The use of nanoparticles as a promising therapeutic approach in cancer immunotherapy

Abstract

Cancer is one of the most important causes of death all over the world, which has not yet been treated efficiently. Although several therapeutic approaches have been used, some side effects such as toxicity and drug resistance have been observed in patients, particularly with chemotherapy. The nanoparticle-mediated drug delivery systems (DDS) have a great potential to improve cancer treatment by transferring therapeutic factors directly to the tumor site. Such a treatment significantly decreases the adverse effects associated with cancer therapy on healthy tissues. Two main strategies, including passive and active methods, have been considered to be effective techniques which can target the drugs to the tumor sites. The current review sheds some light on the place of nanotechnology in cancer drug delivery, and introduces nanomaterials and their specific characteristics that can be used in tumor therapy. Moreover, passive and active targeting approaches focus on antibodies, particularly single chain variable fragments (scFv), as a novel and important ligand in a drug delivery system.

Keywords::

• cancer therapy
• drug delivery
• nanoparticle
• scFv
• tumor targeting

Introduction

Cancer has been considered as the second cause of death after heart disease, accounting for around 23% of all deaths in the United States (Jemal et al. 2008). Breast and prostate cancers are the most common cancers in women and men, with the probability of 29% and 28% respectively (Hajdu 2011). The precise etiology of cancer is unknown. Although the genetic factor is involved in 5–10% of cancers, other factors including the use of tobacco, poor diet, certain infections, lack of physical activity, obesity, and pollution may also directly damage genes and ultimately lead to carcinogenic mutations (Anand et al. 2008).

Surgery, as a primary therapeutic approach to treat solid tumors, can lead to pain relief and patients’ survival (Miller et al. 1981). Radiation and chemotherapy, which destroy cancerous cells, are other therapeutic approaches for tumors (Brannon-Peppas and Blanchette 2012). The tumor-specific therapy is the main objective in the treatment of different cancers, which has not been addressed in previously mentioned tumor therapeutic approaches (Perez 2005). As these drugs do not act selectively against the cancer cells, toxic effects of the chemotherapeutic drugs are expected (Benita 2005). Moreover, non-specific distribution of drugs results in the need for administration of higher drug volumes, which increases the cost and toxicity (Perez 2005).

The improvements in cancer therapy are developing not only in the production of new agents against cancer but also in designing new drug delivery methods for both old and new agents. It is expected that such an advancement can be effective in decreasing the required doses of therapeutic drugs in cancer therapy (Brannon-Peppas and Blanchette 2012). Drug delivery systems (DDS) have recently been developed to reduce the side effects of chemotherapy and to improve the efficiency of the existing drugs. In an organized DDS, the drug is carried to the target and therefore its influence on non-targeted tissues and unwanted side effects can be avoided. Moreover, a DDS reduces the rapid degradation or clearance of the drug and increases drug concentration in the target tissues (Nevozhay et al. 2006). Among these systems, it seems that nanocarriers are a promising option in the field of drug delivery (Park et al. 2009). The unique physicochemical and biological features of nanoparticles make them excellent candidates in cancer therapy (Brannon-Peppas and Blanchette 2012).

In this review, we have tried to discuss the nanoparticles as DDS and their great potential to destroy tumor cells. However, we will describe single chain variable fragment (scFv)-targeted nanoparticles in more detail compared to other nanoparticles.

Nanoparticles and drug delivery

Nanoparticles have various physicochemical characteristics which make them interesting in the treatment of cancer. Since the size of the nanomaterials is smaller than the size of a cell, they can transport large amounts of agents, multiple drugs, or probes onto the surface or to the inside of the cell, without disturbing its function. The small size of such particles enables them to cross through small vessels and penetrate tissues rapidly. These particles improve the therapeutic index and half-life of the drug in blood circulation, with reduced side effects and no platelet activation. Additionally, nanoparticles are able to increase the bioavailability of water-insoluble drugs and protect the therapeutic agents from physiological barriers. Such small particles provide sufficient surface area and strategies for rapid drug release (Conti et al. 2006, Hu et al. 2010, Swami et al. 2012). Therapeutic drugs can be adsorbed, covalently attached, or even encapsulated in nanoparticles. When the drug-loaded nanocarrier reaches the site of action, the therapeutic agents will be released (Wilczewska et al. 2012).

An important issue in the production of nanoparticles is their stability and rapid clearance in blood circulation. Therefore, particular parameters, including physicochemical properties which can affect the distribution of nanomaterials, have to be investigated precisely (Shenoy and Amiji 2005). Generally, nanocarriers ranging in size from 10 to 100 nm have desirable pharmacokinetic properties in vivo. Smaller nanoparticles are removed through tissue extravasations and renal clearance, whereas larger particles are rapidly opsonized with plasma proteins and captured by phagocytes. It has been shown that the particle size plays a crucial role in the blood bio-distribution of nanoparticles (Cole et al. 2011). Moreover, the surface charge of the nanocarriers may affect their uptake by macrophages in the liver and spleen. It was found that neutrally charged particles have lower clearance rates than charged particles (Swami et al. 2012). However, the cationic surface enhances the interaction of the nanoparticles with the cells and improves the rate and extent of internalization (Shenoy and Amiji 2005). Prolonged circulation half-life can be achieved by the conjugation of nanoparticles with hydrophilic polymers such as polyethylene glycol (PEG), which has favorable properties including low toxicity, immunogenicity, high flexibility, and hydrophobicity (Swami et al. 2012). PEGylation results in the creation of a cloud of chains around the nanoparticles, which ultimately repels plasma proteins and inhibits the opsonization process (Owens III and Peppas 2006).

Liposomes, solid lipid nanoparticles, dendrimers, polymers, silicon, carbon, and magnetic particles are the various nanocarriers assessed as DDS. Although several nanoparticle-based chemotherapeutic delivery systems are available in the market, few nanoparticles are currently used in various stages of clinical or preclinical studies (as shown in Table I).

Table I. Examples of nanoparticles being marketed or undergoing clinical studies.

Name	Nanoparticle	Target	Drug	Therapeutic activity	Status	Company	References
BIND-014	Polymeric (PLGA-PEG)	PSMA	Docetaxel	Metastatic cancer, Solid tumor	Phase I	BIND Bioscience	Shi et al. (2011)
CALAA-01	Polymeric	Transferrin receptor	siRNA	Solid tumor	Phase I	Calando Pharmaceuticals	Zuckerman et al. (2014), Mellet al. (2011)
MBP-426	Liposome	Transferrin receptor	Oxaliplatin	Gastric, esophageal,	Phase Ib/II	Calando	Riggio et al. (2011), Senzer et al. (2009)
				gastroesophageal
				adenocarcinoma		Pharmaceuticals, Inc
2B3-101	Glutathione PEGylated liposome (GSH-PEG)	Glutathione transporters	Doxorubicin	Solid tumors, brain metastases, glioma	Phase I/IIa	BBB-Therapeutics B.V	Gaillard et al. (2014), *
CD74-IL-DEX	Liposomes	CD74	Dexamethasone	B-cell malignancies	Preclinical	Immunomedics, Inc.	Mao et al. (2013)
Doxil (Caelyx)	PEGylated Liposomes		Doxorubicin	Ovarian/breast cancer	Approved (1995)	Ortho Biotech, Schering-Plough	Pillai and Ceballos-Coronel (2013)
Oncaspar	Asparaginase (PEG)		Asparaginase	Acute lymphoblastic leukemia	Approved (1994)	Enzon Pharmaceuticals	Silverman et al. (2010)
DaunoXome	Liposome		Daunorubicin	HIV-related Kaposi's sarcoma	Approved (1996)	Gilead Science	Stewart et al. (1998)
Myocet	Liposome		Doxorubicin	Breast cancer	Approved (2000)	Elan/Sopherion Therapeutics	Rivera (2003)
Abraxane (ABI-007)	Albumin		Paclitaxel	Metastatic breast cancer	Approved (2005)	Abraxis BioScience, AstraZeneca	Miele et al. (2009)
Marqibo	Liposome		Vincristine	Philadelphia chromosome–negative lymphoblastic leukemia	Approved (2012)	Talon Therapeutics, Inc.	O’Brien et al. (2013)
Genexol-PM^®	Polymeric micelle		Paclitaxel	Breast and lung cancers	Approved (2004)	Samyang Co.	Guo and Huang (2013)
				Bladder, ureteral Cancer	Phase II	IGDRASOL Inc	**
				Ovarian Cancer	Phase II	Samyang Biopharmaceuticals Corporation	***
Xyotax, CT-2103	Polyglutamic acid (PGA)		Paclitaxel	Naïve advanced non-small cell lung cancer	Phase III	Cell Therapeutic, Inc.	Guo and Huang (2013)
Docetaxel-PNP	Polymer		Docetaxel	Advances solid malignancies	Phase 1	Samyang Pharmaceuticals	Svenson (2012)
NK-105^®	Polymeric micelles		Paclitaxel	Breast Cancer	Phase III	Nippon Kayaku Co.	Cabral and Kataoka (2014)
NK911	Polymeric micelles		Doxorubicin	Metastatic pancreatic cancer	Phase II	Nippon Kayaku, Co.	Li and Wallace (2008)
Aurimmune (CYT-6091)	Colloidal gold		TNF-α	Head and neck cancer	Phase I	CytImmune Sciences, Inc.)	Jain et al. (2014), Visaria et al. (2007)
			All trans-retinoic acid (ATRA)	Acute promyelocytic leukemia	Phase II	Aronex Pharmaceuticals, Inc.	Douer et al. (2001)

*ClinicalTrials.gov Identifier: NCT01386580.

**ClinicalTrials.gov Identifier: NCT01276548.

***ClinicalTrials.gov Identifier: NCT01426126.

PSMA, prostate-specific membrane antigen; mCRPC, Metastatic Castration-Resistant Prostate Cancer; TNF, tumor necrosis factor; PLGA, Poly(lactic-co-glycolic acid); PEG, Polyethylene glycol

Liposomes

Liposomes (80–300 nm) are the first particles designed as nanocarriers (Sunderland et al. 2006) which contain amphiphilic lipid molecules (phosphatidyl ethanolamine and phosphatidyl choline) with cholesterol to enhance their stability and rigidity accumulated into bilayer vesicles. External energy from sonication, homogenization, shaking, or heating is required for the assembly of these particles (Couvreur and Vauthier 2006, Hu et al. 2010). It has been demonstrated that liposomes increase the solubility of drugs and enhance their in vitro and in vivo anticancer activity, reduce the undesirable side effects, and increase pharmacokinetic properties such as the therapeutic index of chemotherapeutic agents (Santos Giuberti et al. 2011). The drug release in liposomes, by adsorption, fusion, endocytosis and lipid transfer, depends on the liposome's features such as pH and osmotic gradient, and the surrounding environment (Santos Giuberti et al. 2011, Wilczewska et al. 2012).

Various drugs including anti-cancer drugs, antibiotics, anti-inflammatory drugs, neurotransmitters (serotonin), and anti-rheumatic drugs can be encapsulated in liposomes (Wilczewska et al. 2012). Recent liposomal formulations such as Doxil, DaunoXome^®, DepoCyt^® and ONCO-TCS are in the world market, and are loaded with Doxorubicin, Danarubicin, Cytarabine and Vincristine, respectively (Haley and Frenkel 2008, Hu et al. 2010). In addition, they have a particular structure which can simultaneously load hydrophilic drugs in the aqueous core and hydrophobic drugs in the lipid membrane (Hu et al. 2010).

The main problem of liposomes as DDS is their non-specific accumulation outside the target tissues, which may lead to the death of normal cells (Daemen et al. 1995). Modified liposomes containing specific proteins, antigens, or other biological substances can be applied to selectively deliver drugs for a particular tissue. Due to these unique characteristics, they have attracted great interest from the researchers. Furthermore, cationic liposomes (CLs) would be a great choice in gene delivery compared to natural or anionic liposomes (Wilczewska et al. 2012).

Nanoparticles based on solid lipids

The solid lipid matrix-based nanoparticles are types of nanocarriers applied in ocular, pulmonary, rectal, dermal, per-oral and parenteral delivery (Wilczewska et al. 2012). Some examples of these structures are SLN (solid lipid nanoparticles), LDC (lipid drug conjugates) and NLC (nanostructured lipid carriers) (Wissing et al. 2004). SLNs, or lipospheres or nanospheres, are made from solid lipids consisting of highly purified triglycerides, waxes or complex glyceride mixtures stabilized by various surfactants which have specific properties such as suitable physical stability and tolerability, controlled drug release, site-specific targeting, and protection of labile drugs from degradation. However, there are some disadvantages regarding these nanoparticles, including their limited loading capacity and drug leakage after SLN crystallization (Kovacevic et al. 2011, Wilczewska et al. 2012, Wissing et al. 2004).

LDC and NLC are modified lipids in which some SLN restrictions are resolved. The main purpose in the development of LDC is to improve the applicability of lipid-based carriers to lipophobic molecules. NLCs are lipid carriers containing a certain structure for the enhancement of payload and the prevention of drug expulsion. As a result, most of the applications of such nanolipids are topical (Davis 2008).

Polymeric nanoparticle

Polymeric nanoparticles (PNPs) have a diameter ranging from 10 to 100 nm, containing natural (albumin, gelatin, chitosan and polysaccharide) or synthetic (polyacrylamide, e-poly caprolactone and polyacrylate) polymers (Hu et al. 2010, Wilczewska et al. 2012). Some polymers, such as poly (lacticoglycolic acid) (PLGA) and polycaprolactone (PCL), have also been approved by the Food and Drug Administration (FDA) to be used as synthetic polymers (Hu et al. 2010).

Drug-loaded polymeric nanocarriers bear a significant impact on the clinical approach by improving the pharmaceutical efficacy, controllable drug-release profiles, and high loading capacity for relatively insoluble drugs. Due to these features, polymeric nanoparticles have attracted academic, clinical, and industrial interest, although they still are in the early stages of development (Davisand Shin 2008, Hu et al. 2010).

PNPs are classified as biodegradable (poly (L-lactide) and polyglycolide) and non-biodegradable (Wilczewska et al. 2012). Biodegradable nanocarriers have numerous advantages, such as high stability and less immunogenicity (des Rieux et al. 2006). Although a minimal systemic toxicity has been assigned by Kumari et al. (Kumari et al. 2010) for using PLGA in drug delivery applications, it has been shown that PLGA is a suitable choice for the delivery of hydrophobic drugs, since they can degrade in the body and produce biodegradable metabolite monomers, such as lactic acid and glycolic acid (Hu et al. 2010, Wilczewska et al. 2012).

Magnetic nanoparticles

Among the wide range of nanomaterials, magnetic nanoparticles (MNPs) have attracted considerable attention in biomedical applications due to their ability to be handled by an external magnetic field, their great potential in imaging, high uptake by the target tissue, and improving the treatment in therapeutically optimal doses (Arruebo et al. 2007, Sun et al. 2008).

Normally, MNPs are composed of a magnetically active core covered by various shells to anchor target ligands or implement extra imaging modalities. Therapeutic drugs can then be set in the shell structure or chemically attached to their surface (Veiseh et al. 2010).

Based on their magnetic features, MNPs can be divided into pure metals (e.g. manganese, iron, cobalt, and nickel), their alloys, and oxides (Wilczewska et al. 2012). Iron oxide nanoparticles have desirable properties such as chemical stability and probable chemical modification by coating the iron oxide cores with various shells (e.g. polymeric, gold, silane) and are also considered as the only type of MNPs that have been approved by the FDA for clinical applications (Fidalgo et al. 2009, Wilczewska et al. 2012). Furthermore, iron oxide can be produced naturally in the human heart, spleen, and liver (Grassi-Schultheiss et al. 1997) which indicates its non-toxicity and biocompatibility in the body.

The effect of iron oxide on cellular function, and its toxicity, have been also investigated. Depending on the injection route, MNPs can interact with extracellular agents, macrophage cell membrane, endothelial cells, skin epithelium, and respiratory tracts (Wilczewska et al. 2012). Studies have shown that iron oxide nanoparticles may induce oxidative stress by increasing ROS production, which results in augmented expression of hemoxygenase-1 and superoxide dismutase as antioxidant enzymes, followed by the release of proinflammatory components. These events can lead to apoptosis and mutagenesis (Naqvi et al. 2010).

Silica materials

Silica materials are divided into two categories, including xerogels and mesoporous silica nanoparticles (MSN) (Hu et al. 2010). Among inorganic nanomaterials, silica particles are the most promising choice in biological applications. These nanoparticles contain several advantages, such as biocompatibility, a highly porous structure, and ease in terms of functionalization (Amato 2010).

Silica xerogels include an amorphous structure with high porosity. The shape and size of the pores are variable, depending on the parameters of synthesis (Echeverría et al. 2010). For example, sol-gel techniques are commonly applied to form silica xerogels embedded with drugs, therefore a change in production conditions, including the ratio of reagents, temperature, and concentration of catalyst, may alter the properties of xerogels (Wilczewska et al. 2012).

MSN, on the other hand, form homogenous structures with higher surface area, which is favorable for the adsorption of therapeutic drugs (Di Pasqua et al. 2009). These particles possess the ability to load large and small molecules, adsorption of DNA, and gene transfer (Wilczewska et al. 2012). A variety of drugs including antibiotics (Li et al. 2010), anticancer drugs (Di Pasqua et al. 2009), and heart disease drugs (Popovici et al. 2011) are loaded into MSNs via chemical or physical adsorption mechanisms (Di Pasqua et al. 2009).

Although several studies have indicated that silica particles are biocompatible and have favorable diagnostic and therapeutic properties, some studies have however shown in vitro and in vivo toxicity of silica nanoparticles (Wilczewska et al. 2012). Depending on the type of cells and the size of the nanoparticles, silica nanoparticles can induce oxidative stress through the generation of reactive oxygen species (Liu and Sun 2010), reduction of glutathione levels (Kyung et al. 2009), and induction of anti-oxidant enzymes (superoxide dismutase (SOD) and hemooxygenase-1 (OH-1)) (Park and Park 2009), which ultimately disturbs the cell membrane (Lin et al. 2006).

Dendrimer Nanocarriers

Dendrimers are unique polymers with repetitively branched structures. Glycogen, amylopectin, and proteoglycans are examples of such structures (Svenson and Tomalia 2012). Dendrimers are spherical compounds and are generally symmetrical around their core. The biocompatibility and biochemical properties of the tree-shaped structure (dendron), which contains a chemical group called the focal group, depend on the surface dynamic groups (Caminade et al. 2005). The amount of drug loaded on dendrimers can also be varied, based on the drug properties. Toxic, unstable, or poorly soluble agents encapsulated in a carrier will enable the possibility of controlling the quantity of drugs on the nanoparticle surface through the control of the number of covalent bonds (Singh et al. 2008).

Polyamidoamine (PAMAM) is one of the most important dendrimers, and has a great potential to carry small drug particles (Wilczewska et al. 2012). The cationic form of PAMAM is a very potent vehicle for the delivery of macromolecular agents such as DNA, through cellular and subcellular barriers (Patri et al. 2005, Yellepeddi et al. 2009).

Carbon nanomaterials

Carbon nanoparticles used in DDS are divided into two categories, including carbon nanotubes (CNTs) and carbon nanohorns (CNH). Nanotubes have a special structure composed of one or more layers of graphite tubes (SWNCTs – single-walled carbon nanotubes, or MWCNTs – multi-walled carbon nanotubes) with an extended surface area and exceptional electronic and thermal conductivity (Beg et al. 2011). The biocompatibility of nanotubes could be improved by chemical modification of their surface (Foldvari and Bagonluri 2008). PAMAM dendrimers can covalently attach to the surface area of nanotubes and amplify their functions (Zhang et al. 2010).

Drugs can be loaded through different methods, such as encapsulation, chemical adsorption on the surface or in the space between the nanotubes, and attachment of active elements to functionalized carbon nanotubes. Drug encapsulation in a carbon nanotube is more applicable compared to the other methods, since it protects the drug from degradation during its crossing to the cells, and the drug will be released only in specific conditions (Wilczewska et al. 2012). Furthermore, in order to prevent the undesirable drug release, the tips of CNTs can be closed with polypyrrole (PPy) films (Luo et al. 2011).

Nanohorns (a type of single-layer nanotube) have properties similar to those of nanotubes; however, they can be produced with lower cost and higher purity (Shiba et al. 2006). Drugs can be loaded on these carbon nanoparticles through surface adsorption (Murakami et al. 2008) or nano-precipitation. It has been demonstrated that nano-precipitation is more effective compared to the adsorption method, with respect to the nanohorns (a 3-fold increase in the number of molecules was observed in nanohorns) (Ajima et al. 2008).

The toxicity of carbon nanoparticles depends on their exceptional geometric structure, which is well-defined (Jia et al. 2005). For instance, the toxicity of nanotubes depends on the length to diameter ratio of the particle, and also the toxicity of the graphite. Additionally, some impurities, like residual metal and amorphous carbon, contribute to the increased levels of reactive oxygen species (ROS) and induced oxidative stress in cells (Dobrovolskaia and McNeil 2007).

Targeted drug delivery

As mentioned earlier, conventional chemotherapeutic drugs are distributed nonspecifically in the human body where they affect both cancerous and normal tissues, which ultimately lead to inadequate treatment and high toxicity. Targeted therapy has proved to be an effective method in overcoming the lack of specificity of conventional chemotherapeutic drugs (Ross et al. 2004).

The strategy of nanoparticle targeting is of great importance in targeted therapy. In order to maximize drug delivery in cancer therapy, the administered drugs are required to pass through the body barriers with minimal loss in their activity and volume in the blood circulation. After reaching the desired region, the drugs should be able to selectively destroy the tumor cells, without affecting normal cells, in a controlled release manner (Cho et al. 2008).

Cell-specific targeting with nanocarriers may be accomplished by using active or passive mechanisms (Allen 2002, Cho et al. 2008, Maeda 2001). In the next section, we will discuss passive and active mechanisms of drug delivery.

Passive targeting strategy

The optimization of the size and surface characteristics of nanoparticles will increase their ability to circulate in the bloodstream for a longer time, and consequently, provide greater chance for reaching specific tumor sites. The unique pathophysiologic features of tumor vessels enable macromolecules, including nanocarriers, to selectively accumulate in tumor tissues, which is the main goal of a passive targeting strategy (Cho et al. 2008).

One of the critical features of tumors is the inherent leaky vasculature present in cancerous tissues (Brannon-Peppas and Blanchette 2012). Fast-growing cancer cells require the employment of new vessels (neovascularization) or rerouting of existing vessels near the tumor mass, to supply necessary materials. The imbalance of angiogenic regulators can cause the tumor vessels to appear disorganized, with enlarged gap junctions between endothelial cells, and compromised lymphatic drainage (Carmeliet and Jain 2000). These features mentioned are expected to enhance the permeability and retention effect (EPR effect), which establish an important mechanism by which nanocarriers can extravagate into the tumor tissues rapidly by the EPR effect. Moreover, poor lymphatic drainage in tumors retains the accumulated nanomaterials and allows the release of toxic agents into the vicinity of the tumor cells (Peer et al. 2007a). By means of these features, concentrations of drug-loaded nanoparticles in tumor tissue can reach levels 10 to 100 times higher compared to the levels achieved in the administration of the free drug (Sinha et al. 2006).

The unique tumor microenvironment, which is different from that of normal cells, is also another major contributor to passive targeting. Some drugs can be administered as pro-drugs or inactive forms and remain inactive until they reach the tumor. The tumor environment is then able to convert the drug into an active form, which is called tumor-activated pro-drug therapy. Furthermore, fast-growing cancer cells show a high metabolic rate, for which oxygen supply and sufficient nutrients are not usually satisfactory. Consequently, tumor cells use glycolysis to obtain extra energy, resulting in an acidic environment (Pelicano et al. 2006, Sinha et al. 2006). The pH-sensitive nanoparticles such as liposomes are designed to remain stable at a physiologic pH (7.4), but degrade in the acidic environment of tumor cells and release the active form of drugs (Yatvin et al. 1980). In addition, most linkers in nanocarrier-conjugated drugs are peptidase-sensitive or acid-labile (Suzawa et al. 2002). For example, the overexpression of the matrix metalloproteinase-2 in melanoma has been observed in preclinical and clinical studies. Matrix metalloproteinase-2 plays a critical role in the degradation of basement membranes and the extracellular matrix (Sinha et al. 2006). Based on the studies reported by Mansour et al., a water-soluble maleimide derivative of doxorubicin, which is an important chemotherapeutic drug, incorporates a matrix metalloproteinase-2-specific octapeptide sequence between the drug and the carrier. This polymer-drug conjugate was found to have a high affinity for the cysteine-34 position of circulating albumin. The albumin-bound form was efficiently and specifically cleaved by the matrix metalloproteinase- 2 that led to the release of free doxorubicin (Mansour et al. 2003).

Another approach that allows the passive delivery of therapeutic agents through nanocarriers, is the localized administration of drug-loaded nanoparticles. Various methods have been observed to improve the tumor delivery of anticancer drugs, like intrapretoneal and intravesical injections, which directly transfer the drug to the tumor tissue, without circulating throughout the body. Direct inoculation of the drug into tumors is also an attractive approach that has been examined (Yockman et al. 2003). For instance, direct administration of mitomycin into target tissue resulted in an enhanced concentration of the drug at the tumor site and declined toxicity (Nomura et al. 1998).

The disadvantage of localized drug delivery through intratumoral administration is that such approaches may result in exposure to higher concentrations of therapeutic agents, which is not always feasible and can be highly invasive. Additionally, sometimes the site of the tumor is not clear, and access to some cancers such as lung cancer would be complicated in such cases (Sinha et al. 2006, Yockman et al. 2003).

In addition, although passive targeting strategies incorporate the basis of clinical therapy, they have several limitations that may not bring about desirable clinical outcomes. Moreover, targeting cells within a tumor is not always possible. Since some drugs cannot diffuse efficiently, the random nature of the approach makes it difficult to control the process, which ultimately leads to the encouragement of multiple-drug resistance (MDR). It has been also observed that certain tumors do not show the EPR effect, and the permeability of tumoral vessels may not be homogenized in a single tumor. A few limitations were also observed due to the lower potency of some drugs after being linked to target moieties when the targeting portion is not cleaved correctly (Peer et al. 2007a, Suzawa et al. 2002, Yockman et al. 2003).

Active targeting strategy

In order to overcome the limitations of passive drug delivery, various active strategies have been developed. In such techniques, nanocarriers will actively bind to specific cells after extravasation, which can be accomplished by conjugating the surface of the nanoparticle to a targeting moiety (Ferrari 2005, Torchilin 2005). Nanocarriers can identify target cells through a receptor-ligand binding (as shown in Figure 1) and the internalization of bound carrier occurs prior to the drug separation, allowing a selective accumulation of the drug in the tumor tissues (Sinha et al. 2006). In the preparation of nanoparticles with a ternary structure (a targeting ligand, a polymer or lipid carrier, and a chemotherapeutic agent), some aspects must also be considered to achieve more efficient delivery systems (Cho et al. 2008).

Figure 1. Passive targeting by nanoparticles injected in blood circulation. Nanopolymers increased in the target tissue due to EPR effect. Active tissue targeting of nanoparticles occur as conjugated by the ligand recognized target specifically.

First, cell-surface antigens and receptors should be expressed only on tumor cells and not on host cells (Cho et al. 2008), or overexpressed on target tissues compared to normal tissues (Peer et al. 2007a). Although most of the receptors are beneficial for tumor targeting and are often expressed on various types of cells (Sinha et al. 2006), the folate receptor is a recognized tumor marker that is highly overexpressed on the surface of different cancers, such as breast, kidney, lung, brain and ovarian cancers. These receptors bind to folate (vitamin B9), and then the folate-targeted nanoparticle transfers via receptor-mediated endocytosis (Leamon and Reddy 2004). Cho et al., (Cho et al. 2008) showed the expression of the folate receptor in 53% of human head and neck primary and metastatic tumor tissues compared to bone marrow tissues as normal cells which never expressed this receptor. Transferrin is another active targeting approach, because transferrin receptors are overexpressed in various tumor cells (Cho et al. 2008, Qian et al. 2002). It would be ideal if the regulation of the target marker or receptor happens through its attached ligands. Hence, endocytosis occurs quickly and thoroughly, by which the receptors will return to the surface of the cancer cells as soon as the active form of drug is released (Sinha et al. 2006).

Moreover, active targeting is particularly critical for the delivery of bio macromolecules such as DNA and siRNA, in which intracellular delivery for bioactivity is needed (Farokhzad and Langer 2009). Therefore, internalization of the drug-carrier complex can be achieved through the interaction of certain ligands and their surface receptors (Allen 2002, Pastan et al. 2006, Peer et al. 2007b). The drug release may occur in lysosomes by lysosomal enzymes, causing the release of free drug into the cytoplasm. The receptors or antigens released from their ligands should return to the cell membrane to start the second cycle of transport by binding with new ligand-targeted nanoparticles (Sinha et al. 2006). It has been shown that immunoliposomes targeted to B-cell lymphoma, labeled by an internalizing anti-CD19 ligand, have more significant therapeutic effects in comparison with a non-internalizing anti-CD20 ligand. However, it should be noted that targeting non-internalizing receptors to nanocarriers may be valuable in solid tumors, where cells lacking the target receptor can be eradicated via drug release in the area of the neighboring cells (bystander effect) (Allen 1994, Sapra and Allen 2002). To reach the optimal conditions, some variables such as cell specificity, ligand biocompatibility, binding affinity, and avidity should be considered (Allen 2002). Ligands used in drug-carrier conjugates require high specificity and avidity for cell surface receptors. Kato et al. (Kato et al. 1996) reported that ligands should have high affinity for their receptors in order to be effective and greatly stimulate the internalization of polymeric particles.

Although high binding affinity will increase the targeting specificity, there is evidence which shows that the higher binding affinity may result in reduced penetration due to the ‘binding-site barrier’ which is created when the nanocarrier binds to its target so firmly (Adams et al. 2001, Allen 2002). There are other targeting ligands such as proteins (mostly antibodies and their fragments), nucleic acids (aptamers), and others agents (carbohydrates, vitamins, and peptides) (Peer et al. 2007a). Among these ligands, antibodies and antibody fragments contain specific sites incorporated into drug carrier systems with high specificity for cellular receptors and a broad range of binding affinities, for more effective and less intrusive cancer therapy (Torchilin 2008). Other features, including immunogenicity, biodegradability, internalization rate, intracellular routing, and physiochemical properties, have also been studied for antibody-dependent drug delivery systems. Thus, the effective antibodies for optimum-targeted immunotherapy should easily integrate into a nanoparticle, specifically bind to tumor cells, have the ability to efficiently lead to internalization of the drug, and most importantly, be biodegradable (Sinha et al. 2006).

The antibody and its single chain Fv fragment in drug delivery

The first report regarding the tumor-targeting ability of monoclonal antibodies was published in 1975 (Köhler and Milstein 1975). However, the application of antibodies in cancer therapy was determined 20 years later (Abou-Jawde et al. 2003). During the past decade, the possibility of antibody targeting has been revealed clinically with the number of monoclonal antibodies (mAbs) approved by the FDA (Abou-Jawde et al. 2003, Glennie and van de Winkel 2003). Recent developments in antibody engineering have led to the development of chimeric, humanized, and fully human mAbs to reduce their immunogenicity (Wang et al. 2008).

On the other hand, mAb-conjugated nanoparticles show some limitations, since mAbs are complex and large molecules (∼150 kDa), which particularly restrict their penetration into solid tumors and require significant engineering at the molecular level to be suitable for drug delivery (Brennan et al. 2004, Weinberg et al. 2005). Consequently, mAbs have been engineered to improve their therapeutic abilities, which has led to the production of antibody fragments such as Fab (fragments of antigen binding), Fab'2, single chain Fv fragments (scFv), diabodies, and minibodies (Pavlinkova et al. 2001). They are smaller in size and have lower molecular weight and complement activation potential, while they have potent antigen-binding specificity (Carter 2001). Examples of antibody fragments used in nanoparticle structures as cancer therapy are shown in Table II.

Table II. Tumor-targeted nanoparticles with antibody fragments in clinical applications.

Brand name	Nanoparticle	Target ligand	Application	Stage	Agent	References
SGT53	Liposomes	Anti-transferrin receptor scFv	Solid tumors	Phase Ib/II	p53 gene	Senzer et al. (2013)
SGT-94	Liposomes	Anti-transferrin receptor scFv	Solid tumors	Phase I	RB94 gene	Matsumura et al. (2004)
MCC-465	PEGylated liposomes	F(ab’) 2 fragment	Metastatic stomach cancer	Phase I	Doxorubicin	Matsumura et al. (2004)
C225-ILS-DOX	PEGylated liposomes	Anti-EGFR Fab	Advanced solid tumors	Phase I	Doxorubicin	Mamot et al. (2012)
F5scFv-PEG Immunoliposomes	PEGylated liposomes	Anti-Her2 scFv	Breast cancer	Preclinical	Doxorubicin	Nellis et al. (2005)
CDP791	PEGylated	Anti-VEGF Fab	Cancer (antiangiogenesis)	Phase 1		http://www/nektar.com
MM-302	liposomes	Anti HER2 scFv	Breast cancer	Phase I	Doxorubicin	Maynard and Georgiou (2000)

PEG, polyethylene glycol; EGFR, Epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; VEGF, Vascular endothelial growth factor

At first, scFv fragments were mainly obtained from hybridoma cells of immunized animals, with amplification of the mRNA of VH and VL domains to couple with each other through a polypeptide linker, and inserted in a certain vector (Tassew et al. 2009). Recently, in vitro display technologies such as phage and yeast have been considered to surpass the traditional hybridoma technique, with better scFv binding properties in a great diversity of population (Bradbury et al. 2011, Sidhu and Fellouse 2006). These in vitro exhibitions could be achieved from natural and synthetic libraries of antibodies (Sidhu and Fellouse 2006). Synthetic libraries especially remove the need to make animals suffer in order to obtain antibodies through immunization, and also provide excessive diversity in scFv construction by manipulating the CDR regions of synthetic DNA (Bradbury et al. 2011, Sidhu and Fellouse 2006). The Phage display technique as a successful method has been developed to display recombinant antibodies at the tips of the phage, and consequently, facilitates the generation of scFv antibodies (Wu et al. 2010) easily and inexpensively just by infection of the E. coli (Wang et al. 1995). Phage display as a well-established and widely used method can generate mutants of scFv with higher affinity by manipulating their genes (Ahmad et al. 2012).

Advantages and disadvantages of single chain Fv fragments (scFvs)

The scFv fragments have provided great potential compared to full length antibodies in diagnostic and therapeutic applications, with 35% of antibody fragments used in clinical trials (Monnier et al. 2013). ScFv fragments are single polypeptide chains with low functional antigen-binding regain of an antibody (∼30 kDa), in which the variety of heavy (VH) and light (VL) chains are linked together by a flexible peptide (Maynard and Georgiou 2000). Minimized fragments have several advantages over the full-length antibody in clinical applications. The scFv fragments show better penetration through solid tumors, reduced retention time in non-targeted tissues, faster renal clearance, and more importantly, lower immunogenicity. ScFv antibodies can be constructed easily and their functions can be improved by manipulation of their structures which leads to the production of fragments with low commercial cost in large-scale yields. Furthermore, scFv fragments could be genetically manipulated to generate multivalent and multifunctional derivatives with more avidity, like diabodies, triabodies and tetrabodies (Brannon-Peppas and Blanchette 2012). Owning to their small size, scFv antibodies have lower half-life in circulation, resulting in restricted exposure to radionuclide or drug molecules, which is desirable in imaging applications (Ahmad et al. 2012, Monnier et al. 2013). In addition, the Fc domain of a mAb can also bind to the Fc receptor-expressing normal cells. This may be due to the undesirable activation of the Fc receptor on non-target cells and increased toxicity through cytokine release (Holliger and Hudson 2005). Recombinant fragments like scFv fragments lacking the Fc domain have another advantage over the intact antibodies (Ahmad et al. 2012).

However, one disadvantage of the in vivo application of these fragments is low retention in target tissues due to their monovalent binding (Holliger and Hudson 2005). Although the small size of scFv fragments confer several properties in tissue penetration resulting in short half-life circulation and rapid off rate, the exposure of the scFv fragment to the target is ultimately limited. To solve these problems, multivalent and multifunctional fragments derived from scFv have been engineered. Genetic manipulation has led to the design of diabodies, triabodies and tetrabodies that have higher avidity and increased maintenance of circulation, thus promoting tumor uptake for a long time without compromising tissue penetration properties (Hudson 1998). It has been demonstrated that the optimal structure which has both high tissue penetration and enough retention with rapid clearance would be a diabody (55 kDa), which is constructed by non-covalently linking two scFv fragments (Robinson et al. 2005, Sundaresan et al. 2003).

scFv fragments in targeted cancer therapy

The scFv fragments have gained significant attention in both diagnosis and therapy due to their advantages over conventional antibodies (Wan et al. 2013). Therefore, most clinical research studies in the United States, China, and the United Kingdom are focused on scFv fragments, particularly cancer therapy (Pucca et al. 2011).

The ScFv antibodies, with an ability to bind a specific antigen which is exclusively expressed on tumor cells, can be conjugated to toxins, small interfering RNA (siRNA), and nanoparticles, and therefore can be considered as a promising approach in DDS (Monnier et al. 2013). For instance, in 2007, Zhou and coworkers demonstrated that the liposome, coupled with single-chain antibody fragment against epidermal growth factor receptor (EGFR), can specifically recognize cancer cells overexpressing EGFR and increase the uptake of these anti-EGFR immunoliposomes (Zhou et al. 2007). Yang et al. obtained similar results by using functionalized quantum dots or magnetic iron oxide nanocarriers targeted with scFv directed against EGFR (scFv EGFR) into an orthotropic pancreatic cancer (Yang et al. 2009). In another study, the scFv antibody (scFv-CM6) has been isolated against an extracellular part of a protein expressed on newly formed blood vessels, cancer cells, and tumor endothelial marker 1 (TEM1), by the phage display technique. ScFv-CM6 was coupled to a liposomal nanoparticle loaded with N4- octadecyl-1-beta-d-arabinofuranosylcytosine-(5′-5′)-3′-Cethynylcytidine as a cytotoxic drug. The result shown was an increased toxicity in the TEM1-expressing IMR32 tumor cell, compared to the control liposome (Marty et al. 2006). Similarly, Lu and colleagues identified a scFv fragment binding specifically with the c-Met protein, the receptor of hepatocyte growth factor (HGF), that is aberrantly expressed and implicated in malignancy and human lung cancer. The anti-c-Met scFv conjugated with PEGylated liposomes carrying doxorubicin was proved to be an efficient DDS in tumor cells which induces the apoptotic effects. Moreover, better tumor accumulation of cytotoxic drugs and anticancer properties have been estimated through the delivery of anti-c-met immunoliposomes directed by scFv in a tumor xenograft model (Lu et al. 2011).

Angiogenesis plays an important role in tumor invasion and metastasis. Cancer cells can produce vascular endothelial growth factor (VEGF), which binds to the receptors on the surface of the endothelial cell, resulting in biological effects and tumor progression. The study of poly(lactic acid) poly(ethylene glycol) (PEG-PLA)-conjugated scFv against the VEGF-2 receptor which was loaded with As2O3 showed that scFv can significantly inhibit angiogenesis and also prevent the progression of the liver cancer xenograft (Xiangbao et al. 2014).

In another study, it has been demonstrated that hydroxyethyl chitosan modified with anti-human death domain 5 (DR5) scFv antibodies led to cell death in various types of tumor cells, and tumor growth suppression, both in vivo and in vitro (Yang et al. 2014).

In general, patients with Her2-overexpressing (Her2⁺) breast cancers do not have a certain prognosis due to the high resistance to chemotherapy. In recent years, for the delivery of small interfering RNA (siRNA) against polo-like kinase 1 gene (Plk1 or siPlk1) to Her2⁺ breast cancer through anti-Her2 single-chain variable fragment antibody (ScFv_Her2), PEG-PLA nanoparticles along with siPlk1 encapsulation (ScFv_Her2-NP_si Plk1) have been designed. Based on the current investigations, ScFv_Her2-NP_si Plk1 is able to promote the cellular uptake, achieve efficient Plk1 silencing and increased cancer cell apoptosis, in comparison with non-targeted NP_siPlk1 in Her2⁺ breast cancer (Dou et al. 2014). It seems that scFv-targeted nanoparticles have great potential for a broad application in cancer therapy.

Conclusion

Nanoparticles applied in DDS are able to improve the therapeutic index of drugs which are used in cancer therapy. Various types of nanocarriers have been designed with different features, conjugating with drug molecules to accumulate such molecules at the site of action via passive and active targeting, to decrease the toxicity or other undesirable effects in healthy tissues, as well as to protect them from clearance or deletion in blood circulation. Single-chain fragment antibodies have been a subject of interest in novel target therapy, particularly in cancer. Also, over the past few years, an increasing demand for the application of these fragments is being observed.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.