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Targeted nanocarriers are one of the most efficacious methods of drug delivery systems available in cancer imaging, diagnosis, and therapy. Due to their nanoscale dimensions and hydrophilic outer membrane, nanomedicine can remain in the bloodstream for a prolonged period of time but surface modifications are necessary to maintain the stability and function of the nanocarrier. The incorporation of biomolecues such as antibodies, peptides, aptamers, and other small molecules are examples of ligands that enhance the functions of nanoparticles and increase its biodistribution.
4.2.1 Antibody Fragments
Antibodies and their functional fragments are commonly used in medicine as shown by its extensive research history and low production costs. The conjugation of nanoparticles with antibodies or antibody fragment combines the properties of the nanoparticles themselves with the specific and selective recognition ability. Also, the improvement in the cellular uptake as well as the major intracellular stability may be two of the major advantages of using antibody conjugated nanoparticles. (Arruebo et al., 2009) MCC-465 and SGT-53 are two antibody-nanocarriers that are enhanced with fragmented antibodies allowing the nanocarrier to effectively reach its target cell.
MCC-465 showed positive results with F(ab')2 GAH antibody fragment ligands in preclinical studies with great biodistribution and high efficiency in doxorubicin delivery to stomach cancer cells (Jain, 2010). As well, the nanomedicine was well tolerated in phase I of clinical trials due to its stability in the blood plasma. However, judging by the lack of speculations on the MCC-465 it seems as though the nanomedicine will not be moving onto phase II of clinical trials. The reason behind this could be the complexity of the antibody-nanoparticle platform, which may contribute to the increased costs and low antitumour activity of MCC-465 in patients with metastatic stomach cancer (Matsumura, 2004).
Another nanocarrier with antibody fragment ligands is SGT-53, a nanomedicine that targets Tf receptors on tumour cell surfaces by targeted delivery of the p53 suppressor protein (Swami et al., 2012). With evident tumour growth inhibition in multiple cancers including head and neck, prostate, and breast, there is potential for future clinical trials and development of new nanomedicine using this nanocarrier and single-chain variable fragment (scFv) ligand (Nemunaitis, 2008). Though the application of antibody modified nanomedicine is still in its early stage, the study of the mentioned antibodies opened the doors to discovering new usage of antibodies in nanoparticle drug delivery and diagnosis.
Nanomedicine with modified antibody ligands can improve drug delivery due to its high specificity and affinity for target molecules. Due to the chemical nature of antibodies, they have high selectivity and binding abilities with their respective receptors and target proteins. Antibodies are readily found in nature due to their high biological activity in the immune system. However, due to the antibody's role as toxin neutralizers, there is a tendency for them to elicit immune responses by the binding of their Fc domain to Fc receptors on circulating macrophages. This causes increased cellular uptake and thus decreased antibody-nanoparticle half-life. Antibody fragments have demonstrated the efficiency of utilizing antibody fragments over monoclonal antibodies due to its small size and minimal immunogenicity.
In addition extra steps in the ligand preparation process to extract antibody fragments from large monoclonal antibodies are required to overcome steric hindrance and to maintain the efficient small size of nanocarriers. As well antibody selection takes longer than cultivation of other ligands for complete modification under biological processes. Temperature sensitivity and proneness to conformational changes under slight environmental alterations (eg. high salt concentrations) and enzymatic degradation are issues to consider when utilizing antibodies (Raoof, 2012) (Arruebo, 2009). These factors greatly limit the function and application range of antibody-nanoparticles and the chance of FDA approval in addition to increasing the costs of antibody ligand utilization.
Since antibody and its fragments often lose their functions in organic solvents and they are too large to participate in the nanoparticle polymer self-assembly process, nanoparticles are usually formed before ligands are conjugated onto the nanoparticle surface., A simple method of using bifunctional agents to couple hydrophilic peptides to the hydrophilic surface of liposomal nanoparticles can also be performed. However, bioconjugation is commonly used for antibody-nanoparticle formation (Yu et al., 2012). A series of chemical coupling reactions is required to conjugate the antibody ligand onto the drug-encapsulated nanoparticle. Amine coupling is possible with N-hydroxysuccinimide (NHS) and 1-Ethyla-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), producing carboxylic groups on the nanoparticle that will form a bond with the antibody's amine groups (Murcia et al., 2005). Similarly, a stable thioether bond is formed between the antibody's thiol group and the nanoparticle's maleimide group. However these covalent linkages often cause conformation changes in the antibodies that affect their function and binding abilities (Kamaly, 2012). The binding ability of antibody ligands to a nanoparticle surface can also be altered depending on the amino acid composition of the antibody fragment. Antibodies possess positive or negative charges which may create repulsion between the ligand and the nanoparticle. In this situation, a streptavidin-biotin interaction may be required to overcome it (Gao, 2010). For the bioconjugation between groups that are not naturally occurring such as alkyne and azide groups a chemical catalyst such as cyclooctyne can be used to drive the reaction. Bioconjugation often results in low product yield and inconsistent batch-to-batch nanocarrier quality, but it is the most efficient method of antibody-nanoparticle formation.
The usage of peptides in nanomedicine has increased greatly over the past decade due improved screening techniques. Screening of a greater number of peptide-binding domains allow for stronger target cell specificity and detection of new peptide ligand sequences. In addition, the ability of nanoparticles to have high peptide ligand densities increases its functionality without sparing its stability or nanoscale size. This allows for a stronger nanocarrier-target cell affinity due to cooperative binding (Fraysse-Ailhas et al., 2007).
Unlike antibodies, the small chain size of peptides, consisting of less than 20 amino acids, allow it to be efficient cell penetrators and to escape immunogenicity. The varying amino acid composition of peptides increases its biocompatibility and affinity for target cells (Delehanty et al., 2010). The process of peptide production is facile as it undergoes large-scale laboratory synthesis, permitting low costs and consistent quality.
The clinically used nanocarrier, Abraxane, has been successful in drug delivery for vasculature tumours in vivo with the usage of peptides such as LyP-1 and RGD (Delehanty et al., 2010). The addition of RGD ligands to nanoparticles increases its target cell affinity and allows greater controlled drug release than non-ligand or untargeted nanomedicine. This allows researchers to effectively diagnose tumour sites and to deliver anti-tumour drugs to vasculature vessels with overexpression of RGD integrins. The potential of using RGD ligands in nanomedicine research is extensive as the protein is present in a vast number of extracellular matrix proteins thus affecting a range of diseases (Delehanty et al., 2010). Similarly, LyP-1-nanoparticles target the lymphatic tumour cells with high binding affinity and increase cellular uptake of the nanocarrier by 8-folds compared to non-targeted nanoparticles (Luo et al., 2010). In addition, Lyp-1 is found to be internalized by its targeted tumour cells indicating its cell-penetrating abilities (Karmali et al., 2009). These factors show how promising it is to utilize LyP-1 in targeted drug delivery to lymphatic tumour cells with direct delivery of drugs into targeted cells via the peptide.
Transferrin conjugated nanoparticles have shown strong therapeutic efficacy in nanocarrier drug delivery to multiple cancer cell lines. This polypeptide is worth mentioning as transferrin liganded nanoparticles showed increased antiproliferative activity of encapsulated drugs and cancer cell apoptosis (Sahoo et al., 2005).In regards to peptide ligand density, high transferrin density plays an important role in target cell specificity as high ligand density increases the target cell binding ability of liganded nanocarriers (Bae et al., 2012).
A method of peptide-nanoparticle attachment depends on specific ligand-receptor interactions (Delehanty et al., 2010). Nanoparticles coated with streptavidin or avidin have complementary pairing with biotinylated peptide ligands. Biotin is commonly used due to its small molecule size allowing it to not inhibit or change the functions of the peptide whereas streptavidin and avidin both have strong affinities for biotin (Fischer, 2012). This creates one of the strongest non-covalent bonds and thus a naturally stable peptide-nanoparticle that can be used in vivo (Shi et al., 2009). It can be deduced that ligand-nanoparticles formed by streptavidin-biotin interactions results in greater immunogenicity in comparison to using other conjugation methods due to the nanoparticle size increase from the additional bulky groups and potential cross-linking between the nanocarrier constituents.
Bioconjugation of the peptide to the nanoparticle through direct covalent bonding between amines and carboxyls and maleimide and thiol groups are two other methods of peptide-nanoparticle formation (Delehanty et al., 2010). This method often creates steric hindrance as the peptides are in close contact with the nanoparticle surface but it is the most robust conjugation method. Another method of bioconjugation, "click chemistry", outlines a single step reaction that involves heteroatom bonds and a chemical catalyst that drives the reaction. Alkyne groups on the peptides will readily form a bond with the azide group on the nanoparticle without formation of side reactions. Recently, non copper catalyzed click-chemistry has been developed using cycloadditions such as cyclooctyne, tri(benzyltriazolylmethyl)amine, and sulfonated bathophenathroline to eliminate the risk of peptide cleavage and DNA degradation by copper (Shi et al., 2009). The usage of cycloadditions eliminates copper toxicity so bioactivity of the peptides will not be affected. There is also the risk of copper altering the conformation of the peptide and nanoparticle structure. Nevertheless the advantage of this method is the ability of conjugating ligand-nanoparticle under organic and aqueous conditions.
The simplest way to conjugate peptide-nanoparticles is allowing the ligand-polymers and other nanoparticle constituents to self-assemble. In aqueous solutions, the nanocarrier will form an inner hydrophobic core surrounded by the hydrophilic PEG and ligands. This one-step process reduces the risks of side reactions and forms favourable bonds between the ligands and nanoparticle. The self-assembly method allows greater control over nanocarrier properties and drug release.
Aptamers are the newest edition to the nanocarrier ligand family with more than 200 variations to date (Swami et al., 2012). Quality consistency is one of the main advantages of using aptamers in drug delivery systems as these synthetic molecules are produced readily and can be modified to function under a vast range of conditions. Similarly to antibody fragments and peptides, aptamers can be used in conjunction with nanoparticles to target specific cells with little immunogenicity.
The abilities of aptamers to fold into complex conformations and to conjugate multiple aptamer sequences on a single nanoparticle are two strong advantages of using aptamers as ligands (Yu et al., 2012). An example of a successful prostate cancer drug delivery aptamer that showed increased therapeutic effect is the Apt10 aptamer-conjugated PLGA-PEG nanoparticle (Swami et al., 2012). The ability of Apt10-nanoparticles to result in close to 2-folds increase in survival rates in comparison to mice survival in non-targeted nanocarriers shows its high binding affinity to overexpressed prostate-specific membrane antigens (Farokhzad, 2006). The aptamer-nanoparticle's high accumulation percentage in tumour cells shows great potential in tumour imaging and cancer diagnosis for the future. However, development of accumulation control of Apt10-nanopartices is necessary to reduce accumulation in other organs such as the liver.
Oligonucleic acid aptamer production undergoes the systematic evolution of ligands via exponential enrichment (SELEX) method. A large library of oligonucleotides are screened, selected, and amplified until the desired oligonucleic acid is obtained (Medley et al., 2011). The SELEX method simplifies the screening process when applying new target cells and allows for selection of aptamers with low immunogenicity or toxicity (Stoltenburg et al., 2007). Peptide aptamers connect to the ends of protein scaffolds and function to inhibit the nanoparticle from interacting with other proteins and molecules. This contributes to the high specificity of aptamer-nanoparticles but peptide aptamers are constrained to scaffold proteins and less flexible in nature.
Again, the streptavidin-biotin interaction is a common method of aptamer-nanoparticle attachment due to the strong ligand-receptor interactions that are involved. This method is more reliable in comparison to bioconjugation of carboxylic-coated nanoparticles with amine-coated aptamers. Direct covalent bonding has a high tendency to inhibit or alter the natural functions of the aptamer thus affecting its target cell binding ability. As well, there is a high tendency for aptamer-nanoparticles to be degraded by proteolytic enzymes as shown by the ethanol precipitation assay that showed oligonucleotide degradation in the blood and rapid blood clearance in the TTA1 experiment (Hicke et al., 2006). Despite these disadvantages, bioconjugation is the most preferred method because of its high stability, strong chemical bonding, and simple chemical synthesis.
In addition, aptamers can be attached to nanoparticles via self-assembly. Recent studies on the research of Apt10-nanoparticles utilized this method by preconjugating aptamer ligands onto nanoparticle polymers and then allowing the polymers to self-assemble with other necessary nanoparticle components (Shi et al., 2011). This innovative methodology allows a one-step process to reduce the risk of side reactions whilst favouring the binding orientation of the aptamer ligands and nanoparticle polymer. The increased stability will allow future aptamer-nanoparticles to remain in blood circulation for prolonged periods of time and to achieve more efficient drug delivery.
4.2.4 Small Molecules
Small molecules are used in nanomedicine due to their low production costs and facile conjugation with nanoparticles (Swami et al., 2012). Synthetic and natural small molecules such as folate and flavin mononucleotide are common ligands used to target specific cancer cells as folate and flavin receptors are usually overexpressed in breast, ovarian, and other type of cancers (Yoo, 2004).
The incorporation of ACUPA, a small molecule ligand moiety in clinical development, into nanoparticles encapsulating docetaxel allowed docetaxel concentrations in the blood circulation of the nanomedicine increases the chances of nanomedicine-PSMA binding and thus more effective drug delivery (Shi et al., 2011) (Hrkach et al., 2012) (Sekhon, 2012). As PSMA is a validated prostate tumour receptor and present in most vasculative vessels of tumours, the conjugation of small targeting molecule ACUPA and perhaps other small molecules with other types of nanoparticles will allow researchers to have a more defined method of tumour imaging and drug delivery to cancer sites. The nanocarrier BIND-014 is currently undergoing phase I of clinical trials and has already shown prolonged nanocarrier circulation (Hrkach et al., 2012).
The process of small molecule bioconjugation to nanoparticles is similar to that of peptides and aptamers. Immobilization of small molecules to nanoparticles is common via amine coupling since amine groups are ubiquitously found in nature and it is a robust reaction. It is important to note that inhibition of the small molecule's targeting specificity and side reactions between EDC and NHS with other molecules are concerns of utilizing this method. Self-assembly of the functionalized small molecule polymers and nanoparticle constituents is the recommended method of small molecule-nanoparticle preparation since small molecules are not inhibited or destroyed by organic solvents. The usage of small molecules as targeting ligands also maintains the purification and functions of the nanoparticle.
Table 1: Clinical Nanomedicine Using Ligands
Type of Ligand
F(ab')2 fragment of GAH
Stomach and colon cancer cells
Single-chain antibody fragment
Tf receptors on tumour cell surfaces
Vasculature tumour cells
p32 protein on lymphatic tumour cells
(Wang, 2012) (Davis, 2008)