targeted NPs exhibit a very promising tool to treat CD44 overexpressing lung cancer cells (243). Interestingly, Yang et al. treated multidrug resistance (MDR) ovarian cancer mice model using MDR1specific siRNA loaded HA-PEI/HA-PEG NPs which showed a great potentials to overwhelm MDR in ovarian cancer (244). Many cancer cells also overexpress the receptor RHAMM (CD168), which is involved in cell motility and cell transformation. The RHAMM pathway is thought to induce focal adhesions to signal the cytoskeletal changes required for elevated cell motility seen in tumor progression, invasion, and metastasis (245). Overexpression of RHAMM is prognostic of poor disease outcome for colorectal, stomach, and breast cancers, and therefore, it is an appropriate target to improve drug delivery to cancer cells (223). Overview of the above discussed carbohydrate-based active targeting studies indicates that they are potent targeting moieties for anti-cancer drug delivery, however, the wide spectrum expression of their ligands on normal cells obligates us to perform more investigations before their application in trials.

4.6. Folate receptors Folic acid (FA, folate or vitamin B9) is a crucial nutrient for the synthesis of purines and pyrimidine in all living cells. Living cells take FA through the high affinity folate receptors (FRs) in a non-destructive, recycling endosomal manner (246). Regarding the low expression of FRs on normal tissues and their overexpression on cancer cells, it is suggested that FA can be used as a promising targeting molecule for active drug delivery to tumor cells (247, 248) (Table 6). Small size and potent binding affinity facilitate the

use of FA as targeting molecule. FA conjugation accompanied with using pH sensitive linker led to faster drug release in pH 5.0 inside the cancer cells (249). Active targeting of micelles with FA also enhanced specific drug delivery to cancer cells (250, 251). Moreover, it is reported that conjugation of FA with polymeric micelles used for PDT significantly reduced the phototoxicity of meta-tetra(hydroxyphenyl)chlorin (m-THPC) (252). Several studies have been shown that coupling of albumin NPs with FA significantly increase site-directed drug delivery in cancer cells (253-258). Small size of albumin-FA acid complex helps to passive targeting and FA enhances active targeting in the same time which lead to reduced drug dosage used for anti-cancer therapy (259, 260). Combining the albumin-based NP therapy with hyperthermia can potently enhance the therapeutic effect of antitumor drugs (261). Gold NPs can also exert higher specific drug delivery function when conjugated with FA (262-264). Pandey and colleagues showed that berberine hydrochloride (BHC)-loaded gold NPs conjugated with FA could markedly deliver their cargo to FR-expressing HeLa cells (265). FA coupled gold NPs are also used for delivery of DOX (266) and siRNA (267), and even for PDT (268). FA decorated magnetic NPs are also successfully used for delivery of several anti-cancer drugs such as PTX, methotrexate, mitoxantrone, and DOX to cancer cells (269-272). Based on above mentioned studies, it seems that FA-targeted NPs can markedly target FR-expressing cancer cells, thereby can be considered as worthy tools for cancer imaging and treatment.

3.7. Epidermal growth factor receptors Growth factors are crucial factors for several physiologic functions including cell survival, apoptosis, differentiation, cell-cell communications, embryonic tissue induction, fate determination, and cell migration (273). These processes are regulated through growth factor receptors. Epidermal growth factor (EGF), which is also known as ErbB or HER, is the member of the receptor tyrosine kinases (RTKs) (274) and has documented relation with cancer progression (275). EGFRs include four structurally related members; EGFR (ErbB1, HER1), ErbB2 (HER2, neu in rodents), ErbB3 (HER3) and ErbB4 (HER4). Signaling of EGFR can stimulate various tumor promoting processes such as the proliferation, angiogenesis, invasion, and metastasis (276, 277). High expression level of EGFR was also associated with a poor prognosis and reduced recurrencefree or overall survival rates in cancer patients (278). An importance of EGFR signaling in the tumorigenesis process is now well-known and several studies have tried to target EGFRs or their downstream signaling molecules to treat cancer patients (279) (Table 7). High ErbB signaling in the consequence of receptor overexpression, mutations or autocrine stimulation is frequently observed in various tumors (280, 281). Regarding the important role of the EGFR signaling in cancer progression, several anti-EGFR-based therapeutics such as cetuximab and erlotinib have been clinically approved in recent years (282). Therefore, majority of EGFR-targeted NPs are conjugated with antibodies and antibody fragments against EGFR such as cetuximab and trastuzumab. On the other hand, small size of EGF makes it an attractive option of targeting molecule for nanocarriers. Accordingly, conjugation of EGF with gelatin NPs led to higher accumulation of NPs in the tumor site compared to unmodified NPs (283, 284). Administration of EGF-coupled PTX-loaded polymeric lipid-based NPs into tumor bearing mice was associated with significant growth inhibition, in vivo (285). Using EGF moiety, concurrent delivery of drugs and imaging agents is also performed by Tam and colleagues for imaging and surface-enhanced raman spectroscopy of EGFR-overexpressed tumors (286). EGF-coupled polyamidoamine (PAMAM) Generation 4 dendrimers labeled with quantum dots are also used for targeted delivery of nucleic acid and imaging factors (287). Similarly, EGF functionalized PAMAM/DNA NPs could effectively uptaken by EGFR-expressing HepG2 cells in vitro. Moreover, these NPs were significantly accumulated in the tumor site, in vivo (288). Conjugation of EGF with stearoyl gemcitabine NPs was also associated with significant ameliorative effects in the breast cancer bearing mice (289). The

intravenous administration of EGF targeted iron oxide NPs into models of subcutaneous melanoma (cloneM3) and MN-22a hepatoma cells led to potent MRI contrast in areas of accumulated cancer cells (290). Similar results were achieved when these NPs were administrated into brain tumor bearing mice (291). Regarding the stimulatory effects of EGF or EGF-derived peptides on the EGFR-expressing target tumor cells, AEYLR, small peptide derived from the carboxyl terminal of EGFR, which has no stimulatory effect is used as a targeting molecule of nanocarriers (292). Some small peptides such as EEEEpYFELV (EV peptide) can not only target EGFR, but also suppress its signaling via competitive interaction with EGFR (293). Moreover, thiolated gelatin-PEG NPs conjugated with the EGFR-binding peptide loaded with the wild-type p53 tumor suppressor gene DNA could rapidly penetrate into Panc-1 cells (294). D4 (LARLLT) is another promising EGFR-binding peptide which is generated based on the crystal structure of EGFR. Targeting EGFR-overexpressing NCI-H1299 cells by D4 conjugated liposomes was significantly effective when compared to non-modified NPs (295). Similarly, conjugation of this peptide with manganese ferrite (MnFe2O4)-PEG NPs enhanced the binding and entry into EGFR expressing tumor cells as assessed by MRI studies (296). GE11 (YHWYGYTPQNVI) is another potent peptide that can specifically target EGFR without receptor stimulation. It is demonstrated that GE11 coupled polyethylenimine (LPEI)-PEG NPs could potently transduce a non-viral sodium iodide symporter (NIS) gene both in vitro and in vivo (297). Similarly, GE11 modified PEG-distearoylphosphatidylethanolamine (PEG-DSPE) micelles containing PTX were efficiently uptaken by EGFR expressing Hep-2 and EGFR negative U-937 tumor cell lines (298). In another study, GE11 functionalized biocompatible PEG-PCL micelles loaded with a photosensitizer phthalocyanine Pc4 potently targeted EGFR-overexpressing cancer cells which was associated with enhanced PDT (299). GE11 conjugated nanoscale filaments formed by the plant virus potato virus X (PVX) potently targeted several EGFRexpressing cancer cell lines, in vitro (300). There are also similar EGFR-binding peptide targeting studies which reported similar results (301). Aptamers are novel promising tools for specific targeting of tumor biomarkers in order to targeted drug delivery using NPs. Accordingly, Li and colleagues have generated gold NPs which selectively targeted EGFR on A431 cancer cells by the 80-residue aptamer, J18, which was associated with efficient receptormediated internalization (302, 303). Based on the above mentioned studies, it seems that targeting EGFR using various targeting molecules can be promising targeted drug delivery approach for treatment of EGFR over-expressing cancers.

5. Conclusion There are several anti-cancer immunotherapeutic approaches such as monoclonal antibodies, immune checkpoint inhibitors and cancer vaccines. However, the efficacy of immunotherapy has been limited by some critical issues such as targeted delivery and controlled release. Majority of monoclonal antibodies and vaccines were not as successful as expected, which was in part related to their toxic effects due to high doses of administration. Systemic and nonspecific administration of immunotherapeutic drugs such as cytokines and monoclonal antibodies can be associated with the risk of systemic toxicity. Similarly, the adoptive transfer of anti-tumor T cells may lead to autoimmunity at off-target sites (304, 305). To address and solve the above mentioned limitations, extensive studies have been performed in the field of nanomedicine for effective and specific drug delivery in cancer therapy. The most important advantages of NPs for cancer therapy include their stability, high carrier capacity, the ability to load with both hydrophilic and hydrophobic drugs, controlled drug release, and variable routes of administration (Table 8). It is also possible to load multiple drugs at the same time and monitor NPs in the body. Reducing the administration dose of current anti-cancer therapeutic agents is one of the main goals of several investigators, to date. Application of various NPs could partially help to reach to this goal. Surface modified targeted NPs can control the transport kinetics and biodistribution of cargo. Therefore, several ligands against various tumor

specific or associated markers were conjugated with NPs in order to specific drug delivery into cancer cells. These ligands should meet the necessary characteristics in order to use as an efficient drug delivery tool in tumor targeting. Moreover, the ligand should bind with high affinity to its receptor and internalized following the ligation with tumor specific biomarker. Several ligands against various tumor biomarkers have tested for specific drug delivery into tumor tissues. Among them, TfRs, CPPs, LDLRs, integrins, carbohydrates (lectin ligands), EGFRs, and FRs are extensively studied for specific drug delivery into cancer tissues. However, very limited studies are performed on humans in clinical trials. Moreover, there is no comprehensive study regarding the comparison of various targeting molecules in the same pathologic condition. Therefore, it is necessary to design the comparative studies accompanied with initial clinical trials in various cancer diseases to reach a rational decision.

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Table1: Studies related to the anti-tumor application of Tf-coupled NPs. NPs

Outcome

Cell lines

Ref.

DOX-liposome-Tf

The Tf-conjugated NPs were 4 to 5 times more potent than free drug in doxorubicin-sensitive tumor cell lines, in vitro.

HL60, Hep2 and L292

(73)

PTX-(γ-PGA-MALPLA-DPPE)copolymer-Tf

Tf modification mediates specific targeting and enhances cellular uptake of the NPs, in vitro.

C666-1 and Hela

(74)

PEG-PLA-micelle-Tf DOX-Fe3O4@SiO2Tf Core-shell NPs (containing sorafenib in albumin shell and DOX in PVA core)-Tf DOX-lipid-coated PLGA-Tf DNR-PLGA-PLLPEG-Tf

Cellular uptake of the Tf–PEG-PLA micelles was significantly higher than non-targeted micelles, in vitro and in vivo. Tf-decorated dual-function magnetic NPs enhanced cellular uptake and exerted potent cancer cell cytotoxicity, in vitro.

Intra-cranial rat tumor model of C6 glioma HeLa and K562

Tf increased NP uptake and cytotoxicity in ~tumor cells in both 2D and 3D cultures, in vitro.

Hepatocellular carcinoma (HCC)

(77)

A549 tumor-bearing mice

(78)

K562 xenografts BALB/c nude mice

(79)

Tf-modified NPs arrested tumor growth in the lung cancer-bearing nude mice, in vivo. Tf-functionalized NPs improved the efficacy of DNR in the treatment of leukemia, in vitro and in vivo.

A431tumor bearing immunodeficient BALB/c mice Bel-7402 tumor bearing nude mice, Human hepatocellular carcinoma cells (Bel-7402)

Plasmid DNA-DAB G3 dendrimer-Tf

Potent DNA transfection by Tf modified NPs, in vivo.

DOX-PAMAM-PEGT7/ADR-Tf

The higher cellular uptake and lesser IC(50) following Tf functionalizing, in vitro and in vivo.

SPIONs-Tf

Significant contrast enhancement of brain glioma using Tfmodification, in vivo.

C6 glioma rat model

NPs-ZOL-Tf

NPs-ZOL-Tf treatment resulted in higher in vitro cytotoxic activity than free ZOL

Mice intramuscularly bearing U373MG-LUC xenografts

Tfpep-Au NPs loaded with the photodynamic prodrug, Pc

A significant increase in cellular uptake for targeted conjugates as compared to untargeted particles.

In vitro studies of human glioma cancer lines (LN229 and U87)

Gd-HSA-Tf DOX-albumin-PEG liposomes-Tf Quinine dihydrochloride SLNs-Tf

Gd-HSA-NP-Tf achieved a significantly higher CE than non-targeted NPs in the blood, cardiac muscle, liver, and brain, in vivo. Increased gliomal DOX uptake was achieved by Tf-conjugated liposomes, in vitro. Tf-coupled SLNs enhanced the brain uptake of quinine, in vitro and in vivo.

Diphtheria toxin-Tf mutants Diphtheria toxin-Tf mutants

Tf-lipoplex exhibited high stability, gene transfer efficiency, and long-term efficacy for systemic p53 gene therapy, in vitro and in vivo. Tf- targeted NPs effectively downregulated Bcl-2 in K562 cells and significantly accumulated in the tumor site which led to tumor growth inhibition and prolonging mouse survival, in vitro and in vivo. Tf-conjugated NPs could markedly inhibit tumor growth, in vitro and in vivo. Site-directed mutagenesis in Tf provides an alternative method for improving the drug carrier efficacy of Tf, in vitro. Higher cellular uptake and toxin delivery and tumor regression by Tf mutants, in vitro and in vivo.

coumarin-6-PEGpoly(lactide)-Lf

Higher cellular uptake of targeted NPs, in vitro, and higher brain accumulation of drug, in vivo.

bortezomib-PLGA-Tf

Effective targeted drug delivery, in vitro.

DNA-liposome-Tf G3139-LP-Tf DOX-PLGA-PEG-Tf

(75) (76)

(80)

(81) (83) (84)

(90)

?

(92)

Rat C6 glioma cell line

(93)

Albino rats, brain tissue

(94)

DU145 prostate cancer model K562 xenograft model

(95) (96)

PC3 and A549, in vivo (PC3 xenograft models)

(97)

HeLa

(98)

U87 and U251 glioblastoma cell lines/ GBM xenografts bEnd.3 the immortalized mouse brain endothelial cell line, KM mice HPNE and S2–013

(99) (100) (306)

Tf: transferrin, DAB: diaminobutiric, HAS: human serum albumin, Gd-DTPA: gadolinium-diethylenetriamine, G3139: an antisense oligonucleotide for Bcl-2, LPs: lipopolyplexes, Lf: lactoferrin, DNR: Daunorubicin, PLGA: Poly (lactic-co-glycolic acid), DOX: doxorubicin, PTX: paclitaxel , SPION: superparamagnetic iron oxide NPs. PAMAM: polyamidoamine dendrimer, SLN: solid lipid nanoparticles , PGA: poly γ-glutamic acid, MAL: maleimide , DPPE :1,2-dipalmitoylsn-glycero-3-phosphoethanolamine, SiO2: Silicon Dioxide, PLA: Polylactide, PVA: Poly(vinyl alcohol), T7: HAIYPRH peptide a unique targeting agent, GBM: glioblastoma multiforme.

Table 2: Studies related to the anti-tumor application of CPP-conjugated NPs. NPs TAT-G3-Gd

Outcome Effective magnetic resonance-guided regional gene delivery by a tatcoupled tumor stroma-permeable NPs in pancreatic cancer, in vivo.

Cell lines PaCa-2 tumor model

Ref. (307)

TAT-modified liposome (TAT-LIP)

TAT-LIP was a promising brain drug delivery system due to its high delivery efficiency across the BBB.

Brain capillary endothelial cells (BCECs) of rats

)143(

Dox-conjugated TAT-Au NPs

Treatment of mice bearing intracranial glioma xenografts with pHsensitive Dox-conjugated TAT-Au NPs via a single intravenous administration leads to significant survival benefit when compared to the free Dox

U87 mouse model

ALMWP-NP

ALMWP-functionalized NPs exhibited an enhanced accumulation in HT-1080 cells, in vivo. The cellular uptake and transfection efficiency were greatly improved by the LMWP-modified quaternary NPs, in vitro. TAT increased the DOX uptake of A431 cells, in vitro.

HT-1080 tumor-bearing BALB/c nude mice B16

(308)

A431, SK-BR-3, MCF7/WT, MCF7/ADR, and MBT2 MDCK-MDR model

(310)

Caco-2

(312)

N2a and MCF-7

(313)

CHO, U2OS, U87, and HEK293, Balb/c mice C26 murine colon carcinoma

(314)

C26

(316)

Nude BALB/c mice, MCF-7 and MDA-MB231 cells CHO cells and H9C2

(317)

HUVEC and A549 cells, A549 and U87 xenografts bearing nude mice Murine lymphocytes and

(319)

LMWP/PGA/PEI/DNA quaternary NPs DOX-liposomeTAT/PEN PEG-LA-Penetratin

Penetratin-Ag NPs

TP10-ds decoy ODNsMyc (oncogenic protein) Stearyl-TP10/plasmid NPs CP-DOX

CP-DOX PEGA-pVECchlorambucil MAP-PNA

RLW-NP-docetaxel

TAT-Dextran-SPION

Penetratin- conjugated NPs exhibited a significantly enhanced brain uptake and reduced accumulation in the non-target tissues, in vitro and in vivo. Ag NPs conjugated with a penetratin exhibited a significantly greater amount of cellular uptake than Ag NPs with a silica capping agent, in vitro. Reduced proliferation was observed when tumor cells were treated with TP10-PNA conjugate hybridized to Myc decoy, in vitro. The high transfection efficacy of stearyl-TP10 without toxicities, in vitro and in vivo. CP-conjugated NPs had higher tolerated dose than free drug, and induced nearly complete tumor regression after a single dose, in vivo. The CP-DOX NPs demonstrate the high stability and cytotoxicity, in vitro. Conjugation of chlorambucil with the PEGA-pVEC chimeric peptide increased the efficacy of drug, in vitro and in vivo. Conjugation of PNA with the synthetic peptide MAP led to higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, in vitro and in vivo. RLW-coupling enhanced the specific cytotoxicity of docetaxel in the A549 xenografts model and improved antitumor effect. TAT-modified particles were internalized into lymphocytes more efficiently than non-modified particles, in vitro.

TAT-SPION

TAT-conjugated particles are efficiently internalized into hematopoietic and neural progenitor cells in immunodeficient mice.

TAT-FITC-CLIO

The biodistribution of FITC-CLIO-TAT loaded T cells can be monitored in vivo over time by MRI.

TAT-Dextran-SPIOFITC TAT-MPLs-HES-NARGSH CPP-LNP-siRNA

TAT-peptide conjugates primarily migrate to the cytoplasm rather than the nucleus. TAT-conjugated MPLs act as an effective drug delivery system for crossing the blood brain barrier, in vitro. CPP-modification of LNP enhances internalization of siRNA in the cytoplasm and thereby to induces gene silencing, in vitro. The transfection efficacy was significantly increased following TAT

TATp-liposome–DNA

)144(

activated cells

human

(309)

(311)

(315)

(318)

(110)

NK

C17.2 and mouse splenocytes, immunodeficient NOD/SCID mice C57BL6 (B6) and the Thy1.1 congenic strain of B6 mice In vitro (T cells, B cells, and macrophages) bEnd.3

(320)

B16F10 and HT1080

(115)

NIH/3T3 and H9C2

(116)

(321)

(112) (114)

complexes TAT-C-PTX-LP R6H4-L-PTX/HA

conjugation, in vitro. TAT-modified NPs markrdly inhibited the proliferation of murine melanoma B16F1 tumor cells in vitro and in vivo. After hyaluronidase treatment, R6H4-L conjugated NPs exhibited a stronger cytotoxicity toward the hepatic cancer (HepG2) cells at pH 6.4 relative to at pH 7.4.

NFL-TBS.40-63-PTXLNC

NFL-TBS.40-63 peptide enhanced the delivery of PTX entrapped in LNC to glioma cells.

psCPP/NGR-NLC

psCPP- NGR coupling promoted the cellular uptake of NLC by cancer cells.

TAT-Dextran- liposome

TAT-conjugated NPs localize for the most part in the cytoplasm with only a small amount of nuclear localization, in vitro. DNA-loaded TATp-L increased the in vitro transfection efficiency in dendritic cells, in vitro. TAT peptide enhanced the skin permeation of NPs by translocating across the skin layers.

TATp-DNA-liposomes TAT- DID-oil-FNLCNT

TAT-DOX-polymeric micelle(PLLA-b-PEG) Penetratin-deferasiroxmicelle TAT-rhodaminephosphatidyl ethanolamine/FITCdextran-liposome R8-c(RGD)-PTXliposome Tf-TAT-PTX/DOXliposome Tf-TAT-DOX-liposome

SR9-QD CPP-InP/ZnS QDs

TAT-QDs encapsulated in PEGylated phospholipid micelles TAT-QDs@mSiO2DOX TAT-gold-NPs TAT-gold nanostars VG-21-gold NPs

CPP-DOXgold nanospheres F3-cisplatin-hydrogel NPs

Higher uptake of TAT-micelles at pH 6.6 indicating deshielding compared to pH 7.4, in vitro. Potential of CPP-drug conjugates for use as nanocarriers for hydrophobic drugs, in vitro. TAT-targeting facilitates internalization of liposomes by cancer cells, in vitro.

Targeted NPs exhibited an efficient antiproliferation effect on brain cancer stem cells, in vitro and in vivo. Double-drug loaded targeted liposomes exhibit both enhanced targeting efficiency and increased therapeutic efficacy, both in vitro and in vivo. Tf/TAT-lip is a promising brain drug delivery system due to its high delivery efficiency across the BBB. SR9 peptide facilitates the delivery of noncovalently associated QDs into A549 cells, in vitro. carboxylated and PEGylated bifunctionalized QInP are biocompatible NPs with potential for use in biomedical imaging and drug delivery, in vitro. When quantum dot-labeled cells were injected through the carotid artery of MCaIV tumor bearing mice, their recruitment by the tumor vasculature could be tracked, in vivo. The enzymatic cleavage of peptide exposes TAT residues on the QDs@mSiO2 surface and facilitates the DOX delivery into cellular nucleus. TAT-conjugation allowed particles to transfer across the cell membrane and locate in the nucleus, in vitro. TAT-modified NPs demonstrate the enhanced intracellular delivery and efficient photothermolysis in cancer therapy, in vitro. Conjugation of gold NPs with a CPP (VG-21) from vesicular stomatitis virus enhanced intracellular translocation and biodistribution of NPs, in vitro and in vivo.

Higher cell death induction by the CPP-conjugated NPs, in vitro. F3-targeted NPs are effective therapeutics that bind to both murine and human ovarian tumor endothelial cells, in vivo.

B16F1, B16F1 murine melanoma tumor models In vitro (HepG2), in vivo (murine hepatic carcinoma, Heps, tumor xenograft models) In vitro (GL261 glioma cells and primary astrocytes), in vivo (C57Bl6 mice bearing GL261 glioma) In vitro ( MCF-7 and HT1080) and in vivo (BALB/c nude mice and Sprague–Dawley (SD) rates) Calu-3

(118)

Dendritic cells

(117)

In vitro (rat skin) CD®(SD) hrBi hairless rats MCF-7

(123)

RBE4

(322)

BT20 and H9C2

(125)

C6 cells, BALB/c mice

(127)

B16 cells, C57BL/6 mice

(128)

in vitro (U87 cells and BCEC cells) and in vivo (BALB/c nude mice) A549

(129)

A549

(131)

In vitro (MS1 cells) and in vivo (C3H mice)

(132)

A549, NIH-3T3, A2780, and A2780/Adr

(133)

hTERT-BJ1

(136)

BT549

(137)

In vitro (HEp-2, African green monkey kidney (Vero and Cos-7) and HeLa and in vivo (BALB/c mice) HeLa and A549 cells

(323)

Animal tumor models of ID8, SKOV3, A2780GFP, and DsRED HEY1) in either C57Bl6 or nu/nu

(138)

(119)

(120)

(121)

(122)

(124)

(130)

(134)

TAT-DOX-chitosan

TAT- targeted NPs could effectively decrease tumor volume, in vivo.

TAT-siRNA-ChitosanPEG CPP-GSE24.2-PLGAPEG CPP-piggyBac transposase

Ataxin-1 specific siRNA was effectively delivered into cancer cells by TAT-coupled NPs, in vitro. CPP-conjugated NPs enhanced the intracellular uptake, in vitro.

TAT-RGD-PEI-DNAnanocomplex CPP-dendrimeroligonucleotide NPs

CPP was able to synchronously deliver covalently linked piggyBac transposase and noncovalently linked a cis plasmid into human cells, in vitro. RGD and TAT enhanced the cellular uptake and gene transfection efficiency of primary neurons, in vitro. CPP-conjugated NPs can be taken up by cells, in vitro and in vivo.

mice. BALB/c mice bearing subcutaneous tumors Neuro 2a

(139) (140)

HeLa, VA13, and MEF

(141)

A549

(145)

SH-SY5Y

(324)

MES-SA/Dx5, transgenic (325) mice harboring an EGFP gene PEG: poly(ethylene glycol), ODNs: oligodeoxynucleotides, MPLs: Magnetic poly lipids, HES: hesperidin, NAR: naringin, GSH: glutathione, LNC: lipid nanocapsules, FNLCN: nano lipid crystal nanoparticles, InP: indium phosphide, ZnS: zinc sulfide, QD: quantum dot, Gd:gadolinium, ALMWP : activatable Low molecular weight protamine, PGA: poly(g-glutamic acid), LMWP: low molecular weight protamine, PEI: a cationic polymer, PEN: penetratin, TAT: HIV trans-activating transcriptional activator protein a type of CPP, LA: lactic acid, TP10: a cell-penetrating peptide, CP: chimeric polypeptide, PEGA: a homing cyclic peptide cCPGPEGAGC , pVEC : a cell-penetrating peptide, MAP: a cell-penetrating a-helical amphipathic model peptide KLALKLALKALK AALKLA-NH2 , PNAs: peptide nucleic acids, RLW : a cell penetrating peptide , CLIO, cross-linked iron oxide , SPIO: superparamagnetic iron oxide, MPL: Magnetic poly (D,L-lactide-co-glycolide) (PLGA)/lipid nanoparticles, LNP: Lipid nanoparticles, PTX :paclitaxel, R6H4-L :R6H4-modified liposomes, HA: hyaluronic acid, LNC :lipid nanocapsules, NFL-TBS.4063: neurofilament light subunit-tubulin-bindingsite.40-63 , psCPP: photon-sensitive cell penetrating peptides, NGR: Asn-Gly-Arg, FITC: Fluorescein isothiocyanate, DID-oil: a Fluorescent dye, FNLCN: Fluorescent dye encapsulated nano lipid crystal nanoparticles, PLLA-b-PEG: Poly(L-lactic acid)-b-poly(ethylene glycol), mSiO2: mesoporous silica nanoparticles, PLGA :polymer poly(lactic-co-glycolic) acid.

Table 3: Studies related to the anti-tumor application of LDL-coupled NPs. NPs

Hematoporphyrin-DOXNPs LDL-osthole-NSuccinyl-chitosan

Outcome LDL targeting reduced the DOX-induced cardiotoxicity in the host, in vivo. The phototoxicity was enhanced using hematoporphyrin-coupled NPs, in vivo. LDL-coupled NPs had high targeting efficacy and inhibited tumor growth, in vivo.

LDL-DOX/siRNA-NSuccinyl-chitosan

LDL-modified NPs were significantly accumulated in tumor site, in vivo.

LDL-PTX-SLNs-PEG

LDL-modified NPs was effective in inhibition of tumor growth, in vivo.

LDL-DOX

Apolipoprotein Eloperamide-albumin-NP

Apolipoprotein E-conjugated NPs markedly enhanced drug transport into the brain

LDL-DOX-PEGliposome

LDL-conjugated NPs across the BBB higher than free DOX in cells exposed to statins, in vitro.

Apolipoprotein Edalargin/ loperamidepolysorbate 80- PBCA

NPs coupled with polysorbate 80 or apolipoprotein E induced an antinociceptive effect, in vivo.

peptide-22-PTX/PNPNPs

LDLR-mediated peptide-22-conjugated NPs were effective for dual-targeting therapy of brain glioma, in vitro, ex vivo and in vivo.

5-FU-entrapped LDL NPs

LDL facilitated the cellular uptake of drug, in vitro and in vivo.

LDL-DHA NPs LDL- pyropheophorbide cholesterol oleate SPIO@ apolipoprotein B lipoparticles, M4N@ apolipoprotein B lipoparticles

LDL-DHA NPs exhibited enhanced physical and oxidative stabilities compared to native LDL and free DHA, in vitro. Photosensitizer-reconstituted LDL could be internalized via LDLR by cancer cells, in vitro. Apolipoprotein B lipoparticles can be monitored directly with fluorescence microscopy, in vitro and in vivo.

Cell lines HepG2 tumor bearing nude mice

Ref. (158)

HepG2 tumor-bearing mice

(160)

HepG2 tumor bearing nude mice

(162)

In vitro (HepG2, L-02, and HepG2/ADM) in vivo (a liver tumor model by orthotopic inoculation. Balb/c nude mice NCI-H1975, NCI-H1650, NCIH520, and PC9. SK-BR-3 . C57BL/6 mice

(163)

(164)

UKF-NB-3rVCR10), ICR (CD1) mice

(165)

hCMEC/D3, U87-MG, SJKNP cells, MDA-MB-231, and A549 cells

(168)

ICR and C57BL/6J mice

(170)

H92c(2-1), BCEC and C6 cells , BALB/c mice, C6 orthotopic glioma model Ehlrich’s ascites carcinoma (EAC) cells were cultured in the peritoneal cavity of Swiss mice TIB-73, BNL CL.2, TIB-75, and BNL 1ME A.7R.1

(172)

(173) (174)

HepG2

(175)

In vivo (BALB/c mice) and in vitro THP-1 and A549

(176)

PBCA: Poly(butyl cyanoacrylate), DOX :doxorubicin, LDL: Low density lipoprotein, SLNs: solid lipid nanoparticles, PBCA: Poly(butyl cyanoacrylate) nanoparticle, LDLR: Low-density lipoprotein receptor, 5-FU: 5-Fluorouracil, DHA: docosahexaenoic acid, SPIO : superparamagnetic iron oxide.

Table 4: Studies related to the anti-tumor application of integrin-targeted NPs. NPs Polyglutamic acid-tetrameric H2009.1 peptide-DOX cRGDyk-cisplatin-liposome

RGD-albumin nanoparticle

cRGD-H40-DOX and cRGDH40-DOX-64Cu

cRGDfC-PLGA-4-arm-PEG branched

PGA-PTX-E-[c(RGDfK)(2)] cRGD-platinum-polymeric micelle c(RGDyK)-PTX-PEG-PLA [c(RGDyK)](2)-PTXcRGD-albumin nanospheres c(RGDfK)-HSA- monomethylauristatin-E cRGD-PEG-gold NPs c(RGDfC-PEG-SPIO nanocarriers-PET ⁶⁴Cu cRGDfK-gold nanorods

cRGDfK-Cy5.5-small rigid platforms cRGD--Rhodamine123-PEG cRGDfK-technetium-99m-gold NPs

RGD-USPIO-3-APTMS cRGDyK-microparticles of iron oxide RGD-TRAIL

RGD4C-64Cu-DOTA-TNF

Outcome Conjugated NPs are effective for treatment of αvβ6 integrin expressing tumor cells, in vitro. cRGDyk conjugated liposomes showed significantly higher cellular uptake and higher cytotoxicity in vitro and in vivo. Uptake of targeted NPs by pancreatic cancer cells was enhanced by RGD peptide, in vitro and in vivo. cRGD-coupled micelles (H40-DOX-cRGD) exhibited a higher cellular uptake in U87MG human glioblastoma cells. In tumor bearing mice, H40DOX-cRGD-64Cu also exhibited a higher tumor accumulation. cRGD conjugated NPs were taken up more efficiently by tumor cells over-expressing the αvβ3 integrin compared with the non-targeted NPs, in vitro. Targeted NPs had augmented antitumor activity and reduced systemic toxicity for PTX, in vitro. cRGD-conjugated micelles (cRGD/m) accumulated into the tumor parenchyma, in vivo. The presence of c(RGDyK) enhanced the antiglioblastoma cell cytotoxic efficacy, in vivo. The PTX-RGD conjugate markedly inhibited cell proliferation. RGD-anchored nanospheres were significantly effective in the prevention of lung metastasis, angiogenesis and regression of tumor, in vivo. Targeted NPs demonstrated high specificity for αvβ3expressing tumor cells, in vitro. High affinity and binding to αvβ3-positive cancer cells, in vitro. cRGD-conjugated SPIO nanocarriers exhibited a high cellular uptake and tumor accumulation, in vitro and in vivo. Targeted NPs was effectively uptaken in vitro, but not in vivo due to a fast clearance of the NPs from the blood. Targeted NPs demonstrated a strong association with αvβ3 integrins, in vitro and in vivo. RGD-coupled NPs effectively target αvβ3 integrin expressing cancer cells. RGD-modified NPs showed specific recognition for αvβ3 integrins expressing tumor cells, in vitro and in vivo. RGD-anchored NPs were accumulated in tumor cells, in vitro and in vivo. RGD-functionalized NPs were markedly concentrated in the tumor site. The fusion protein RGD-TRAIL bound to microvascular endothelial cells in a dose-dependent manner and showed enhanced apoptosis-inducing activity in cancer cells, in vivo. RGD-anchored NPs had significantly higher activity accumulation in integrin-positive tumors, in vitro and

Cell lines

Ref.

H2009 and H1299

(183)

RM-1 cells, RM-1 tumor bearing C57BL/6 mice

(184)

In vitro (BxPC3, SW1990, PANC-1and CFPAC-1,) and in vivo BALB/C-nu/nu mice with pancreatic cancer xenografts

(185)

In vitro (U87MG cells) and in vivo (athymic nude mice)

(188)

In vitro (U87MG and Panc-1)) and in vivo (Panc-1 tumor bearing athymic nude mice )

(192)

In vitro (U87-MG, 4T1, and HUVEC), in vivo (4T1 bearing mice) Mouse model of U87MG( human glioblastoma cell line)

(193) (189)

U87MG-bearing nude mice

(190)

MDA-MB-435, athymic nude mice

(194)

HUVEC, B16F10 tumour-bearing BALB/c mice

(186)

HUVEC, in vivo (C26 murine colon carcinoma model)

(187)

MDA-MB-231 and PC3

(198)

In vitro (U87MG) and in vivo (U87MG tumor bearing athymic nude mice) In vitro (DU145 and HUVEC) and in vivo (prostate tumor bearing athymic mice) In vitro (HEK293) and U87MG) and in vivo (U87MG bearing nude mice)

(191)

(199) (210)

-

(211)

In vitro (C6) and in vivo (athymic mice with C6-induced tumors)

(212)

In vitro (HUVEC, HaCaT-ras-A5RT3 and A431) and in vivo (Human squamous cell carcinoma xenografts nude mice In vitro (HUVEC), in vivo (B16F10 or MC38 tumor bearing mice)

(213)

(214)

In vitro (MDA-MB-231 and COLO205), in vivo (COLO-205 tumorbearing mice)

(195)

In vitro (U87MG, MDA-MB-435 and C6), in vivo MDA-MB-435

(197)

RGD-177Lu-AuNP PR-b-5-FU-PEG-liposome

PR-b-DOX-PEG-liposomes PR-b-(Polymersomes)[ poly(1,2-butadiene)-bpoly(ethylene oxide)] encapsulating siRNA Aka H2009.1 peptide-DOXSPIO RGD-siRNA-PEG-chitosanpoly(ethylene imine) hybrid Fmp-IO-Pc 4 (fibronectin mimetic peptide- iron oxide NPs RGD-modified liposome (monomeric RGD (moRGDLP), dimeric RGD (diRGD-LP) and (P-diRGD-LP))

in vivo. RGD-modified NPs demonstrated the highest tumor radiation absorbed dose, in vivo. PRb-targeted liposomes show significantly higher cytotoxicity against the α5β1 integrin expressing tumor cells, in vitro. PRb-functionalized liposomes were able to specifically bind to tumor cells and induced higher cytotoxicity than the non-targeted liposomes, in vitro and in vivo.

tumor bearing Athymic nude mice. In vivo (C6 gliomas bearing athymic mice)

(215)

CT26, HCT116 and RKO

(200)

In vitro (MDA-MB-231 cells) and in vivo (MDA-MB-231 tumor bearing mice)

(201)

T47D and MCF10A

(202)

H2009 and H460

(204)

H1299

(206)

Both nontargeted IO-Pc 4 and targeted Fmp-IO-Pc 4 NPs accumulated in xenograft tumors.

In vitro (HNSCC, M4E, M4E-15, 686LN, and TU212) and in vivo (M4E tumor bearing nude mice)

(208)

P-diRGD-LP demonstrated strongest interaction with B16 cells and highest cellular uptake. P-diRGD-LP demonstrated the best targeting effect, in vivo.

In vitro (B16 and MCF-7), in vivo (B16-tumor bearing mice)

(209)

PR-b-anchored vesicles induced high cytotoxicity in cancer cells, in vitro. Targeted multifunctional micelles showed significantly increased αvβ6-dependent cell targeting in cancer cells, in vitro. The internalization of RGD-conjugated NPs was highly dependent on the surface concentration of the ligand, in vitro.

RGD enhanced siRNA delivery to cancer cells, in In vitro (HUVEC and N2A) and in (207) vitro and in vivo. vivo N2A tumor bearing nude mice) HAS: human serum albumin, APTMS: aminopropyltrimethoxysilane, cRGDyk: cyclic arginine-glycine-aspartic acid-tyrosine-lysine peptide, PLGA: poly(lactide-co-glycolide), PGA: polyglutamic acid, PET: positron emission tomography, SPIO: superparamagnetic iron oxide, TRAIL :(TNF) – related apoptosis-inducing ligand, PTX: paclitaxel, TNF: tumor necrosis factor, DOTA: a macrocyclic RGD-PEG-PEI/siRNA

chelating agent, DOX: doxorubicin, PRb:a fibronectin-mimetic peptide, IO: Iron Oxide Nanoparticle, Fmp: fibronectin-mimetic peptide, PEI: polyethyleneimine.

Table 5: Studies related to the anti-tumor application of carbohydrate-targeted NPs. NPs

Outcome

HPMA-DOX galactosamine

Galactosamine-modified NPs showed enhanced in vivo targeting.

ASGP-R Ligands-Curcumin Gantrez NPs

ASGP-R Ligands enhanced the Hepatic Targeting of Curcumin Gantrez NPs, in vivo. Higher blood concentration and lower liver concentration of PES-DOX-PUL, indicating the long circulating time of these NPs and improved targeting to hepatocytes. M-SLNs deliver a higher concentration of PTX as compared to PTX-SLNs in an alveolar cell site.

In vivo Sprague–Dawley rats

(227)

In vivo Sprague–Dawley rats

(228)

A549

(229)

Different regions of breast tissue expressed particular types of carbohydrates.

In vitro(normal breast tissue and fibroadenoma (benign) and invasive ductal carcinoma (malignant)

(231)

(thiol-triazole-mannose derivative)/ HYNIC-GGC – AuNP- Technetium-99m

(99m)Tc-AuNP-mannose) specifically recognized mannose receptors in rat liver tissue.

In vito(Liver rat tissue) in vivo (Wistar rats)

(232)

M-SLNs-DOX

M-SLNs were able to deliver a higher concentration of DOX in the tumor mass.

M-PEG-QDs

M-PEG-QDs were uptaken by cancer cells through mannose receptors, in vitro.

In vitro (A549) and in vivo (A549 tumor bearing mice) Primary peritoneal macrophages or HepG2 cells and in vivo (ICR mice)

PES-DOX-PUL M-SLNs- PTX MSA-CdTe QDs-Con A MSA-CdTe QDs -UEA I

Glucose-DOX-chitosan PcGal16 Galectin-1-siRNA-gold nanorod Anx/RGD dual-conjugated liposomes branched polyester copolymers of hydroxy-acid and allyl glycidyl ether/selectin ligand C11Pc-PEG gold nanoparticle/ jacalin/mAb Lectins (ConA, RCA and WGA)-Fe2O3@ Au NPs

Glucose-anchored NPs had better endocytosis and anti-tumor ability than non-targeted NPs, in vitro. The photoactivation of PcGal16 induces cell death by generating oxidative stress, in vitro. The nanoplexes significantly decreased gene expression for galectin-1, in vitro. Targeted NPs were effectively accumulated in tumor site, in vivo. Strong adhesion of the ligand decorated NPs was demonstrated, in vitro. Non-conjugated C11Pc-PEG gold NPs were only minimally phototoxic. Lectin-conjugated NPs had a capacity not only for dual mode MR and CT imaging in vitro and in vivo.

Cell lines Patients with histologically confirmed solid hepatic neoplasms

Ref. (226)

(230) (233)

MEF and 4T1

(234)

HT-1376 and UM-UC-3

(235)

Human monocyte derived macrophage B16F10 melanoma bearing mice

(326) (236)

HUVEC

(238)

HT-29 and SK-BR-3

(239)

SW620 tumor bearing nude mice, (SW620) In vitro (B16F10 and A549 cells) and in vivo (B16F10tumor bearing mice)

(240)

HA-PTX-SLNs

HA-anchored NPs effectively deliver PTX into cancer cells, in vitro and in vivo.

HA-DTX-PLGA-PEI

HA-modified NPs demonstrated an increased time-dependent uptake in cancer cells, in vitro.

A549 and Calu-3

(243)

HA-PTX-MDR1 siRNAPEI-PEG

HA-PEI/HA-PEG NPs can efficiently deliver MDR1 siRNA into MDR ovarian cancer cells, in vitro and in vivo.

In vitro (SKOV3TR, OVCAR8TR) and in vivo (SKOV- 3TR tumor bearing nude mice)

(244)

(242)

HPMA: Hydroxypropyl methacrylamide, PES: Polyethylene sebacat, M-SLN: Mannosylated solid lipid nanoparticles, ASGPR: asialoglycoprotein receptor, PUL: Pullulan, QD: Quantum dot, MDR:multidrug resistance, Gantrez: a polymer , PTX: paclitaxel, MSLNs: mannosylated solid lipid nanoparticles, MSA: Mercaptosuccinic acid, CdTe: Cadmium Telluride, Con A: concanavalin A, UEA I: Ulex europaeus agglutinin I, HYNIC-GGC: Hydrazinonicotinamide-Gly-Gly-Cys-NH2 is a peptide, AuNP: gold nanoparticles, PEG :polyethylene-glycol, PcGal16 :Pc decorated with sixteen molecules of ga lactose in a dendritic manner, Galectin1: an adhesion molecule, Anx: anginex, C11Pc: zinc phthalocyanine photosensitizer, jacalin: a lectin specific for the cancerassociated Thomsen–Friedenreich (T) carbohydrate antigen, mAb: monoclonal antibody, RCA: Ricinus communis agglutintin, ConA: concanavalin A, WGA: wheat germ agglutintin, HA: hyaluronan, DTX: docetaxel, PLGA: poly(lactic-co-glycolic) acid.

Table 6: Studies related to the anti-tumor application of FR-targeted NPs. NPs FA-DOX-hyd-PEG

Outcome Addition of FA to NPs increased the intracellular accumulation of DOX in cancer cells, in vitro.

Cell lines

Ref.

KB, A549, and HepG2

(249)

In vitro (4T1) and in vivo (4T1 bearing BALB/c mice)

(251)

FA-PLLA-b-PEG

FA-anchored micelles have the ability to suppress cancer cell proliferation and retard tumor growth and cancer cell metastasis.

FA-5-FU-PNVCL-bPEG micelles

FA-conjugated micelles exerted a cytotoxicity effect in cancer cells, in vitro.

EA.hy 926 and 4T1

(250)

FA-DOX-PLA-PEG

FA-modified micelles showed higher cytotoxicity than that of nontargeted NPs.

SKOV3

(327)

FA-DOX-mixed polymeric micelles

FA-conjugated micelles effectively delivered DOX to cancer cells, in vitro. FA-functionalized micelles exhibited much better efficiency of cellular uptake and higher cytotoxicity to tumor cells, in vitro and in vivo. DOX-loaded targeted micelles effectively killed both wild-type sensitive and multidrug resistance cancer cell lines, in vitro. FA-conjugated m-THPC-loaded micelles are taken up and accumulated by folate receptor-overexpressed KB cells in vitro and in vivo. FA-modified NPs could get efficiently transferred into the cells through the folic acid-mediated endocytosis, leading to higher antitumor efficacy, in vitro. FA-decorated NPs targeted a human prostate cancer cells effectively, in vitro. FA-modified NPs showed enhanced cellular uptake, increased targeting capacity, and increased cytotoxicity against cancer cells, in vitro and in vivo. FA-decorated NPs exhibit stronger inhibition rate and induce apoptosis in FR expessing cancer cells, in vitro. FA-conjugated NPs was effectively incorporated into tumor cells, in vitro.

HeLa, and HT-29

(328)

In vitro (KB and A549), in vivo (BALB/c mice)

(329)

A2780) and A2780/DOXR

(330)

In vitro (KB cells and HT-29) and in vivo (KB or HT-29 tumor bearing mice)

(252)

SMMC-7721

(253)

PC3

(254)

In vitro (KB and 4T1) and in vivo (KB and 4T1 bearing mice)

(255)

SMMC-7721 and Hela cells

(256)

HeLa and AoSMC

(257)

Murine ascites hepatoma H22 tumor-bearing ICR mice

(258)

Hela cells

(259)

FA-DOX-PEG-poly(εcaprolactone) FA-DOX-PLLA-bPEG FA-m-THPC micelles

FA-2-ME-BSA NPs FA-PTX-BSA NPs

FA-ergone-BSA NPs FA-5-Fu-CM-β-CDBSA NPs FA-DOX-AN NPs FA-DOX-BSA-dextran FA-VBLS-BSA NPs FA-TMX-BSA FA-DOX-magnetic iron oxide-BSA NPs FA-4Atp-AuNP FA-BSA-Au NPs FA-Au-SMCC-DOX FA-BHC-gold NPs FA-Au-P(LA-DOX)-bPEG-OH micelles FA-PEG-HAuNSsiRNA FA-gold NPs FA/FITC-Fe3O4-DPAPEG NPs FA-dextran-DOX retinoic acid magnetic NPs

FA-functionalized NPs decreased the toxicity of DOX, in vivo. FAIanchored NPs ould be effective in targeting VBLS-sensitive tumors, in vitro. FA-anchored NPs caused tumor remission whereas non-targeted NPs could only attenuate the tumor development. FA-conjugated NPs showed greater inhibition of tumors than in the absence of FA in vitro and in vivo. FA-modified NPs can be used to selective targeting of FR expressing tumor cells, in vitro. FA-functionalized NPs exhibit a great tumor targeting and dualmodality imaging, in vitro. FA-anchored NPs exerted enhanced drug accumulation and retention in cancer cells, in vitro. FA-modified complexes were found to be active against FRexpressing cancer cells, in vitro. Cellular uptake of the FA-conjugated micelles facilitated by the FR-mediated endocytosis, in vitro. Injection of FA-anchored NPs into tumor bearing mice led to higher tumor uptake of the targeted NPs compared to the nontargeted ones. FA-attached NPs induce apoptosis in cancer cells, in vitro. FA-conjugated magnetic NPs could potently detect the FRexpressing cancer cells, in vitro. FA-conjugated magnetic micelles showed the IC₅₀ of the targeted drug to about 10 times lower than the free drug, in vitro.

MCF-7 and MCF-7 bearing athymic nude mice In vitro (KB cells) and in vivo (KB tumor bearing BALB/C nude mice)

(260) (261)

Hela and MCF-7

(262)

MGC803

(263)

HDF, C0045C, and HepG2

(264)

Vero and HeLa

(265)

4T1

(266)

In vitro (HeLa cells) and in vivo (HeLa bearing Nude mice) KB cells

(267) (268)

MCF-7 and A549

(269)

MCF-7 and MDA-MB-468

(270)

FA-DOX-magnetic NPs FA-SPIONs-DOXPEG-lipid shell

FA-anchored NPs enhanced the DOX-induced apoptosis in cancer cells, in vitro. FA-modified core-shell NPs exhibited the possibility of codelivering drugs and SPIONs to the same cells, in vitro.

C30 and CP70

(271)

HeLa cells

(272)

VBLS: vinblastine sulfate, TMX: tamoxifen, BHC: berberine hydrochloride, DOX: doxorubicin, hyd: a hydrazone bond, PNVCL: Poly(N-vinylcaprolactam), m-THPC: meta-tetra (hydroxyphenyl)chlorin, 2-ME: 2-methoxyestradiol, PTX: paclitaxel, CM-b-CD: carboxymethyl-b-cyclodextrin, 5-Fu: 5-fluorouracil, AN: albumin nanospheres, TMX: tamoxifen , 4Atp: 4-aminothiophenol , AuNP: Gold nanoparticles, SMCC: Succinimidyl 4-(Nmaleimidomethyl) cyclohexane-1-carboxylate, BHC: berberine hydrochloride, PLA: poly(L-aspartate), FITC: fluorescein isothiocyanate, DPA: dopamine hydrobromide ,PEG: ethylene glycol ,PLLA: poly(Llactide), FA: folic acid, BSA: Bovine serum albumin, HAuNS: hollow gold nanospheres, siRNA :Small interfering RNA, SPIONs: superparamagnetic iron oxide nanocrystals.

Table 7: Studies related to the anti-tumor application of EGFR-targeted NPs. NPs Biotinylated EGFNeutrAvidin(FITC)-gelatin NPs EGF-PTX-poly [MPC-co-n-butyl methacrylate-co-pnitrophenyloxycarbonyl-PEGmethacrylate] EGF-Raman-phospholipid gold NPs EGF-PAMAM-QDs- vimentin / yellow fluorescent protein siRNA

Outcome EGF-conjugated NPs was mainly distributed in cancerous lungs.

EGF-DNA-PAMAMFITC/Lss670 NPs

EGF-functionalized NPs showed a significantly higher cellular uptake into cancer cells.

EGF-stearoyl gemcitabine NPs

The extent of drug uptake tumor cells was correlated to the EGFR density on the tumor cells.

EGF-Magnetic NPs EGF-SPION FITC-NLC, FITC-AEYLR-NLC, or FITC-RALEL-NLC EEEEpYFELV (EV)- PEGLiposome-protamine-heparin NPs EGFR binding peptide- wildtype p53 tumor suppressor genePEG-thiolated gelatin NPs D4-PEG-liposome D4-MnFe2O4-PEG

The cytotoxicity and antitumor effect of EGFmodified NPs were significantly greater than those of non-targeted ones. An efficient contrast agent with in vitro confocal reflectance microscopy on lung carcinoma. EGF-modified NPs can localize within cells that express the EGFR in a receptor-dependent manner, in vitro.

EGF-modified NPs enhanced the MR imaging contrast at the tumor site. EGF-modified conjugates in animals provided receptor-mediated targeted delivery across the blood–brain barrier and tumor retention. FITC-AEYLR had high EGFR targeting activity mediated by tumor cells with high-expression of EGFR. EGFR-targeted NPs were taken up by the tumor cells and trafficked to the cytoplasm with high efficiency, in vitro.

Cell lines

Ref.

A549 tumor bearing nude mice

(283)

In vitro (BT-20, A431, and H69) and in vivo (BALB/c, nu/nu, athymic mice)

(285)

A549 and H460

(286)

HN12, NIH3T3, and NIH3T3/EGFR

(287)

In vitro (Hela, HepG2 and MDAMB-231) and in vivo (HepG2, MDA-MB-231-luc bearing BALB-c nude mice) In vitro (MDA-MB-468, HTB132, MDA-MB-231, HTB-26, MCF-7, and HTB-22) and in vivo (MDA-MB-468 tumor bearing athymic Nu/Nu mice) Murine tumor models of melanoma and hepatoma

(288)

(289)

(290)

In vitro (C6) and in vivo (C6 glioma bearing mice)

(291)

NCI-H1299 and K562

(292)

NCI-H460

(293)

EGFR-targeted NPs efficiently transferred into cancer cells, in vitro.

Panc-1, Capan-1, SKOV3, and NIH-3T3

(294)

D4-attached liposomes accumulated in EGF-Rexpressing tumor site. Up regulation of EGFR expression was associated with the increased MR contrast, in vitro.

In vitro (H1299) and in vivo (H1299 xenograft mouse models )

(295)

SKBR-3, PC3 and HEK-293

(296)

GE11-NIS-LPEI-PEG

NIS gene transfer using polyplexes coupled with an GE11 induced tumor-specific iodide uptake.

In vitro (HuH7, FTC-133 , MCF-7 and SKOV-3) and in vivo (HuH7 bearing nu/nu mice)

(297)

GE11-PEG-DSPE-PTX micelle

Tumor cell proliferation was significantly inhibited by GE11-coupled micelle, in vitro.

Hep-2 and U-937

(298)

Active EGFR targeting enhances intracellular uptake of the drug, in vitro.

A431

(299)

GE11-Pc 4-PEG-PCL

Successful targeting and partitioning toward the A-431, HT-29, MDA-MB-231 and cancer cells was happened by EGFR-targeted NPs, (300) BT-474 in vitro. EGFR-targeting peptide-EGFPPeptide-modified NPs were internalized efficiently Panc-1 (301) N1 plasmid DNA-gelatin NPs by receptor-mediated endocytosis, in vitro. Aptamer-anchored NPs were effectively Aptamer J18-gold NPs internalized by EGFR-expressing cancer cells, in (303) A431 and MDA-MB-435 vitro. QDs: quantum dots, NLCs: Nanostructured lipid carriers, EGF: epidermal growth factor, PTX: paclitaxel, MPC: 2Methacryloyloxyethyl phosphorylcholine, PAMAM: polyamidoamine, Lss670: a near-infrared (NIR) dye, SPION: Superparamagnetic iron oxide nanoparticles, NLC: nanostructured lipid carriers, FITC: fluorescein isothiocyanate, MnFe2O4: manganese ferrite nanoparticles, PEG: polyethylene glycol, NIS: nonviral polyplex-mediated sodium iodide symporter, LPEI: linear polyethylenimine, PEG-DSPE: Poly (ethylene glycol)-distearoylphosphatidylethanolamin, PVX: potato virus X. fluorescently labeled PVX-GE11 filaments

Table 8: Advantages and disadvantages of various active targeting approaches using NPs. Advantages Disadvantages Transferrin conjugated NPs 1. Well studied 1. A nonspecific biodistribution and targeting 2. Overexpression of Tf receptor on metastatic and drug resistant tumors 2. A exogenously-supplied transferrin can lead to 3. Transferrin (Tf) can be conjugated easily to a an overdose of iron transport into brain variety of materials for cancer targeting 4. The high intracellular uptake of Tf-conjugated NPs 5. A significantly delayed blood clearance, the longest tumor residence time and the greatest tumor accumulation Cell-penetrating peptides (CPPs) conjugated NPs 1. The ability of crossing the cell membrane via energy-independent processes 2. The capacity to efficiently internalize the associated biomolecules without compromising their biocompatibility 3. The capacity of peptides to protect the bioactive conjugates from protease or nuclease degradation, thereby increasing the serum halflife of cargoes 4. High biological safety and low cytotoxicity LDL conjugated NPs

1. In some cases, it may alter the biological activity of conjugates 2. The transmembrane mechanism of CPPs is still not precisely clear

1. LDL NPs are natural carriers and are therefore biocompatible. 2. LDL particles are non-immunogenic, so they escape recognition by phagocytes . 3. Hydrophobic drugs can be loaded in the hydrophobic core while amphiphilic drugs can be loaded in the amphipathic shell of LDL 4. LDL has a large core capacity 5. LDLR is highly expressed in most tumor cells 6. Uptake of LDL particles occurs through both receptor- and non-receptor-mediated pathways . Integrin modified NPs

1. existence of LDL receptors on normal cells 2. Many tumors do not overexpress the LDLRs whereas some normal tissues express 3. LDL particles are derived from human blood, so, there is concern about the introduction of pathogens

1. Integrin signaling control diverse functions in tumor cells, including migration, invasion, proliferation and survival 2. Some integrins are highly over-expressed on many cancer cells

1. Integrins also exist on normal cells

3. Some integrins such as αvβ3 and αvβ5 are induced on endothelial cells during angiogenesis 4. well accessible cell surface receptors Carbohydrate modified NPs 1. Glycotargeting, which is based on endogenous lectin interactions with carbohydrates 2. Effective oral delivery system to improve the absorption and bioavailability of poorly absorbable drugs 3. Cytoadhesive properties of the lectin and the protective effects of the nanocarrier result in improved bioavailability of the formulation Folate modified NPs

1. A glycotargeting often requires multiple interacting carbohydrates to achieve strong enough binding strength 2. Lectin elicits an immune response and some degree of toxicity

1. The folate receptor is overexpressed on the vast majority of cancer tissues, while its expression is limited in healthy tissues and organs 2.Folate is small, stable over a broad range of temperatures and pH values, and thus amenable for site-specific chemical modification. 3. It is inexpensive, nonimmunogenic, and binds to the FR with high affinity even after conjugation to a diagnostic or therapeutic cargo 4. Folate is transported across the cellular membrane through various ways EGF modified NPs

1. Folate uptake can promote cancer cell proliferation, migration and loss of adhesion through downregulation of the cell-cell adhesion molecule, E-cadherin, promoting cellular motility and metastasis