Recent advances in biocompatible nanocarriers for delivery of chemotherapeutic cargoes towards cancer therapy.

Volume 12 Number 27 21 July 2014 Pages 4765–5040

Organic & Biomolecular Chemistry www.rsc.org/obc

ISSN 1477-0520

REVIEW ARTICLE Yanli Zhao et al. Recent advances in biocompatible nanocarriers for delivery of chemotherapeutic cargoes towards cancer therapy

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Recent advances in biocompatible nanocarriers for delivery of chemotherapeutic cargoes towards cancer therapy

Published on 18 March 2014. Downloaded on 20/06/2014 08:47:24.

Chung Yen Ang,†a Si Yu Tan†a and Yanli Zhao*a,b Cancer is currently one of the major diseases that has gained a lot of scientific attention. Conventional cancer therapeutics involve surgical removal of tumors from patients followed by chemotherapeutic treatment. In the use of anticancer drugs during the chemotherapy process, patients often suffer from a variety of undesirable side effects including damage to normal organs. Thus, there is an urgent need for the development of novel strategies to overcome these side effect issues. Among several strategies, the utilization of nanocarriers for anticancer drug delivery has shown improved therapeutic efficiency of the drugs with minimization of the undesirable side effects. In this review, we discuss various types of nanocarriers recently reported in the literature for application in cancer therapy. We introduce some targeting Received 21st January 2014, Accepted 18th March 2014 DOI: 10.1039/c4ob00164h www.rsc.org/obc

ligands that have been functionalized on nanocarriers in order to impart specificity to the nanocarriers for targeted drug delivery. We also highlight some therapeutic cargoes that are commonly used and their therapeutic mechanisms in cancer treatment. Finally, we summarize some interesting stimulus strategies for controlled release of therapeutic cargoes at tumor sites. This review is expected to inspire new ideas and create novel strategies in advancing efficient cancer therapy using nanomedicine approaches.

1. a

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, 637371 Singapore, Singapore. E-mail: [email protected]; Tel: +65 63168792 b School of Materials Science and Engineering, Nanyang Technological University, 639798 Singapore, Singapore † These authors contributed equally to this work.

Chung Yen Ang was born in Singapore in 1985. He received his BSc. (Hons.) degree with First Class Honours in Chemistry from Nanyang Technological University, Singapore in 2012. Currently, he is pursuing his Ph. D. studies under the supervision of Prof. Yanli Zhao at Nanyang Technological University and cosupervision of Dr Tamil Selvan Subramanium at Institute of Materials Research and EnginChung Yen Ang eering. His research focuses on developing organic materials for various biological applications including drug delivery, photodynamic therapy, and bio-detection.

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Introduction

Cancer is currently the leading cause of death worldwide.1 Based on the number given by the World Health Organization (WHO), it accounted for a total death toll of 7.6 million (about 13% of all deaths) within the year of 2008. Though there is always a perception that a high cure rate for early-detected cancer, such as breast cancer, cervical cancer, and colorectal

Si Yu Tan

Si Yu Tan was born in Singapore in 1988. She obtained her BSc. (Hons.) degree in Chemistry from Nanyang Technological University, Singapore in 2011. Currently, she is pursuing her Ph.D. studies under the supervision of Prof. Yanli Zhao at Nanyang Technological University. Her research interests include the development of mesoporous silica based materials for theranostic applications in cancer therapy.

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cancer, can be reached, many cancers only show signs and symptoms at a late stage when the chance of recovery is tremendously reduced. Hence, cancer is still a major medical problem facing society and it requires immediate attention in developing new strategies for combating this type of disease. Traditional cancer therapeutic techniques include the surgical removal of the tumor, radiation therapy and chemotherapy. However, the chance of recovery, particularly for late stage cancer patients, still remains slim. During the terminal stage of cancer, the cancer cells metastasize to various organs of the body2,3 to further complicate the problem. These cancer cells often show multidrug resistance (MDR),4 making the present anticancer drugs ineffective in treating the disease. Furthermore, some therapeutic agents have limited solubility in the biological environment, which greatly reduces their pharmacokinetic properties. In addition to discovering new drugs,5,6 a lot of effort has been devoted to developing novel therapeutic techniques such as photodynamic therapy7–9 and gene therapy10,11 for improved cancer treatment. On the one hand, these techniques often require the delivery of therapeutic agents (drugs, photosensitizers, and gene) into the target tumor sites so as to maximize the desired therapeutic effect. On the other hand, these techniques also suffer from various major drawbacks such as the lack of specificity that may result in the delivery of therapeutic agents into healthy tissues to produce side effects. Thus, ideal therapeutic techniques have been sought after by scientists over the past decade. A promising solution for improved cancer therapy is to load therapeutic agents into nanocarriers for target-specific drug delivery (Fig. 1).12–21 In addition to imparting target specificity towards the tumor, the nanocarriers have the ability to improve the bioavailability of the cargos, thereby reducing the

Yanli Zhao is currently a Nanyang Assistant Professor and a National Research Foundation Fellow at Nanyang Technological University, Singapore. He received his BSc. degree in Chemistry from Nankai University in 2000 and his Ph.D. degree in Physical Chemistry there in 2005 under the supervision of Professor Yu Liu. He was a postdoctoral scholar with Professor Sir Fraser Stoddart at University of Yanli Zhao California Los Angeles (October 2005 to November 2008) and subsequently at Northwestern University (January 2010 to August 2010). In between (December 2008 to December 2009), he was a postdoctoral scholar with Professor Jeffrey Zink at University of California Los Angeles. He has published over 100 scientific papers. His current research focuses on biocompatible nanoparticles for diagnostics and therapeutics, and porous materials for energy storage and catalysis.


Review

Fig. 1 Applications of nanocarriers for the delivery of various cargoes for cancer therapy. Properties such as targeting and stimuli-responsive molecular switches can be integrated to customize the nanocarriers for applications.

side effects. These nanocarriers can also be incorporated with molecular triggers to enable the cargoes to be released only at the target tumor sites, further improving the specificity of the treatment. Hence, a carefully engineered design of a delivery system is one of the key aspects considered in cancer treatment. There are many successful examples in the development of delivery systems for cancer therapy. In particular, some of these delivery models have passed through the third stage of clinical trials and have been approved by the US Food and Drug Administration (FDA) as commercially available anticancer drug formulations.13,22–25 One notable example is Doxil®, which has been available in the market since 1998. Developed mainly by Gabizon and Barenholz, Doxil® is liposome-encapsulated doxorubicin (DOX) anticancer drug.26 The development of Doxil® aims to reduce the side effects, especially the cardiotoxicity brought about by the use of free DOX during chemotherapy. The encapsulation of DOX allows the drug to escape from the reticuloendothelial system (RES) and hence prolong the biodistribution of the drug. On account of its suitable size (∼100 nm) the encapsulated drug, Doxil® has the ability to target the tumor sites by the enhanced permeability and retention (EPR) effect. This effect allows the accumulation of the drug-loaded liposome in the tumor sites, thereby releasing the anticancer drug into the tumor. Doxil® is by far is one of the best anticancer formulations that are available in the market, and the strategic design of this encapsulated drug enables chemotherapy to achieve a high efficiency with less side effects. Thus, it is of utmost importance to understand the various components of the encapsulated drugs or any other therapeutics before designing a customizable nanocarrier for efficient cancer therapy. In this review, we provide an in-depth discussion regarding various

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components of delivery systems for cancer therapy. We first outline a variety of nanocarriers that have been developed over the past decades for delivery applications. Thereafter, we introduce several targeting ligands and outline how these ligands have been used together with the nanocarriers. The next section covers therapeutic cargos and their mode of action towards the elimination of cancerous cells. Then, we address various triggering systems that have been employed for controlled release of the loaded cargos in the target sites. Finally, we conclude the review by summarizing some important aspects discussed.


2. Nanocarriers The nanocarriers that are commonly used in cancer treatment include polymer-based nanoparticles, liposomal based nanopackages, and inorganic nanomaterials such as gold nanoparticles and mesoporous silica nanoparticles (MSNPs). As mentioned in the introduction, these materials can be loaded with anticancer therapeutic agents, thereby improving the bioavailability of the agents. Each of these carriers has advantages over others, while having certain drawbacks over other carriers. Over the past decades, various synthetic techniques have been developed to functionalize these carriers, aiming to overcome the drawbacks over the pre-existing techniques or to improve the quality of the pre-existing carriers. In this article, we will review the synthetic approaches used for some representative carriers and discuss how these carriers have been employed for delivery applications. a.

Polymer based nanocarriers

Polymer based nanocarriers have received a wide variety of attention over the past decades owing to their versatility in terms of synthesis and functionalization.12,27–30 Furthermore, some of the polymer backbones are biodegradable, and hence are well accepted by the medical community, particularly in dealing with concerns pertaining to their fate after delivery. Thus, polymer based nanocarriers are often chosen as the delivery vehicle for various agents ranging from gene to drugs.31–35 Polymer based nanocarriers can exist as liposomes, polymersomes, and polymer based NPs. In this review, we discuss the applications of these nanocarriers with reference to some groundbreaking work in the literature. Although polymersomes or liposomes are mainly made from the self-assembly of polymers, the main principles behind the formation of these nanocarriers are the same as those made from simple molecules. Hence, we only discuss them later in the liposome section. We highlight the formation and applications of the polymer NPs in this section. Polymer based NPs are often synthesized via the self-assembly technique, which consists of various polymer based components.36–39 One notable example is the IT-101 system developed by Davis and co-workers. This system is the first therapeutic design that has achieved clinical trials for cancer

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Fig. 2 Chemical structure and synthetic scheme for the formation of IT-101 nanocapsule in the delivery of camptothecin anticancer drug. Inset shows the electronic micrographs of the NPs. (Reproduced with permission from ref. 40. Copyright 2009 Elsevier.)

therapy (Fig. 2).40,41 The polymer system consists of alternate segments of cyclodextrin and polyethylene glycol (PEG) together with functional carboxylate groups. Thereafter, the drug camptothecin (CPT) was conjugated onto the carboxylate group to form the polymer–drug conjugate. The polymer was then dispersed in water where the CPT formed an inclusion complex with the cyclodextrin ring, and hence the polymer self-assembled into polymer based NPs. The as-prepared NPs are spherical in shape and have a size range from 30 to 40 nm. The drug delivery system as mentioned above relies on the formation of an inclusion complex between the drug and the cyclodextrin ring that is pre-conjugated onto the polymer chain. Due to the presence of a hydrophobic core, the cyclodextrin ring is capable of hosting a great variety of hydrophobic guest molecules such as azobenzene,42 heterocyclic aromatics43,44 as well as bulky alkanes.45 In this way, the NPs can be prepared by the self-assembly of cyclodextrin-containing polymers with guest-containing polymers or drugs. In a recent work reported by Li et al., β-cyclodextrin was first conjugated onto a branched polyethylenimine (PEI), followed by the complexation of adamantine conjugated doxorubicin with the cyclodextrin on PEI (Fig. 3a).32 Thereafter, the doxorubicin grafted PEI was combined with a DNA plasmid to achieve both drug and gene delivery both in vitro and in vivo. In this work, the authors have used transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to characterize the shape and size of the as-prepared NPs. From the electron microscope images, it was observed that the NPs have a polypetalled shape, having particle sizes from 200 to 500 nm with a relatively high polydispersity (Fig. 3b). Another interesting concept of using the supramolecular self-assembly technique for the preparation of polymer based NPs was reported by Tseng et al. In this system, the NPs were prepared by the self-assembly of β-cyclodextrin conjugated PEI, adamantane conjugated PEG, and adamantane conjugated poly(amido amine) (PAMAM) (Fig. 4).46 Interestingly, this system has the advantage of controlling the particle size by changing the ratio of different components. The work showed the successful preparation of NPs with sizes of 30 nm and 100 nm in a good polydispersity. Furthermore, the as-prepared


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Fig. 5 (a) Computer simulated conformation of the DOX–poly-L-lysine complex. (b) Conformation diagram of the DOX–poly-L-lysine complex with the interpolated charges. The blue surface represents the positive charges of poly-L-lysine. (Reproduced with permission from ref. 64. Copyright 2013 American Chemical Society.)

Fig. 3 (a) Scheme showing the self-assembly process of PEI–adamantine conjugate for the delivery of drugs and genes. (b) SEM and TEM images of the polymeric nanocarriers. (Reproduced with permission from ref. 32. Copyright 2011 Royal Society of Chemistry.)

molecules (e.g., drugs, targeting ligands and solubilizing molecules) with different functionalities in a controlled fashion, thereby improving their delivery efficiency.52,62,63 In addition, the presence of a variety of functional groups on dendrimers imparts conjugation flexibility, such that the guest entities could be functionalized onto dendrimers in many ways ranging from covalent to non-covalent methods.52,57 In one study reported by Al-Jamal and co-workers, a poly-Llysine dendrimer was used as a delivery vehicle for anticancer drug DOX.64 The authors proposed that the DOX molecule complexes with the dendrimer through hydrogen bonding interaction (Fig. 5). In addition, the authors used molecular simulation and 1H NMR spectroscopy to confirm the complexation between the drug and the dendrimer. Thereafter, the drug delivery efficiency of the drug/dendrimer complex was investigated both in vitro and in vivo. The obtained results indicate that the complex could exhibit an enhancement in therapeutic efficiency as compared with that of DOX or dendrimer alone. It was further concluded that the dendrimer has good potential as a delivery vehicle for different types of therapeutic cargoes. b. Liposome based nanocarriers

Fig. 4 Schematic illustration for the supramolecular self-assembly of polyamine based NPs. (Reproduced with permission from ref. 46. Copyright 2009 John Wiley and Sons.)

NPs served as good vectors for drugs and genes towards various interesting applications.47–49 Dendrimers have received a great deal of attention over the past decades in terms of synthesis and applications.50–60 Recently, this class of materials have emerged as promising candidates for biomedical applications such as for drug and gene delivery.57,59–61 As compared with linear polymers, controlled multivalent dendrimers have the ability to host guest


Liposomes are vascular NPs that are formed from the dispersion of amplifiers in aqueous environment. Due to the entropy effect, the water molecules are expelled from the hydrophobic region to form a well defined hydrophilic domain. In order to further minimize the interaction of the nanocarriers with the solvent molecules, the nanocarriers adopt a spherical shape having a maximum surface tension on the surface of the nanocarriers. Liposomal nanocarriers contain an aqueous core separated by a bilipid layer from the external aqueous solution, and the cargo is loaded within the aqueous core of the liposome. To date, there are several US FDA approved drugs that are actually in the form of liposomes, including the famous Doxil® as mentioned in the introduction.22,23,65 The reason for this is due to the ease fabrication of the liposomes, and very often they are made of biodegradable materials, which renders them suitability for in vivo applications.66

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Fig. 6 Chemical structures of (a) poly(lactic-co-glycolic acid) (PLGA) and (b) polyethylene glycol (PEG).

One example of a liposome vesicle is poly(lactic glycolic acid) (PLGA) based nanomaterials. As a hydrophobic polymer, PEG modified PLGA could self-assemble into a liposome effectively. Being a polyester polymer, PLGA is biodegradable in nature, and hence becomes a popular polymer for designing a liposome delivery vehicle (Fig. 6a). On top of imparting hydrophilicity to the liposome, the PEG moiety (Fig. 6b) also provides the liposome with steric hindrance to prevent the interaction of the liposome with blood proteins, thereby improving the bioavailability of the liposome.67–70 One useful concept was designed by Langer et al. in creating a liposome based nanocarrier.71 In this work, lidocaine drug was loaded into the liposome, and then its bioavailability was investigated in mouse models. From these studies, they found that loading the drug into the liposome could greatly reduce the accumulation of the drug in the liver and dramatically increase the blood circulation time. In addition, a remarkably high loading capacity of the drug up to a value of 45% by weight was reported. In the same work, they also synthesized a series of liposomes by varying the PEG length. Interestingly, they observed that the blood circulation time of the liposomes increases when increasing the length of the PEG unit. Other than relying on PLGA and PEG based polymers for the preparation of the liposomes, other biocompatible materials could also be employed. A recent work reported by Sung and co-workers used a mixture of hydrogenated soy phosphatidylcholine (HSPC), cholesterol and 1,2-O-diocta-decenyl3-trimethylammonium propane (DOTMA) in phosphate buffered saline (PBS) for the preparation of liposomes (Fig. 7).72 In this work, a simple inorganic reagent, NH4HCO3, was loaded into the liposome, which was internalized into the intracellular domain of cancer cells. The as-prepared liposome was characterized accordingly and the results showed that the size of the liposome was around 295.0 nm with a surface potential of 41.1 ± 1.3 mV. Another work reported by Szoka et al. utilized a disterol modified phospholipid for the preparation of a liposome based nanocarrier.73 In this work, they successfully loaded doxorubicin into the liposome, and demonstrated remarkable therapeutic effects of doxorubicin-loaded liposome, in which the efficiency was equivalent to Doxil. c. Inorganic nanocarriers Inorganic nanocarriers have also attracted a great deal of attention for their properties ranging from synthesis to appli-

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Fig. 7 Schematic representation of HSPC, cholesterol and DOTMA based liposomes for inducing necrosis of cancer cells. Top left figure shows a TEM image of NH4CO3 loaded liposome. (Reproduced with permission from ref. 72. Copyright 2012 John Wiley and Sons.)

cations. Due to the relative ease of synthesis, inorganic nanocarriers are emerging as key candidates for biomedical applications. Many exciting results have been reported regarding their applications as drug delivery vectors. This section introduces some representative inorganic nanocarriers. i. Mesoporous silica nanoparticles. Mesoporous silica NPs are a class of inorganic nanomaterials possessing porous structure and high surface area. This class of nanomaterials have been widely used as delivery vehicles for a number of therapeutic cargoes in cancer therapy.15,74–80 Mesoporous silica NPs are synthesized via the template assisted technique, where cetyltrimethylammonium bromide (CTAB) serving as the template for the pore formation of the NPs arranges itself into cylindrical arrays and directs the formation of a silica wall surrounding the arrays (Fig. 8). Thereafter, the micelle templates are removed and the resulted channels are used for drug loading towards delivery. The main advantage for the utilization of mesoporous silica NPs as delivery vectors is their remarkably high surface area (>1000 m2 g−1), which implies that a large amount of cargo can be loaded into mesoporous silica NPs for delivery applications. In addition to the large surface area, the silanol groups on the surface of the NPs allow for the grafting of various functional groups on the surface. Thus, various interesting ligands and gatekeepers can be conjugated. The purpose of using the gatekeepers is to keep the loaded cargos within the pores of the NPs, and then release them upon a certain trigger or activation. We will discuss various applications of mesoporous silica NPs in a later section of this review. ii. Gold nanoparticles. Gold NPs (Au NPs) have received a lot of attention in various biological applications.81–83 On top of being non-toxic and non-immunogenic, Au NPs also exhibit interesting optical properties that allow the combination of several functions (e.g., drug delivery, sensing, and photothermal therapy) in a single system. In view of this capability,


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Fig. 8 Schematic diagram illustrating the template assisted synthesis of mesoporous silica NPs.

some Au NP-based systems have reached the clinical trial stage, demonstrating their incredible application potential.84 Au NPs are typically synthesized by a simple reduction of an aqueous gold salt (such as tetrachloroaurate) in a controlled manner, yielding NPs with various sizes and shapes.85,86 Since Au NPs have the surface Plasmon resonance effect, the size of the NPs can directly influence their UV-Vis absorption.87 Hence, one can correlate the UV-Vis absorption or color of an Au NP sample to the size of the NPs (Fig. 9). The as-synthesized Au NPs are usually stabilized by ligands added during the synthesis process. These ligands can either be used directly for further functionalization or be easily replaced by strong chelating ligands.88–90 In many cases, mercaptans are used as the ligands for the Au NP functionalization, since the thiophilic properties of Au could result in relatively strong bond formation between the thiol ligands and the Au NP surface. Thus, mercaptan ligands with various functionalities or properties could be easily introduced onto the Au NP surface by simple ligand exchange reactions. For instance, in a study reported by Brown et al., the anticancer drug oxaliplatin was anchored onto the Au NP surface through the use of lipoic acid ligand.91 The authors have demonstrated that the Au NPs could successfully deliver oxaliplatin into the nucleus of cancer cells, indicating that the Au NPs have the capability to serve as the drug delivery vehicle. iii. Iron oxide nanoparticles. Another class of inorganic nanomaterials that has been employed as drug carriers is iron oxide NPs.92–94 Since the physical size of iron oxide NPs is much smaller than the ferromagnetism domain size of its bulk material, iron oxide NPs exhibit superparamagnetism properties.95,96 When an external magnetic field is applied to the NP samples, there would be an immediate saturation of magnetization, which is in alignment to the vector of the external field (Fig. 10).97,98 In addition, the primary elemental compositions of iron oxide NPs are none other than iron and


Fig. 9 (a) TEM images of Au NPs of various sizes: (i) 20 nm, (ii) 40 nm, (iii) 60 nm and (iv) 80 nm. (b) Photographs of Au NP solutions with different sizes. (c) UV-Vis absorption spectra of Au NPs with different sizes. (Reproduced with permission from ref. 87. Copyright 2007 American Chemical Society.)

oxygen. In view of this, iron oxide NPs are known to be biocompatible and even biodegradable.99,100 Thus, iron oxide NPs could serve as excellent contrast agents for magnetic resonance imaging (MRI) applications.101–103 Iron oxide NPs exist in two main forms, namely maghemite (γ-Fe2O3, Fig. 10a) and magnetite (Fe3O4, Fig. 10b). These two types of NPs could be synthesized by three methods including the co-precipitation method, thermal decomposition of organoiron reagents, and the microemulsion technique.95,98,104–107 These methods produce iron oxide NPs in the size range from 2 to 17 nm, depending on the synthetic method and conditions used. In this review, we do not discuss the synthesis

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Fig. 10 TEM images of (a) γ-Fe2O3 NPs (Reproduced with permission from ref. 106. Copyright 2009 John Wiley and Sons.) and (b) Fe3O4 NPs (Reproduced with permission from ref. 107. Copyright 2004 American Chemical Society.). These two images clearly show that the size of γ-Fe2O3 NPs is significantly smaller than that of Fe3O4 NPs. (c) Magnetization curve of Fe3O4 NPs coated with a layer of mesoporous silica shell. The inset shows the photographs of Fe3O4 NPs captured by a 0.4 T magnet, which indicate that the dispersion and capturing processes of the NPs are reversible. (Reproduced with permission from ref. 97. Copyright 2009 Royal Society of Chemistry.)

and properties of these materials in detail, since they have already been summarized by several excellent review papers.94,104,105 In biological applications, Fe3O4 NPs are often preferred over γ-Fe2O3 NPs as Fe3O4 NPs have the ability to achieve higher magnetization saturation and magnetic susceptibility.108 In other words, the magnetic saturation of Fe3O4 NPs could be achieved with the application of lower external magnetic field. The Fe atoms on the surface of iron oxide NPs could act as an excellent Lewis acid for interacting with a variety of oxygencontaining electron lone pair donors. For instance, when iron oxide NPs are dispersed in an aqueous environment, the water molecules act as ligands to chelate onto the NP surface. The consequence of such chelation changes the properties of the NPs. In addition, other types of functional groups such as carboxylate, phosphate, sulfate and phenoxylate can also bind with the Fe atoms effectively for further functionalizations of the NPs.104 On top of using these molecular ligands for the functionalizations, macromolecules and inorganic materials such as silica are also often used to coat the iron oxide NPs, so

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as to impart higher stability and bio-availability on the NPs.109–112 To further elaborate the applicability of the iron oxide NPs for drug delivery and MRI imaging, a recent work reported by Yu et al. was selected as an example.113 In this work, the authors prepared thermally cross-linked polymer coated Fe3O4 NPs followed by loading the anticancer drug DOX into the polymer network. The obtained nanocomposite was then injected intravenously into mice to perform T2-weighted MRI imaging. The imaging results showed that there was an accumulation of the nanocomposite in the tumor after 4.5 h from the time of the injection, indicating excellent uptake of the nanocomposite by the tumor as well as its high performance as an MRI contrast agent. Thereafter, the authors also studied the therapeutic efficiency of the nanocomposite and observed that the nanocomposite displayed remarkable tumor inhibition efficiency up to 63% relative to the control group. This work indicates that functional iron oxide NPs could perform as not only the MRI contrast agents, but also the drug delivery vehicle. iv. Quantum dots. Semiconductor nanocrystals or quantum dots (QDs) are a class of inorganic nanomaterials that have also been widely used in the biomedical field.114–116 This class of emissive nanomaterials exhibit interesting optical properties and are more stable from photobleaching than classical organic fluorophores.117 In addition, QDs have narrower emission spectra than the organic fluorophores, allowing multicolor imaging without obvious overlapping in the emission signals when QDs with different colors are used simultaneously. Since the physical size of QDs is smaller than the Bohr radius of bulky materials, they exhibit the quantum confinement effect whereby their bandgap is strongly dependent on their size.118–120 Thus, the emission wavelength of QDs could be tuned by simply changing the size of the nanocrystals (Fig. 11).121 Among many QDs developed, Cd-containing QDs, such as CdSe and CdTe, have received a great deal of attention, because the emission wavelengths of Cd based QDs fall in visible region.122–125 Various papers have reported the bioimaging applications of this type of nanomaterials.126–128 Some studies have shown that growing a shell of higher bandgap materials over the QD cores (e.g. CdSe/ZnS) can further improve the luminescence efficiency and photostability.129 Cd based QDs could be synthesized by either the high-temperature organometallic method or the aqueous synthesis method, producing Cd chalcogenide QDs in the size range from 3 to 4 nm.130,131 Similar to Au NPs, the as-synthesized QDs are stabilized by the ligands added during the synthesis process, allowing the QDs to grow in a controlled manner.132,133 These ligands can either be functionalized directly through chemical reactions or be replaced with other ligands for further modifications.134–137 The surface of the QDs contains the Cd atoms, which act as good coordination sites for Lewis bases. Typically, thiol groups are selected as suitable chelators for binding with the Cd atoms on the surface of the QDs.138 However, some studies have indicated that the ligand exchange


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synthesized materials. Since different materials may require different types of characterization techniques, there is no universal technique that can be employed to characterize all kinds of materials. In this section, we briefly highlight some commonly used characterization techniques accompanied with some examples. i. Electronic microscope. Electron microscopy is frequently used for the characterization of nanomaterials. On account of the de Broglie postulate, the wavelength of a fast moving electron is much shorter than that of a photon (eqn (1)),


λe ¼

Fig. 11 (a) Color photographs of UV-excited CdSe QD solutions. The QD solutions were excited by 365 nm UV light. (b) TEM images of CdSe QDs with maximum emission wavelengths ranging from 525 to 590 nm. (c) UV-Vis absorption spectra and (d) emission spectra of QDs synthesized under various conditions. The longer the growth time, the larger QD size produced. (Reproduced with permission from ref. 121. Copyright 2013 IOP Publishing.)

process might cause a decrease in the luminescence efficiency of the QDs, especially for CdSe QDs.139–141 Hence, the compatibility of a ligand with the QDs is an important factor to be considered in functionalizing the QDs for applications. In a work reported by Bagalkot et al., the authors have functionalized the QD surface with the A10 RNA aptamer followed by intercalating anticancer drug DOX within the aptamer network.142 In this system (QD-Apt(Dox)), the energy from the QD emission was transferred to the DOX molecule through the Förster Resonance Energy Transfer (FRET) process, and the absorbed energy from the DOX molecule was then transferred to the RNA aptamer through a secondary FRET process. The net result of this bi-FRET system is total fluorescence quenching, indicating the successful loading of DOX within it. When QD-Apt(Dox) was delivered into cancer cells, the loaded DOX was gradually released from the aptamer network, leading to the fluorescence recovery of both DOX and the QDs. Thus, the QDs in the QD-Apt(Dox) system serve as both a fluorescence tracer and an indicator for the real time monitoring of the DOX release. Most of the QDs used for biomedical applications are Cd based QDs and Pb based QDs (NIR emissive QDs).122 The Zn based QDs are UV emissive143,144 and show little use for biomedical applications. On the other hand, the Cd based QDs have received a lot of concerns pertaining to their toxicity for practical biomedical applications.145,146 Hence, the QDs consisting of non-toxic materials need to be developed in future studies.147–149 d. Material characterization Material characterization plays an essential role in the preparation of the nanocarriers. This process provides useful information pertaining to the quality and identity of the


h me νe

ð1Þ

where λe is the wavelength of the moving electron, h is Planck constant, me is the mass of electron, and νe is the velocity of the moving electron. In this manner, the images produced from the electron microscope should have much higher resolution than that of an optical microscope (eqn (2)), Resolution ðrÞ ¼

1:22λe 2n sin θ

ð2Þ

where n is the refractive index of the medium surrounding the focus point, and θ is the maximum half angle of the light that can enter the lens. Thus, electron microscopy has been used to study the morphology of nanomaterials, overcoming the limitations of normal optical microscope. Two modes of electron microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM), are commonly used for the characterization of the morphology of nanomaterials. These two methods differ in the relative position at which the image is captured. TEM captures the images based on the transmitted electrons, while SEM captures the images based on the scattered electrons (Fig. 12a). Hence, the SEM micrograph provides three-dimensional images, while the TEM micrograph only gives two-dimensional images.150 However, the TEM images normally have better resolution than the SEM images, and the former is particular useful when fine details of a sample, such as nanoporous structures and the crystal fringe of nanocrystals, are required.151,152 Based on the operation principles of an electron microscope, it can be expected that materials consisting of atoms with higher atomic number (e.g. metals or silicon) would be able to scatter the electrons more effectively than materials with atoms of lower atomic number (e.g. nitrogen or carbon). This explains the fact that polymeric nanocarriers normally have lower contrast than that of inorganic nanomaterials. In addition, SEM is often equipped with energy-dispersive X-ray spectroscopy (EDX or EDS), so that the system can also perform elemental analysis and generate an elemental composition map on the micrograph.153 Similarly, TEM could be equipped with electron energy loss spectroscopy (EELS), serving the function of elemental analysis.152 ii. Powder X-ray diffraction ( pXRD). The pXRD technique is often used to study the crystallinity of materials.152,154 When preparing the samples for pXRD, the samples are required to

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Fig. 12 Characterization data of various nanomaterials by different techniques. (a) TEM and SEM images of PLGA liposome particles. Although SEM has the ability to generate three-dimensional images, the detailed morphology of the material can only be observed by TEM. For instance, the polymer shell could be observed from the TEM image but not from the SEM image. (Reproduced with permission from ref. 151. Copyright 2011 John Wiley and Sons.) (b) pXRD diffraction patterns of Fe3O4, γ-Fe2O3 and mesoporous silica NPs. The pXRD diffraction pattern can be used to confirm the identity of crystalline materials. For instance, the pXRD pattern of Fe3O4 NPs is different from that of γ-Fe2O3 NPs. In addition, relatively broader signals from the diffraction pattern of γ-Fe2O3 than that of Fe3O4 are due to the smaller NP size of γ-Fe2O3. Hence, there are insufficient crystal planes to produce sharp signals. (Reproduced with permission from ref. 106 Copyright 2009 John Wiley and Sons, from ref. 107 Copyright 2004 American Chemical Society, and from ref. 79 Copyright 2013 American Chemical Society.) (c) N2 absorption–desorption isotherm and the BJH pore size distribution of mesoporous silica NPs. (Reproduced with permission from ref. 155. Copyright 2012 John Wiley and Sons.) (d) X-Ray photoelectric spectroscopy (XPS) of graphene oxide. The XPS has the ability to analyze the surface element of the materials. (Reproduced with permission from ref. 162. Copyright 2012 John Wiley and Sons.)

be ground into a fine powder so as to randomize the orientation of the crystals in the powder samples. Thereafter, X-rays are irradiated onto the powder samples, and the samples diffract the X-rays at a few characteristic angles (θ) based on Bragg’s Law (eqn (3)), 2d sin θ ¼ nλ

ð3Þ

where d is the distance between the two planes of interest, θ is the angle between the incident ray and the diffracted plane, and n is an integer. The samples with ordered structures or possessing crystallinity are well suited to be characterized by this technique. For instance, the iron oxide NPs have a crystalline lattice within the nanostructure, and thus pXRD can be used to determine the quality of the nanomaterial.95 In addition, the diffraction pattern of a nanomaterial is normally the same as the corresponding bulk material. Hence, the diffraction pattern can be used to match with the database for confirming the identity of the nanomaterials (Fig. 12b).106,107 Another common application of pXRD is to measure the pore size of porous materials such as mesoporous silica NPs. The highly ordered hexagonal structure of the mesopores has 100 and 200 facets, where the distance between the planes (d ) is in the order of 2.4 nm for MCM-41 NPs.79 The signals generated by these facets usually appear in the low angle region based on the relation from the Bragg’s Law. Thus, small angle pXRD is often used to measure the pore size of mesoporous silica NPs.

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iii. N2 adsorption–desorption isotherm. Since highly porous materials usually possess high surface areas, they could serve as good hosts for N2 adsorption at the equilibrium temperature (−196 °C). Thus, the N2 adsorption–desorption isotherm can be measured to study the surface properties of highly porous materials such as mesoporous silica NPs (Fig. 12c).155–157 In this technique, liquid N2 is adsorbed onto the surface of the materials and the instrument generates a plot between relative pressure of N2 versus the amount of N2 adsorbed. There are totally 5 types of isotherm plots, which can be used to correlate to the type (Type I to V) of pores the samples possess.158 Most importantly, the Brunauer–Emmett– Teller (BET) theory can be applied on the adsorption plot to calculate the surface area of the porous materials. In addition, when the desorption measurements are performed, the Barrett–Joyner–Halenda (BJH) theory can be applied to study the pore size distribution of the porous materials. iv. Other characterization techniques. Apart from these techniques abovementioned, there are several other characterization techniques commonly employed to study the properties of the nanomaterials. For instance, zeta potential measurement is normally used to study the surface charge of the nanomaterials, and can be employed to predict the stability of the materials in solution. It is also used as a confirmation tool when the surface modifications are carried out on the materials. In addition to the zeta potential measurement, the dynamic light scattering (DLS) technique is normally used to


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measure the hydrodynamic size of the nanomaterials.159,160 Although the size of the nanomaterials can be measured by electron microscopy, the real-time measurements of the particle size in solution can be realized by the DLS technique. However, dust from the environment or solution may interfere with the measurements, resulting in false information pertaining to the materials of interest. Another characterization technique that is also used in this field is atomic force microscopy (AFM), where the morphology of the nanomaterials can be investigated.161 X-ray photoelectric spectroscopy (XPS) is used at times to study the surface elemental composition of the nanomaterials (Fig. 12d).162–164 In this review, we will not discuss these techniques in detail. It should be noted that the characterization techniques employed to study the properties of the nanomaterials are not limited to the methods mentioned here. Selecting suitable characterization techniques is based on the intrinsical properties of the nanomaterials synthesized.

Review

Fig. 13 Schematic representation of folate mediated endocytosis of micelles for drug delivery and the recyclability of the receptors. (Reproduced with permission from ref. 169. Copyright 2002 Elsevier.)

3. Methods of targeting The use of targeting ligands in the delivery of chemotherapeutic drugs for cancer therapy is important. As chemotherapeutic drugs normally cannot bind specifically to cancerous cells, normal cells in the body also suffer from their harmful effects.155,165–167 Thus, the use of targeting ligands allows the nanocarriers to selectively enter the cancerous cells for the delivery of these drugs. This targeting strategy prevents systemic toxicity and reduces undesired side effects occurring during the chemotherapy. Targeting ligands can be molecules or proteins that bind preferentially to cancerous cells. Some of the commonly used targeting ligands are highlighted in this section. a.

Folic acid

Functionalization of nanocarriers with folic acid is a widely used technique in the area of targeted drug delivery. Folic acid is commonly known as vitamin B9, and is important for cell division and growth.168 When folic acid reaches the cells, it binds to the folate receptors present on the cell membrane for undergoing endocytosis. Thereafter, the receptor releases the folic acid inside the cells, and the receptor returns to the surface of the cells. The movement of the folate receptors in and out of the cells allows the receptors to be reused (Fig. 13).169 As cancerous cells grow and divide uncontrollably, they have a large amount of folate receptors on their cell membrane.170 Owing to this fact, a lot of research has focused on the use of folic acid to provide targeted drug delivery towards cancer cells. One of the first few reports using folic acid incorporated nanoparticles for the delivery was carried out by Couvreur and co-workers.171 They synthesized polymer nanoparticles, which were functionalized with folate to investigate their binding efficiency with folate binding protein. The nanoparticles could bind specifically to the folate binding protein on a sensor chip. Inspired from the above result, Kataoka and


Fig. 14 (a) Flow cytometric analysis and (b) growth inhibitory activity of ADR drug, ADR-loaded micelles (MA), and ADR-loaded micelles containing folate (FMA). (Reproduced with permission from ref. 172. Copyright 2005 Royal Society of Chemistry.)

co-workers prepared folate-containing polymeric micelles for in vitro drug delivery.172 An amphiphilic block copolymer, folate–poly(ethylene glycol)–poly(aspartate hydrazone adriamycin) (Fol–PEG–P(Asp-Hyd-ADR)), was designed to covalently conjugate a folic acid group at one end and an anticancer drug, adriamycin (ADR), at the other end of the polymer. When exposed to water, the polymer self-assembled into micelles for drug delivery in vitro. From the cytotoxicity assay and flow cytometry analysis (Fig. 14), the authors showed that the micelles containing folic acid (FMA) had better growth inhibition of KB cells as compared to the micelles without folic acid (MA). Another work by Gong et al. also made use of folic acid for drug delivery. An amphiphilic hyperbranched block copolymer was conjugated with folic acid to give H40-PLA-b-MPEG– PEG-FA.173 Owing to its ability to form a core–shell structure, the polymer formed micelles in aqueous solution, and anticancer drug doxorubicin was loaded within these micelles for delivery. The fluorescence microscopy images revealed that the micelles containing folic acid showed higher uptake of doxorubicin in 4T1 mouse mammary carcinoma cell line. In addition,

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the cell viability assay indicated that folic acid integrated micelles presented the lowest cell viability in the same cell line. Zhao and co-workers reported a successful example using folic acid conjugated hollow mesoporous silica nanocarriers (HMSNs).166 HMSNs were functionalized with an α-cyclodextrin-based [2]rotaxane bridged through a disulphide bond. DOX drug was loaded within the nanocarriers kept by the [2]rotaxane unit. To demonstrate the drug delivery efficacy of the nanocarriers, the as-synthesized nanoparticles were incubated with cancerous and normal cells, respectively. Based on the flow cytometry data, cancerous cells presented higher uptake of the nanoparticles as compared to the normal cell lines due to the large amount of the folate receptors on the cancerous cell surface. The in vivo tumor therapy also showed the targeting ability of folic acid-containing nanoparticles. b. RGD peptides Although folic acid shows a high specificity towards the tumor cells, another class of targeting ligand, the RGD peptide, is able to target the tumor cells as well as the tumor endothelial cells. A RGD peptide is a tripeptide and is composed of L-arginine, glycine and L-aspartic acid. It binds to most integrin receptors, which participate in signal transduction and cell signaling.174,175 Integrin is involved in early angiogenesis, which is crucial for the formation of cancerous tissues.176 It is overexpressed in malignant tumors and plays an important role in cancer propagation.175 Similar to folic acid, the RGD peptides bind to the integrin receptors and are endocytosed into cells. Upon successful release of the RGD peptides in the cells, the receptors return to the cell surface for subsequent binding to other RGD peptides.177 Due to the recyclability of the RGD receptors, nanocarriers functionalized with the RGD peptides can be constantly consumed by the cells. Duan and co-workers made use of this property for the delivery of polymeric nanoparticles into MDA-MB-231 breast cancer cells. A polymer monomethoxy(poly-ethylene glycol)-poly(D,L-lactide-co-glycolide)-poly (L-lysine) (mPEG-PLGA-PLL) was synthesized by covalently conjugating a cyclic RGD (cRGD) peptide to the amino terminal of the block co-polymer.178 Thereafter, the nanoparticles were prepared by using an emulsion-evaporated method and were internalized into MDA-MB 231 cells. In order to demonstrate the targeting ability of cRGD, the authors incubated the cells with nanoparticles containing cRGD and nanoparticles without cRGD, respectively. From the flow cytometry data, the nanoparticles containing the cRGD peptide showed the cell uptake, reflecting the targeting ability of cRGD. Another work by Wu and co-workers reported hyperbranched amphiphilic copolymer (HPAE-co-PLA–DPPE) based nanoparticles with cyclic RGD peptide and transferrin (Tf ) on the surface for the delivery of paclitaxel.179 The nanoparticles containing RGD and Tf showed higher chemotherapeutic efficiency on human cervical carcinoma (HeLa) cells as compared with human umbilical endothelial cells. Hence, these

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Fig. 15 (a) Schematic representation of RGD-encapsulating hydrophobically modified glycol chitosan (HGC) directly administered into the solid tumor, and (b) quantification of blood vessels in the tumor tissue under different treatments. (Reproduced with permission from ref. 180. Copyright 2008 Elsevier.)

results indicate the importance of using targeting ligands for cancer therapy. In a recent work by Kwon and co-workers, RGD not only served to target cancer cells, but also acted as an antiangiogenic drug (Fig. 15a).180 In this case, the glycol chitosan nanoparticles with bile acid analogues at one end encapsulated the RGD peptide within the hydrophobic core via a solvent evaporation method. The use of a nanocarrier has its advantage, as it allows for a sustained release of drug over a period of 1 week (Fig. 15b), enabling a high concentration of drug to accumulate at the tumor site. The efficacy of these particles (RGD-HGC-i.t.) was tested and compared with other types of treatment methods such as intravenous injection (RGD-i.v.) and intratumoral injection (RGD-i.t.) of RGD. Quantification of the amount of blood vessels as well as the staining of the microvessels in the tumor tissue showed that RGD-HGC-i.t. provided the antiangiogenesis effect. The RGD peptide was also introduced into polymeric micelles by Oku and co-workers. The polycation liposomes containing dicetylphosphate-tetraethylenepentamine were prepared for targeted siRNA delivery towards cancerous cells.181 The RGD peptide was incorporated into the system through the polyethylene glycol chain, and then the transfection


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efficiency was tested. The in vivo imaging of the gene-silencing effect was performed with luciferin, and the system containing the RGD peptide showed a decrease in luminescence over time. The gene silencing was successfully achieved on the mice with specific targeting to the lung metastasis model.


c. Aptamers Another group of targeting ligands that has been widely explored is aptamers. Aptamers can be divided into two classes: nucleic acid aptamers and peptide aptamers. A nucleic acid aptamer is a single-stranded oligonucleotide that is folded and binds to a molecular target. The binding of this class of aptamers occurs through complementary base pairing of the RNA sequence with the protein binding site to form secondary and tertiary structures.182 Peptide aptamers, on the other hand, are simple peptides containing a loop region in the center along with ends tied to proteins. The loop region confers the targeting properties of the aptamer and allows it to achieve the desired effects.183 In order to find suitable aptamers that target specific proteins, combinatorial libraries of oligonucleotides are investigated. Most aptamers are known to have antagonistic effects, but there are a handful of aptamers showing agonist-like effects. These aptamers bind to various proteins on cancerous cells to achieve the desired inhibitory effect.184 In 2004, the US FDA approved the first aptamer based drug, Macugen, for the treatment of age related macula degeneration (AMD). Thereafter, many research groups have adopted the use of such aptamers for nanocarrier-based drug delivery. Tan and co-workers used a DNA aptamer, sgc8, for binding to CEM-CCRF leukemia cells.185 A liposomal system consisting of hydrogenated soy phosphatidyl choline (HSPC), cholesterol (Chol), methoxypoly-(ethylene glycol)-distearoyl-phosphatidylethanolamine (MPEG-DSPE) and maleimide-terminated PEG-DSPE (MalPEG) was synthesized, and the aptamer was incorporated into the system via covalent conjugation with the maleimide group of the MalPEG (Fig. 16). In another work by the same group, a polymeric aptamer was designed to induce selective cytotoxicity inside targeted cancer cells. The poly-

Fig. 17 In vivo fluorescence images overlaid with X-ray images after the mice were incubated with the polymer containing folate (yellow) and the polymer containing aptamer (red) for 24 h. (Reproduced with permission from ref. 186. Copyright 2013 Royal Society of Chemistry.)

meric aptamer consists of a polyacrylamide polymer backbone, a mixture of multiple cell based aptamers and a dye labelled short DNA. As the polymer backbone induces some toxicity within the cells, only the cells that uptake the aptamer show cytotoxicity. The aptamer-based nanocarrier was then tested on two leukemia cell lines, i.e., CEM cells and NB4 cells. The results from the in vitro studies showed that the aptamerbased nanocarrier was readily endocytosed by the CEM cell line that contains the membrane receptor for binding with sgc8. In a recent article by Thurecht and co-workers, peptide aptamers were used together with hyperbranched polymers.186 The targeting ligand is a peptide aptamer that has specificity for heat shock protein 70 (HSP70). The peptide aptamer was conjugated to the polymer via a “click” reaction. The polymer containing the aptamer was then compared with the polymer containing folic acid for in vivo specificity. The in vivo fluorescence images showed that the HSP70 aptamer had higher specificity than folic acid (Fig. 17), further reflecting the application potential of aptamer-based drug delivery. Farokhzad and co-workers also made use of aptamer based polymeric nanoparticles for the delivery of a chemotherapeutic drug, docetaxel.187 The aptamer chosen was based on its ability to be internalized rather than having the highest binding affinity towards the target cells. The results from the confocal images as well as the cytotoxicity assay showed that the cancer cells had higher uptake and lower cell viability when incubated with the nanoparticles containing the aptamer.

d. Passive targeting Fig. 16 Schematic representation of aptamer sgc8 and its incorporation into a liposomal system. (Reproduced with permission from ref. 185. Copyright 2009 Royal Society of Chemistry.)


Passive targeting does not involve the use of any targeting ligands and is achieved by the enhanced permeability and retention (EPR) effect. As the endothelial lining of the tumor

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blood vessels is leakier than that of normal blood vessels, the nanocarriers can easily enter and accumulate at the tumor sites.188 In order for the EPR effect to occur, the nanocarriers must have a size of at least 50 nm and have a long blood circulation time. This can be achieved by the synthesis of appropriate sized nanocarriers and functionalizing them with watersoluble groups to prevent renal clearance. The current US FDA approved anticancer drug, Doxil, makes use of these properties to achieve its anticancer effect. Doxil consists of a liposomal system, which has a size of about 100 nm, to encapsulate the drug and it does not contain any targeting ligands. When Doxil was employed for the delivery of doxorubicin, the nanocarrier system was proven to have better drug retention capability than free drug.13 This successful demonstration of the EPR effect encourages more research in this area. Gu and co-workers employed a multifunctional multiblock polyurethane (MMPU) for the delivery of paclitaxel into A431 squamous carcinoma cell line.189 The nanomicelles prepared were spherically or cylindrically shaped and had diameters of about 100 nm in water. From the in vivo results, the use of these nanomicelles (GH/PTX) showed a significant decrease in tumor size as compared to the use of free Taxol (Fig. 18). Although targeting ligands were used in this work, passive targeting also played a role in reducing the tumor growth. Another work by Chan et al. investigated the effect of nanoparticle size and surface chemistry on passive targeting of


tumors in vivo.190 The results revealed that smaller nanoparticles are able to diffuse throughout the tumor but larger nanoparticles tend to stay at the vasculature. From the histological data (Fig. 19), at 1 hour, all the nanoparticles with different sizes (20 nm, 60 nm and 100 nm) remained at the perivascular space with little penetration into the tumor. At 8 hours, the 20 nm nanoparticles penetrated more, while the 60 nm and 100 nm nanoparticles did less. This work has provided an insight to better engineer nanoparticles for cancer therapeutics. Tseng and co-workers also investigated the effect of EPR using magnetothermally responsive doxorubicin-encapsulated-supramolecular nanoparticles on DLD-1 colorectal adenocarcinoma cell line.48 The magnetic nanoparticles were synthesized in 3 different sizes, namely 70 nm, 100 nm and 160 nm, which were intravenously injected onto DLD-1 tumor bearing mice. From two-dimensional micro-PET ( positron emission tomography) cross sectional images, the magnetic nanoparticles with a size of 70 nm exhibited the best retention within the tumor. The time dependent accumulation of the nanoparticles within the tumor site of the mice further supported the results obtained from the micro-PET images. With this ability to remain at the tumor site, an alternative magnetic field (AMF) was applied for the treatment of the tumor in vivo. The ability to silence cancer can be achieved via active and passive targeting. Both methods have proven to be effective in cancer therapy, and in some cases have reached the commer-

Fig. 18 (a) Schematic mechanism of MMPU entering the tumor cell via the EPR effect and intracellular release of drug, (b) tumor volume and (c) tumor weight when treated with different samples. (Reproduced with permission from ref. 189. Copyright 2013 American Chemical Society.)

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Fig. 19 Histological data of the nanoparticle uptake in tumors when different sized nanoparticles were used in vivo. (Reproduced with permission from ref. 190. Copyright 2009 American Chemical Society.)

cial stage. With the knowledge of cancer targeting, selecting appropriate anticancer drugs is the next step towards achieving effective cancer therapy.

4.

Types of therapeutic cargoes

In addition to the use of targeting ligands, a wise selection of therapeutic cargoes is another important aspect of cancer therapy. Although many cargoes can be used to stop and inhibit the cancer growth, one type of carrier normally cannot be employed to deliver all kinds of cargoes. Cancer therapy aims to silence the cancerous cells in many ways in the hope of completely eradicating the cancer. Hence, combined therapeutics are often used in cancer treatment which makes the search for new drugs and treatment methods important.191,192 Some common anticancer drugs and treatments are discussed in this section. a.

Fig. 20 (a) The structure of DOX, which contains an aromatic group as well as a sugar moiety. (b) Model of DOX intercalation with DNA. The arginine residue at position 45 enters the minor groove of DNA. Similarly, the amino sugar unit in DOX also interacts with the minor groove to communicate with the DNA bases. (Reproduced with permission from ref. 193. Copyright 2013 Nature Publishing Group.)

Doxorubicin (DOX)

Doxil is a well-known anticancer drug and its active anticancer component is DOX.26 DOX is comprised of a sugar and an aromatic moiety, whereby the aromatic moiety intercalates with DNA of the cells. This intercalation causes the disruption of topoisomerase-II-mediated DNA repair and hence results in cell death (Fig. 20).193 Another proposed cause of cell death by DOX involves the production of reactive oxygen species from the oxidized form of DOX. The production of the reactive oxygen species can result in damage of the membrane as well as DNA to trigger apoptosis in the cells.194 Among the few ways of detecting DOX within the target cells, the most commonly used way is to carry out fluorescence measurements. As DOX can be excited at 480 nm to give a maximum fluorescence intensity at 593 nm, the fluorescence intensity at 593 can be measured to determine the amount of DOX in a sample.155 As for quantification of DOX within


plasma or tissues, the treatment of the selected organ followed by analysis of its contents by high performance liquid chromatography (HPLC) might give the concentration of DOX within the organ.195 Though DOX has shown to be effective towards a large number of cancer cell lines, the major drawback of this drug is its cardiotoxicity. It is suspected that the reduced form of DOX interferes with iron to result in a loss of the iron homeostasis. In addition to the interaction with iron, the metabolism of DOX in the mitochondria can disrupt respiration for apoptosis.196 Due to the efficacy of DOX as a drug, a lot of research work has adopted this drug as a cancer-therapeutic model. One recent paper by Li and co-workers employed polymeric micelles for the co-delivery of DOX and disulfiram (Fig. 21).197 In this work, fast release of disulfiram inhibited the activity of P-glycoprotein and restored the cell apoptotic signaling pathway. DOX was then released slowly to ensure the accumulation of the drug at the tumor site. DOX, disulfiram (DSF), a mixture of DOX and disulfiram (DOX + DSF), polymeric micelles loaded with DOX (SAD), a mixture of SAD and disulfiram (SAD + DSF), and polymeric micelles encapsulated with both DOX and disulfiram (DSM) were tested on the MCF-7 and MCF-7/ADR (DOX resistant) cell lines, respectively. Fig. 21 shows that the DOX resistant MCF-7/ADR cells presented almost no DOX internalized in the nucleus. On the contrary, MCF-7 cells showed a large amount of DOX present in the nucleus. This comparison suggests that the MDR mechanism can prevent nuclear localization of DOX within MCF-7/ADR

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Fig. 22 (a) Structure of paclitaxel, and (b) a model of paclitaxel interaction with microtubules. (Reproduced with permission from ref. 203. Copyright 2010 Nature Publishing Group.)

Fig. 21 The cell viability and fluorescence microscopy images of DOX, DSF, DOX + DSF, SAD, SAD + DSF, and DSM on (a) MCF-7/ADR cells and (b) MCF-7 cells. (Reproduced with permission from ref. 197. Copyright 2013 American Chemical Society.)

cells. The results obtained from this combination therapy of polymeric micelles encapsulated with both DOX and disulfiram exhibited a high efficacy for in vitro and in vivo cancer therapy. Gu et al. also adopted a similar system for the delivery of DOX to the 4T1 murine breast cancer model.198 Polymeric nanoparticles with a diameter of 100 nm were synthesized where DOX was covalently attached to the polymer backbone. The CD31 immunohistochemical (IHC) staining of tumor tissues showed that the tumor microvessel density (MVD) was the highest when treated with saline, and the lowest when treated with the polymeric nanoparticles. The side effect of free DOX was observed in the heart via hyperemia and myocardial fiber breakage. When the DOX-attached nanoparticle system was used, however, no tissue and cell lesion was observed. Other than the use of DOX for the treatment of normal breast cancer, Bae and co-workers proposed the use of folate conjugated pH-sensitive polymeric micelles (PHSM/f) for the treatment of resistant MCF-7 tumor.195 Two block co-polymers, poly(L-histidine) and poly(L-lactic acid), were conjugated with PEG and folate for in vitro and in vivo studies. From the in vivo results obtained, the nanoparticles containing folic acid and DOX gave the best reduction in tumor volume.

b. Paclitaxel As combined therapies are often used in the area of cancer therapy, paclitaxel has been also used in the treatment of certain types of cancer. Paclitaxel can be found in Taxol and Abraxane. The Abraxane version of the drug is often used, as it is comprised of albumin bound paclitaxel which is more biocompatible than Taxol which contains Cremophor® EL.199 Paclitaxel is employed as a mitotic inhibitor, which targets tubulin, stabilizes it and stops its disassembly (Fig. 22). This process prevents the cells from progressing from the metaphase to anaphase, eventually causing cell death.200

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Fig. 23 Fluorescence microscopy images of (A) HepG2 and (B) MDA-MB-231 cells with immunofluorescence staining of tubulin. (Reproduced with permission from ref. 204. Copyright 2013 Elsevier.)

As paclitaxel does not contain any fluorescence signal, UV absorption at 227 nm is often used in the detection of paclitaxel.201 In some cases, commercially available paclitaxel conjugated with fluorescent units can be purchased for its detection by flow cytometry. In addition, polymerized and free tubulin can be measured on account of paclitaxel’s ability to stabilize tubulin.202,203 Since paclitaxel is an anticancer drug, some research groups incorporated it within nanoparticle systems for the purpose of drug delivery. Li et al. made use of cyclodextrin based nanoparticles for the delivery of paclitaxel within four different cancer cell lines, i.e., B16F10, HepG2, MCF-7 and MDA-MB-231.204 Immunofluorescence staining was performed to observe the effects of paclitaxel on drug resistant (MDA-MB-231) and sensitive cell lines (HepG2). Extensive microtubule bundles were observed in both cell lines after 24 and 48 hours of incubation with free paclitaxel (PTX) or paclitaxel-loaded nanoparticles (PTX/Ac-aCD NP) (Fig. 23). The extensive microtubule formation is an indication of increasing microtubule stability, which prevents mitosis. When no drug was added to the cells, only a few microtubule bundles were observed. Flow cytometry analysis also revealed that paclitaxel resistant and sensitive cell lines show apoptosis when paclitaxel-loaded nanoparticles were used. Another work by Wooley et al. also adopted paclitaxel as a drug in conjunction with poly(ethylene oxide)-block-polyphosphoester micelles for cancer therapy within a series of cell lines.205 The IC50 value of the paclitaxel-containing nanoparti-


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Fig. 24 Cell viability of LLC cells incubated with (a) nanoparticle formulations with no drug and (b) free drug as well as nanoparticle formulations with drug. (Reproduced with permission from ref. 201. Copyright 2012 American Chemical Society.)

cle was higher than commercial drugs. The low cytotoxicity might be advantageous to increase the blood circulation time as well as to allow high accumulation of drugs within the tumor cells. In a recent work by Ma and co-workers, targeted drug delivery of paclitaxel (PTX) was performed using PEGylated O-carboxymethyl-chitosan (CMC) nanoparticles grafted with RGD peptide.201 High dispersibility in water enabled the nanoparticles to have longer blood circulation time, and the introduction of the RGD peptide enhanced the affinity of the nanoparticles to the tumor tissue. In vivo cell viability experiments on Lewis Lung carcinoma (LLC) were performed using free PTX, Taxol®, CMC nanoparticles with paclitaxel (CNP: PTX), PEGylated CMC nanoparticles (PEG-CNP:PTX), and RGD-PEG-CNP:PTX (Fig. 24). According to the results, the CMC nanoparticles exhibited low cytotoxicity, which implies that the nanoparticles had a good biocompatibility. On the other hand, Cremophor® showed severe cytotoxicity to the cells. When comparing the drug effects of different nanoparticles, free PTX displayed the least cytotoxicity and RGD-PEG-CNP:PTX showed the highest cytotoxicity. These experiments indicate the importance of employing appropriate drug carriers in drug delivery applications. c. Cisplatin Cisplatin is one example of organometallic compounds serving as chemotherapy drugs. It is a neutral square planar inorganic platinum compound, which reacts with a water molecule to achieve an unstable mono-aquated form of the drug. This intermediate can readily interact with DNA to prevent cell division and mitosis, and in turn result in apoptosis (Fig. 25).206 The only drawback in using cisplatin as an anticancer drug is that the cells may become resistant to the drug over time.207 There is evidence suggesting that cell lines that are resistant to cisplatin showed 20–70% reduction in drug accumulation, probably due to an inhibition of drug uptake or an increase in drug efflux or both.208 Owing to the platinum content in cisplatin, the detection of cisplatin is often via the use of inductively coupled plasma optical emission spectroscopy (ICP-OES) and inductively coupled plasma mass spectroscopy (ICP-MS). After the in vitro cell experiments, the cell


Fig. 25 Activation of the cisplatin compound by hydrolysis or reaction with S-donors before interacting with DNA of cells. The DNA could either be repaired or not be repaired for apoptosis after reaction with cisplatin. (Reproduced with permission from ref. 206. Copyright 2012 Royal Society of Chemistry.)

Fig. 26 Percentage of platinum in (a) whole blood and (b) plasma after administration of Pt(IV)-prodrug (1) and Pt-PLGA-b-PEG-NP. (Reproduced with permission from ref. 210. Copyright 2011 National Academy of Sciences.)

pellets are digested to determine the platinum concentration by ICP-OES or ICP-MS.209 There are many groups that make use of this organometallic drug for nanomaterial-based drug delivery. In a work by Farokhzad and co-workers, aptamer targeted PLGA-b-PEG encapsulated Pt(IV) prodrug was used in the treatment of prostate cancer (PCa).210 The nanoparticles were prepared by a nano-precipitation method, and then in vivo studies were carried out. The use of this nanoparticle system (Pt-PLGA-bPEG-NP) achieved an anti-tumor effect at a low drug dosage and also allowed the drug to have long blood circulation time (Fig. 26). In addition, Shin et al. made use of ScFvEGFR-herapin-cisplatin nanoparticles (EHDDP) for the delivery into EGFR-positive tumor cells.211 The nanoparticles were formed by coordination between the carboxyl groups and Pt2+ to give cisplatin-encapsulated herapin nanoparticles containing EGFR targeting ligand ScFvEGFR. The nanoparticles were then tested with EGFR-positive H292 cells and EGFR-negative H520 cells, and the results indicate a low Pt accumulation in H520 cells

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Fig. 28 H&E staining of different organs after treatment with PBS, cisplatin (CDDP) and lipid-coated cisplatin nanoparticles (LPC NPs). (Reproduced with permission from ref. 213. Copyright 2013 American Chemical Society.)

Fig. 27 (a) Platinum concentration in H292 and H520 cells after 24 h incubation with free cisplatin (DDP), herapin encapsulated cisplatin (HDDP), EHDDP nanoparticles, and ScFvEGFR with EHDDP. (b) H292 cells were incubated with the different samples to investigate antitumor efficacy. (Reproduced with permission from ref. 211. Copyright 2011 American Chemical Society.)

but a high retention in H292 cells. Free ScFvEGFR could inhibit the uptake of Pt into cells when a competition experiment was carried out using ScFvEGFR and EHDDP nanoparticles (Fig. 27). With the use of EHDDP nanoparticles, better antitumor effect was achieved in vitro and in vivo with a low cytotoxicity to the spleen and kidney. Other than the use of targeting agents to deliver cisplatin to the tumor site, passive targeting was adopted by Barenholz and co-workers. In their work, low frequency ultrasound (LFUS) was used to trigger the release of cisplatin that was encapsulated within the nano sterically stabilized liposomes (nSSL).212 Various combinations of liposomes were then injected intravenously into BALB/C mice with C26 colon adenocarcinoma tumor. From the results obtained, the liposomal system that incorporates cisplatin together with the use of LFUS showed the best therapeutic efficacy. In addition to the above work, another paper adopting the passive targeting strategy is from Nguyen and co-workers. In this work, the authors used synergistic properties of cisplatin and DOX for co-drug delivery.209 Polymer-caged nanobins were used for this co-drug delivery, where DOX was encapsulated within the liposomal core protected by pH-responsive cisplatin prodrug-loaded polymer shell. A high level of synergism was observed in the nanoparticle system that contains both cisplatin and DOX. In another work by Huang et al., the efficacy of encapsulated cisplatin within a lipid layer was studied in human A375M melanoma tumor cells in vivo.213 Other than allowing

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the water-insoluble drug to be delivered to the tumor site, the release of the drug within the tumor cells generated a neighboring effect. This effect involves the release of active drug from apoptotic cells, and via diffusion, the released drugs enter the neighboring cells that are previously unaffected. This effect was only observed in rapidly proliferating cancer cells, but was not detected in the liver, spleen and kidney tissues via H&E staining (Fig. 28). For comparison, when free cisplatin was used, some nephrotoxicity was observed in the kidney of the mice. Doxorubicin, paclitaxel and cisplatin are drug molecules that are commonly used in nanoparticle carrier-based cancer therapy on account of their high efficacy. In addition to these common cargoes used in the nanoparticle systems, there are many other cargoes that are not drug molecules but serve the same purpose of eradicating cancer cells, which include photosensitizers and nucleotides. d. Photosensitizers for photodynamic therapy Photodynamic therapy (PDT) refers to rational utilization of light, photosensitizer and the oxygen present in the vicinity of the photosensitizer (referred to as type II PDT) to generate cytotoxic singlet oxygen as the therapeutic species. Due to its non-invasive properties, PDT has emerged as an alternative approach for cancer therapy.7,79,214–218 In this manner, the common strategy of PDT is to deliver the photosensitizers to the site of treatment followed by remote irradiation of photons on the site of treatment. This PDT process can reduce side effects especially when other parts of the body are not exposed to the light. As abovementioned, the major components required for a PDT treatment include the photosensitizer, oxygen and photoirradiation. Upon delivery of the photosensitizer into the tumor site, subsequent photo-irradiation on the treatment site


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Fig. 29 General structures of (a) porphyrin and (b) phthalocyanine based compounds. The photosensitizers used for PDT usually differ from the above structures by introducing different functional groups on the peripheral sites of the heterocyclic rings. A coordinated metal center (such as zinc, silicon, aluminium) is usually present.

could induce the excitation of the photosensitizer. The excited electrons then undergo inter-system crossing from the singlet state to the triplet state before final relaxation back to the singlet ground state. During the final relaxation of the photosensitizer, the spin flip of the excited electrons transfers its energy and spin direction to the electrons in the triplet oxygen, thereby converting the triplet oxygen to cytotoxic singlet oxygen.219,220 It has been well documented that high energy singlet oxygen has the ability to induce oxidative damage to biological systems.221,222 To further elaborate on this mechanism, singlet oxygen induces damage to biological systems by means of the oxidation of some biological molecules such as unsaturated lipid, cholesterol, tryptophan, methionine and guanine. Hence, the photosensitizer can be considered as a mediator in generating cytotoxic singlet oxygen for therapeutic purposes. The common photosensitizers that are used in PDT are porphyrin and phthalocyanine as well as their derivatives (Fig. 29). Together with their reasonably high quantum yields (ranging from 36% to 83%) in generating singlet oxygen, their absorption in the red or near infra-red region makes them ideal candidates for PDT applications.222 The red absorption properties of these classes of photosensitizers allow them to absorb light ideally for deep tissue applications. This is especially due to the fact that the biological optical window lies within the range from 800 to 1300 nm where most of the biological components do not absorb.219,223 In order to choose a suitable photosensitizer for PDT application, one should consider not only the quantum yield for the generation of singlet oxygen, but also the region where the selected photosensitizer absorbs for photoexcitation. Over the past decades, various studies pertaining to the synthesis, functionalization and applications of photosensitizers have been reported.224–227 In many cases, the efficiency of these photosensitizers in generating singlet oxygen has been quantified. During the process, the singlet oxygen is detected by measuring its luminescence with a maximum fluorescence wavelength at 634 nm. However, the luminescence of singlet oxygen can be easily quenched by some small molecules including H2, N2, C2H2, and C2H4.228–231 Hence, molecular


Review

Fig. 30 Schematic illustration of the reactions between singlet oxygen and the detection probes: (a) ABDA and (b) DPBF.

probes with unique optical properties, such as changes in their absorption properties or their fluorescence properties upon reaction with singlet oxygen, are used for detection applications.232 A review paper by Gomes et al. summarizes various optical probes used for the detection of singlet oxygen and other reactive oxygen species.233 To briefly describe some examples of such probes for the detection of singlet oxygen, 1,3-diphenylisobenzofuran (DPBF) and 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA) were selected for discussion. Fig. 30 shows the reactions of singlet oxygen with ABDA and DPBF during the detection process. In the case of ABDA, the original state of the molecule emits fluorescence with a maximum wavelength of 431 nm upon photoexcitation at 380 nm. Upon reaction with singlet oxygen, the fluorescence intensity of the probe is quenched significantly. The other probe that is also commonly used for the detection of the singlet oxygen is DPBF. In this process, DPBF first reacts with the singlet oxygen through the 4 + 2 cycloaddition to form a trioxolane intermediate, which is subsequently hydrolyzed into the corresponding diketone compound. Thus, the original absorption of DPBF at 472 nm is quenched significantly. In these two cases, the kinetic measurements of the quenching rates of the probes can be performed accordingly. Together with the concentration of a probe and the irradiation power used during the kinetic measurements, these data can be plotted using eqn (4) to obtain a linear plot.234–236 1 1 β ð4Þ ¼ 1þ ϕprobe ϕΔ ½probe 1 ϕprobe

¼

1 ϕΔ

1þ

1 ϕprobe

¼

β ½probe

F ϕΔ

α þ F½probe α þ ½probe

1þ

β ½probe

ð5Þ

ð6Þ

Eqn (4) depicts the relationship between the concentration of the probe and the quantum yield of the probe consumption, where β signifies the ratio between the rate constant of the natural decay of singlet oxygen and the rate constant of the oxidation of the probe by singlet oxygen. Upon obtaining the linear plot based on the equation, the corresponding quantum yield for the generation of the singlet oxygen can be obtained accordingly. However, some reports have indicated that the

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direct usage of eqn (1) as a model for the determination overestimates the quantum yield for the generation of singlet oxygen, particularly at high DPBF probe concentrations.237 A detailed mechanistic study has shown that the overestimation of the quantum yield is due to the presence of a chain reaction in the oxidation of DPBF. Hence, a more precise mechanism was proposed as shown in eqn (5), where F represents the degree of deactivation of the chain carrier and α represents the ratio of the rate constant of a chain oxidation rate. In the usual case when the value of [ probe] is much higher than the value of α, i.e., [ probe] ≫ α, eqn (5) can be simplified to eqn (6). In this case, the value of F is obtained using Rose Bengal as a standard. The work reported by Krieg provides more information on the determination of F.234 Once the determination of the quantum yield for the generation of singlet oxygen is complete, the photosensitizer is then used for PDT application. Normally, the photosensitizers are directly used in their original molecular form. In some cases, they are restricted to in vitro applications. Hence, by combining the use of photosensitizers with nanocarriers, better retention at the tumor site can be achieved to reduce the dark toxicity on healthy tissues. In an example reported by Lee et al., the chlorine based photosensitizer was directly conjugated onto a polysaccharide chain (chitosan) containing a pH ionizable group (GCS-g-DEAP-g-Ce6-g-PEG) (Fig. 31a).238 Upon dispersion in water, the polymer could undergo a supraFig. 32 (a) Chemical scheme for the synthesis of 2, which was then cocondensed into the silica network of MSN1. (b) Schematic diagram for the post-grafting of MSN1 with mannose for targeting purposes. (c) Images of tumor from control mice treated with saline, MSN1-mannose without photo-irradiation, and MSN1-mannose with photo-irradiation. (Reproduced with permission from ref. 239. Copyright 2011 John Wiley and Sons.)

Fig. 31 (a) Chemical structure of GCS-g-DEAP-g-Ce6-g-PEG (1). (b) Schematic representation by using 1 for PDT application. The scheme shows the turn-on photo-toxicity of 1 in acidic medium. (Reproduced with permission from ref. 238. Copyright 2011 John Wiley and Sons.)

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molecular self-assembly and the photosensitizer was in its self-quenched state. Upon delivery onto the tumor site, the acidic environment of the tumor broke the self-assembly and removed the self-quenching mechanism of the photosensitizer, thus conferring photo-toxicity onto the tumor site upon photo-irradiation (Fig. 31b). Another common way for improving the specificity of PDT applications is the use of mesoporous silica nanoparticles as carriers for the delivery of photosensitizers to the tumor side for the on-site PDT treatment. Durand et al. demonstrated the co-condensation of porphyrin-based photosensitizer within the silica network of mesoporous silica nanoparticles (MSN1 from Fig. 32).239 Thereafter, the surface of the mesoporous silica nanoparticles was functionalized with mannose to impart the target specificity towards MDA-MB231 breast cancer cells by targeting the mannose receptors on the cell surface. The incubation of the mannose functionalized nanoparticles showed much higher photo-toxicity to the cancer cells than the nanoparticles without the mannose functionalization. To demonstrate its in vivo applicability, the system was then injected into the tumor bearing nude mice for the PDT treatment. Fig. 32c shows that the tumors from mice treated


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with the MSN1-mannose under photo-irradiation have smaller mass than those treated with the MSN1-mannose alone. Though PDT has shown a tremendous amount of potential as an alternative to conventional therapy, there are still some drawbacks pertaining to the employment of PDT for the cancer treatment.240 One major issue is the presence of phototoxicity to patients during the course of treatment. Since the tumor specificity can only be achieved when localized irradiation is applied on the tumor site, patients have to avoid sunlight irradiation after the administration of photosensitizing drugs in order to minimize complications from the treatment. Another issue of relying on PDT for cancer therapy is the poor penetrating ability of light. Thus, tumors located in deep tissue regions cannot receive the same light dosage as compared to tumors grown on the surface of the body. Hence, non-invasive PDT treatments are usually effective for the treatment of skin related cancers such as melanoma. e.

Other types of cargoes

We have so far discussed various types of cargoes that are commonly used for cancer therapy. On account of rapid development of scientific research, the cargoes that are used for cancer therapy are not limited to the molecules mentioned in this section. Many anticancer drugs and biomolecules can also serve as cargoes for target-specific delivery into cancer cells. For instance, genes (such as siRNA) are commonly used cargoes, and they have shown a great potential as a promising strategy for cancer therapy.241–246

5. Stimuli-controlled release The integration of targeting ligands on the surface of nanocarriers is an effective technique for enhancing the therapeutic efficacy of the treatment while reducing the side effects brought about by the therapy. In addition to the targeting approach, controlled release techniques can also be used to improve the specificity of the delivery system. Such techniques rely either on physiological differences between cancerous cells and healthy cells or on applying an external localized stimulus, such as light or magnetic field, on the treatment site.247 In this section, we discuss the use of various strategies to perform stimuli-controlled release of cargoes into the tumor site or cancer cells for cancer therapy. a.

Acid stimulated release

A tumor is a tissue formed by uncontrolled cell division of cancer cells. In this context, cancer cells often possess a high metabolic rate, and hence require a high demand of nutrients for cell division. Of these nutrients, glucose is one of the main nutrients for energy production, and glucose metabolism is also possible under anaerobic conditions. This process causes the generation of a high amount of lactic acid in cancerous cells, resulting in a lower pH value (∼5.6) in the intracellular compartment of cancerous cells.248–252 In view of this fact, one can design delivery vectors that release therapeutic cargoes


Review

only under acidic conditions for killing cancer cells specifically. One commonly used strategy is the design of pH-responsive ligands particularly having a pKa value around 5.6. These ligands are used as gatekeepers on the surface of mesoporous silica nanoparticles. These pH-responsive ligands can form inclusion complexes with the cyclodextrin ring to block the pores of silica mesoporous nanoparticles, preventing the prerelease of loaded drugs in normal physiological conditions. When the nanoparticles are localized into the acidic compartment of cancerous cells, the low pH value induces the protonation of the ligand, which causes the dissociation of the inclusion complexes. This process hence leads to the release of the drugs from silica nanoparticles into the acidic compartment of cancerous cells. Such pH-triggered drug delivery has been demonstrated by a number of groups by using functionalized mesoporous silica nanoparticles as the drug carriers. In one example, a nanopiston prototype was developed in conjunction with β-cyclodextrin as the gatekeeper (Fig. 33a).253 The β-cyclodextrin was first functionalized onto the surface of mesoporous silica nanoparticles followed by loading 2,6naphthalenedisulfonic acid disodium (NDAD) as a model drug into the mesopores. In this work, a benzidine derivative was used as a plug, where it blocks the mesopores of the nanoparticles via the complexation with the β-cyclodextrin ring on the surface in neutral pH. When the system was dispersed in an acidic environment, the benzidine unit was protonated to destabilize the inclusion complex, resulting in the removal of the plug from the complex and the release of the content from the mesopores. The authors relied on fluorescence spectroscopy to monitor the removal of the plug by means of conjugation with fluorescent Rhodamine B and the release of the NDAD cargo from the mesoporous silica nanoparticles (Fig. 33b and 33c). In another work reported by the same group, the benzimidazole unit was conjugated on the surface of mesoporous silica nanoparticles, and then the content was loaded into the mesopores. Similarly, the β-cyclodextrin ring was used to form an inclusion complex with the benzimidazole unit, thus blocking the mesopores of the nanoparticles. Upon the dispersion in acidic medium, the β-cyclodextrin ring was removed, and the content was released from the nanoparticles (Fig. 34a and 34b).254 Another strategy in designing pH-responsive systems is the use of hydrazone or Schiff base conjugation. This process relies on the condensation between a ketone/aldehyde and an amine. The resulting Schiff base can then be hydrolyzed under acidic conditions. An example demonstrating the utilization of Schiff base in controlled drug delivery was reported by Kataoka et al.255 In this work, polyamine based micelles were prepared, which were further conjugated with adriamycin (ADR) by means of Schiff base reaction (Fig. 35a). Upon the dispersion in water, the polymer–drug conjugate could self-assemble into a micelle system. When the micelle was dispersed in an acidic medium, the acid catalyzed the hydrolysis of the hydrazone bond to release the ADR drug from the micelle (Fig. 35b).

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Fig. 34 (a) Synthesis scheme for the functionalization of benzimidazole onto mesoporous silica nanoparticles. Upon the functionalization, the pores of mesoporous silica nanoparticles were loaded with cargo followed by capping with β-cyclodextrin. (b) Upon dispersion in acidic medium, the benzimidazole unit is protonated, repelling the β-cyclodextrin ring from the surface. Thereafter, the cargo will be released from the pore accordingly. (c) TEM image of mesoporous silica nanoparticles. (Reproduced with permission from ref. 254. Copyright 2010 American Chemical Society.)

CO2 based on the following equation: NaHCO3 þ Hþ ! Naþ þ CO2 þ H2 O

Fig. 33 (a) Scheme illustrating the mechanism of pH-responsive release of NDAD cargo from the nanopores of mesoporous silica nanoparticles functionalized with the cyclodextrin–benzidine complex. Release profiles of (b) nanopiston and (c) NDAD under various pH. At pH 7, the release profiles showed no release of the nanopiston and NDAD. (Reproduced with permission from ref. 253. Copyright 2010 American Chemical Society.)

In addition, the delivery of this micelle system into the cellular compartment of cancer cells could induce time-dependent toxicity to the cancer cells as compared to free ADR (Fig. 35c). In addition to the two pH-controlled release mechanisms abovementioned, the use of NaHCO3 for stimulated release of drugs was also reported. In a series of results reported by Sung et al., DOX was first dissolved in water containing NaHCO3.151 Thereafter, the aqueous solution was loaded into the PLGA microsphere through the double emulsion technique. The dispersion of this microsphere in acidic medium allowed acid to diffuse into the microsphere to react with NaHCO3 to generate

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The generation of CO2 gas within the microsphere induced the rupture of the microsphere (Fig. 36a), thus releasing the DOX out of the microsphere (Fig. 36b). The utilization of acid to induce the release of cargoes from nanocarriers is not limited to drug-loaded nanocarriers. In gene delivery, some studies have indicated that the acid medium in the intracellular compartment of cancer cells can greatly enhance the transfection efficiency using non-viral gene delivery vectors.256,257 Non-viral gene delivery is commonly achieved through the use of polyamine carriers, and the dispersion of the DNA–polyamine complex in acidic medium induces the protonation of the polyamine backbone. Based on this process, the proton sponge effect was proposed to explain the enhancement of transfection efficiency. In an article by Behr, the author explained that the protonation of the complex results in the accumulation of positive charges in the endosomal environment.257 This then induces a large amount of chloride ions into the endosome with increased ionic strength. In addition, the ionization of the DNA–polymer complex induces the charge–charge repulsion between the complex, thereby expanding the polymeric network. The direct consequence of this expansion is to ease the release of the plasmid


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Review

Fig. 36 (a) SEM images of PLGA liposomes at various pH. (b) DOX release profiles of PLGA liposomes with different loading capacity of NaHCO3. (Reproduced with permission from ref. 151. Copyright 2011 John Wiley and Sons.)

of growing resistance against anti-tumor drugs with the increase of the GSH amount in cancerous cells.263–265 Hence, one can design disulphide bond-containing nanocarriers for GSH-induced release of drugs, when dealing with MDR cancer cells. The general mechanism for GSH-induced disulphide bond cleavage is shown below: RSSR′ þ 2GSH ! GSSG þ RSH þ R′SH

Fig. 35 (a) Chemical scheme indicating the hydrolysis of the hydrazone bond between polymer and ADR drug. (b) Percentage release of ADR from the polymer at various pH and time. (c) Comparison of the cell growth profiles at various concentrations of ADR using free ADR drug and the polymer–drug conjugate. (Reproduced with permission from ref. 255. Copyright 2003 John Wiley and Sons.)

into the endosome and hence enhance the transfection efficiency. b. Thiol induced release Glutathione (GSH) is a common reductant found in eukaryotic cells, where it plays its role as an anti-oxidant.258–262 Usually, GSH is present in normal cells at a level of 5.0 mM. However, the intracellular GSH concentration increases drastically when the cells turn cancerous. Research has shown direct evidence


In an article presented by Zhang et al., DOX was loaded into mesoporous silica nanoparticles followed by capping with β-cyclodextrin through a disulphide bond.155,165 It was demonstrated that GSH could reduce the disulphide bond cleavage between the silica nanoparticles and the β-cyclodextrin ring, thereby releasing the loaded DOX from the mesopores (Fig. 37a and 37b). The designed system was responsive to GSH by monitoring the release of the DOX content in aqueous GSH solution. In addition, the authors also demonstrated the selective release of the drugs into cancer cells but not into healthy cells (Fig. 37d). By the way, this nanoparticle system is also capable of releasing the DOX content under acidic medium, thus making it a dual responsive system (Fig. 37c). The thiol reduction of the disulfide bond for the release of content can also be demonstrated in a polymersome nanocarrier system. In a work reported by Hubbell et al., an amphiphilic polymer containing a disulfide bond was prepared via the living polymerization technique.266 The polymer was then dispersed in water to form a polymersome where calcein was loaded into the polymersome to demonstrate its drug delivery efficiency. The dispersion of calcein-loaded polymersome in the presence of reducing thiol resulted in the cleavage of the

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Fig. 37 (a) Scheme illustrating the release of DOX from the mesopores of functionalized mesoporous silica nanoparticles in the presence of acid and reducing thiol. Release profiles of DOX from mesoporous silica nanoparticles triggered by (b) reducing thiol and (c) acidic medium. (d) Fluorescence images of cancerous HeLa cells and healthy HEK293 cells after treatment with DOX loaded mesoporous silica nanoparticles. The green signal in the images is from FITC-labeled mesoporous silica nanoparticles, and the red signal is from fluorescent DOX. (Reproduced with permission from ref. 155. Copyright 2012 John Wiley and Sons.)

disulfide bond, causing the dissociation of the polymersome for calcein release. The authors showed the positive uptake of the calcein-loaded polymersome and the subsequent release of calcein in cancer cells, proving the feasibility of utilizing the disulfide bond in designing drug delivery systems. The inclusion of disulfide bonds in nanocarriers is not limited to only drug delivery vectors. This strategy can also be extended to the gene delivery using non-viral vectors. Dai et al. reported the utilization of single wall carbon nanotubes (SWNTs) for the delivery and release of DNA and si-RNA into the intracellular compartment (Fig. 38).267 In this system, a phospholipid molecule was first immobilized onto the wall of SWNTs by means of van der Waals interactions. Thereafter, the other end of the phospholipid molecule was functionalized with DNA or si-RNA bridged by a disulfide bond (1-X). Though the authors did not demonstrate the transfection efficiency, they observed positive uptake of DNA by means of a fluorescence technique due to the fact that the DNA was functionalized with a fluorescence dye. Furthermore, successful delivery of si-RNA was confirmed by the down regulation of lamin A/C

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Fig. 38 Schematic illustration of DNA/RNA immobilized SWNTs through two methods. 1-X has the presence of a disulfide bond, while 2-X contains no disulfide bond. (Reproduced with permission from ref. 267. Copyright 2005 American Chemical Society.)

protein in cancerous HeLa cells. In order to prove that the disulfide bond is crucial in the delivery and in liberating the genetic cargoes, a controlled analogue was designed, in which the disulfide bond was absent (2-X). The control analogue showed a reduced efficiency as compared to the disulfide analogue, hence confirming the importance of the disulfide bond for delivery.


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Fig. 39 Chemical scheme showing reversible photo-dimerization of coumarin under two different excitation conditions.


c. Light/photo and magnetic field induced release Localized cancer treatment can be achieved through careful design of the delivery systems, such that the delivery systems can only respond to a stimulated environment for cargo release. In this section, we discuss light and magnetic field driven release of cargoes. One example of photoinduced release of cargoes was demonstrated by Tanaka and co-workers using mesoporous silica nanoparticles.268 The mesopores of coumarin functionalized mesoporous silica nanoparticles were first loaded with steroid cholestane. Thereafter, a UV light centered at 324 nm was used to irradiate the nanoparticle system, causing the coumarin unit to photodimerize through 2 + 2 cycloaddition (Fig. 39). The resulting cyclobutane could serve as a gatekeeper and prevent the loaded cholestane from release. Upon photoirradiation with 250 nm of UV light, the cyclobutane ring was cleaved to open the mesopores of the silica nanoparticles for the release of loaded cholestane. Though the authors did not demonstrate the bioapplicability of this system, they performed a “proof of concept” work for photoinduced release of cargoes from mesoporous silica nanoparticles. Another example of using mesoporous silica nanoparticles for light-controlled drug delivery was demonstrated Zhao et al.269 Mesoporous silica nanoparticles were first functionalized with rotaxanes that consist of a cyclodextrin ring threaded on a rod containing a triazole group, an azobenzene unit and a stopper (Fig. 40a). In the initial state, the azobenzene unit was in its trans conformation and the cyclodextrin ring located on the azobenzene group. In this manner, the bulky cyclodextrin ring was away from the surface of the mesoporous silica nanoparticles, leaving the mesopores opened for the loading of curcumin drug. Upon UV light irradiation at 365 nm, the isomerization of the azobenzene unit from the trans to the cis conformation caused the cyclodextrin ring to shuttle to the triazole position, thereby closing the mesopores. When the curcumin-loaded nanoparticles were localized in biological systems, they were subjected to visible light irradiation to cause the isomerization of the azobenzene unit to its trans conformation. At this stage, the shuttling of the cyclodextrin ring back to the azobenzene group led to the opening of the mesopores for the release of loaded curcumin (Fig. 40b). Interestingly, light-controlled curcumin release was employed on zebrafish models in vivo for heart failure therapy. Though this work did not indicate the applicability of photoinduced drug release from nanocarriers for cancer therapy, the research has clearly demonstrated the feasibility of this delivery system in solving other biological problems.


Fig. 40 (a) Reversible isomerization of the azobenzene-based rotaxane on the surface of mesoporous silica nanoparticles. This process allows curcumin to be loaded into the mesopores for efficient delivery in a zebra-fish model. (b) Release profiles of curcumin from functionalized mesoporous silica nanoparticles at various conditions. (Reproduced with permission from ref. 269. Copyright 2012 John Wiley and Sons.)

Other than using light or photons as an external stimulus for the release of drugs from the nanocarriers, the application of magnetic field (AFM) for stimulated drug release has also become a promising non-invasive technique in cancer therapy. In a recent report, polymer based nanoparticles prepared by means of supramolecular techniques were employed to deliver DOX and magnetic nanoparticles, Zn0.4Fe2.6O4, into the tumor of nude mice by the EPR effect (Fig. 41).48 Upon the delivery of the nano-hybrid into the tumor site, an external magnetic field was applied onto the tumor site to cause the magnetic nanoparticles to heat up. The increase in temperature destabilized the supramolecular assembly to release the loaded DOX into the tumor environment. In this work, this technique can effectively inhibit tumor growth by releasing a substantially low amount of drug dosage into the mice as compared to other reports in literature, demonstrating an excellent non-invasive application of the system for cancer therapy.

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Fig. 41 Development of a supramolecular self-assembly system and its drug delivery application in vivo. The tumor growth profiles show that the treatment of the mice with the system and AFM suppresses the tumor growth as compared to the sole applications of AFM and the system. (Reproduced with permission from ref. 48. Copyright 2013 John Wiley and Sons.)

Another work using remote-controlled release of drugs under magnetic field was reported by Stoddart et al., where iron doped zinc nanocrystals were encapsulated within mesoporous silica nanoparticles.270 The surface of the mesoporous silica nanoparticles was functionalized with an alkyl diamine threaded by a curcubit[6]uril ring to form a pseudorotaxane (Fig. 42a). The mesoporous silica nanoparticles were preloaded with a dye molecule as a drug model and capped by the pseudorotaxane. Upon the delivery of the hybrid into the endosome of cancer cells, an external magnetic field was applied to the cell samples to cause the magnetic nanoparticles to heat up. This process resulted in the dissociation of the pseudorotaxane on the nanoparticle surface, so that the release of the model drug from the mesopore was realized (Fig. 42b).

6. Conclusions In this review, we have highlighted various components and strategies developed towards the design of effective drug delivery systems for controlled and targeted cancer therapy. On top of improving the biocompatibility of cargo-loaded nanocarriers, targeting ligands or stimuli-responsive groups could be post-functionalized onto the nanocarriers, thereby customizing the nanocarriers for a variety of cancer therapy applications. Several successful examples have been discussed in the review, clearly demonstrating the feasibility of the strategies in a synergetic manner towards cancer treatment. In this way, the shortfall of each individual component could be complemented by combination with another component.

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Fig. 42 (a) Scheme illustrating the preparation of the dye loaded mesoporous silica nanoparticles capped with a pseudorotaxane. The application of oscillating magnetic field induces the dissociation of the curcubit[6]uril ring from the pseudorotaxane for the release of the loaded cargo. (b) Release profiles of loaded dye from functionalized mesoporous silica nanoparticles. (Reproduced with permission from ref. 270. Copyright 2010 American Chemical Society.)

Although a lot of efforts have been devoted to developing novel therapeutic systems, only some of the systems have been demonstrated in clinical trials. Hence, the future research in this field will be (1) the further improvement of the existing technologies towards clinical applications, and (2) the development of new techniques for the next generation of therapeutics.

Acknowledgements This work was financially supported by the Singapore National Research Foundation Fellowship (NRF2009NRFRF001-015), the Singapore National Research Foundation CREATE program— Singapore Peking University Research Centre for a Sustainable Low-Carbon Future, and the NTU-A*Star Centre of Excellence for Silicon Technologies (A*Star SERC no. 112 351 0003).

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