PEG is a biocompatible linear synthetic polymer that can be prepared in a range of sizes and with a variety of terminal functional groups. PEG is commonly used to achieve stealth properties, due to its hydrophilicity, flexibility, and neutral charge in biological fluids, all of which properties help disperse nanoparticles and increase their blood circulation times 169,170
. Various methods have been used to attach PEG to magnetic nanoparticles, including in situ
coating under aqueous conditions 171
, silane-grafted PEG coating 172
, fabrication of amine-functionalized 6-armed PEG derivatives 173
, polymerization at nanoparticle surfaces 174
, or modification through sol-gel approaches 175
. Covalent attachment of one end of a heterobifunctional PEG to the magnetic nanoparticles and functionalization of the other end with targeting ligands, imaging probes, or therapeutic agents have been described 176-178
. Jon et al
. developed a multifunctional poly(TMSMA-r-PEGMA-r-NAS) capable of covalent anchoring onto iron oxide surfaces (silane functionality), resisting protein absorption (PEG functionality), and binding to functional moieties (carboxyl functionality) 136
. Interestingly, silane functionalities were employed to cross-link polymer coating layers, formed by heat-mediated condensation of hydrolyzed silane groups (-Si(OH)3
), to yield highly stable nanoparticles. This polymer facilitated fabrication of iron oxide nanoparticles and demonstrated their in vivo
dual imaging (MR/optical) modalities in tumor xenograft animal models.
Copolymers containing PEG residues have been developed as a means of combining the properties of PEG with the desired properties of other moieties. Chen and Shuai synthesized PEG-g-PEI copolymers that condense DNA to facilitate gene delivery (PEI functionality) and display a stealth profile necessary for in vivo
applications (PEG functionality). The copolymers were used to construct PEG-g-PEI-SPIONs as MRI-visible gene carriers 60
. Zhang et al
. prepared a PEGylated chitosan-branched copolymer to coat iron oxide core and develop brain tumor targeting magnetic/optical nanoprobe. Chitosan is a hydrophilic and biocompatible natural polymer that is popular as a theranostic nanocarrier material 179
. In a study by Zhang et al.
, a PEG-grafted chitosan copolymer was prepared using a Schiff base reaction between PEG-aldehyde and amine-containing depolymerized chitosan, and nanoparticles were synthesized by coprecipitation of ferrous and ferric chloride with ammonium hydroxide in the presence of the copolymer. The chitosan amine groups present on the surfaces of the nanoparticles were coupled to the brain tumor targeting ligand, CTX. The presence of surface chitosan sterically stabilized the nanoparticles by preventing aggregation under physiological conditions. Additionally, the positively charged chitosan cations could interact with the negatively charged brain endothelium via electrostatic interactions to trigger adsorptive-mediated transport across the BBB. Jon et al
. developed another multifunctional amphiphilic copolymer, poly(DMA-r-mPEGMA-r-MA), that formed an efficient coating on the surfaces of oleic acid-stabilized SPIONs 180
. Hydrophobic dodecyl methacrylate (DMA) residues on the polymer bound to the oleic acid tail group formed a shell around the SPIONs via hydrophobic and van der Waals interactions. Hydrophilic PEG residues improve the water-dispersibility of nanoparticles and prevent biofouling during in vivo
molecular imaging. Finally, methacrylic acid (MA) residues can be used to introduce additional functionalities. Sonication of the immiscible phases containing oleic acid-stabilized SPIONs in hexane or the amphiphilic polymer in water created an oil-in-water emulsion. Evaporation of the hexane left a homogenous aqueous solution containing amphiphilic polymer-coated SPIONs. The in vivo
diagnostic capabilities were demonstrated in a Lewis Lung Carcinoma cell allograft animal model. In a similar approach, Hadjipanayis et al
. formed oleic acid-stabilized SPIONs using an amphiphilic triblock copolymer consisting of a polybutylacrylate segment (hydrophobic), a polyethylacrylate segment (hydrophobic), a polymethacrylic acid segment (hydrophilic), and a hydrophobic hydrocarbon side chain 52
. Strong hydrophobic interactions between the oleic acid and the hydrophobic residues of the polymer favored spontaneous encapsulation of the SPIONs. The surface-exposed carboxyl groups of the methacrylic acid segments covalently bound to EGFRvIIIAb to target glioblastoma, and PEG stabilized the dispersion under aqueous conditions.
Polysaccharide dextrans are widely used as surface shielding materials for in vivo
applications. The biocompatibility and high affinity of dextrans toward iron oxide surfaces, mediated by polar interactions, including hydrogen bonds, provide excellent properties to SPIONs coated with dextrans, and several of the clinically approved SPION preparations are dextran-coated 181-183
. Conventional dextran polymer coatings do, however, suffer some degree of detachment from the nanoparticle surfaces due to the lability of the hydrogen bonds. Weissleder et al.
developed stable cross-linked iron oxide nanoparticles (CLIO) by cross-linking the dextran polymer using epichlorohydrin or ammonia. Ammonia treatment of CLIOs introduced primary amine groups to the particle surfaces, which facilitated attachment of other functional moieties. The resulting nanoparticles were extensively evaluated in a variety of MRI and theranostic applications 71, 87, 184
. The CLIOs exhibited a long blood circulation time without inducing an acute toxic response 185
; however, the persistence and stability, as well as the presence of trace amounts of the epichlorohydrin reactant, were problematic in the clinical setting, and further development of these agents has slowed 186
Atomic transfer radical polymerization (ATRP) is another common method for coating iron oxide nanoparticles 187
. Li et al
. used the ATRP method to synthesize polystyrene-coated iron oxide nanoparticles using divinylbenzene as a cross-linker 188
. Several other polymers, including polyvinyl alcohol (PVA) 189
, polyvinyl pyrrolidine (PVP) 190
, and polyacrylic acid 191
, were described as coating materials for iron oxide nanoparticles. These polymers provided a steric barrier to prevent nanoparticle agglomeration and enhance the blood circulation time. In addition, a variety of monomeric species, including bisphosphonates 192
, dimercaptosuccinic acid (DMSA) 193
, and alkoxysilanes 194
, has been evaluated as anchors to facilitate attachment of polymers to the nanoparticle surfaces. Cheon et al.
developed water-soluble iron oxide (WSIO) nanocrystals using DMSA as coating materials 195
. The DMSA formed a stable coating on the magnetic Fe3
nanocrystals through carboxylic chelating bonds, then further stabilized the shells through intermolecular disulfide cross-linkages. The remaining free DMSA thiol groups were linked to a breast cancer-targeting molecule, Herceptin. The WSIO-Herceptin probes specifically targeted breast cancer cells to provide a targeted MRI contrast agent, as revealed by T2-weighted MRI. Cheon et al.
also developed a hybrid nanoparticle probe for PET/MR imaging consisting of albumin-stabilized iron oxide nanoparticles 196
. Serum albumins were linked to oleic acid-coated manganese-doped magnetic engineered iron oxide (MnMEIO) nanoparticles via ligand exchange, and the surfaces of the nanoparticles were further stabilized by EDC/NHS-mediated cross-linking between the amine and carboxyl groups of the serum albumin. The resultant MnMEIO nanoparticles showed high colloidal stability over a wide range of pH and at high salt concentrations. The tyrosine residues in the serum albumin could be directly conjugated to 124
I to introduce radioactive properties. This nanoparticle platform was also used to develop all-in-one target cell-specific magnetic nanoparticles for simultaneous molecular imaging and siRNA delivery 197
. To target αvβ3 integrin-positive cancer cells and to inhibit specific protein expression, SH-PEG-RGD and SH-siRNA-Cy5, respectively, were conjugated to MnMEIO via an SPDP cross-linker. In vitro
evaluation of MRI and gene suppression demonstrated that these multimodal nanoparticle systems selectively silenced genes, confirming their theranostic behavior.
Liposomes and micellar dispersions provide another shielding strategy. Spherical assembles of amphiphilic molecules can be used to coat magnetic nanoparticles by incorporating the nanoparticles within hydrophilic or hydrophobic cores to enhance blood circulation time. The amphiphilic substructures can encapsulate additional therapeutic agents or functional molecules within the core to easily achieve multifunctional nanoparticles. Lesieur et al
. developed magnetic-fluid-loaded liposomes (MFLs) by encapsulating maghemite (γ-Fe2
) nanocrystals in the unilamellar vesicles of (DSPE)-mPEG2000 and egg phosphatidylcholine 198
. MFLs with a hydrodynamic size of 195 ± 33 nm were prepared by film hydration followed by extrusion. The particles were stable without the need for ferrofluid flocculation under physiological conditions. In vivo
tests in mice using MR angiography demonstrated that the presence of MFLs enhanced the image contrast significantly, and the particles persisted in the blood 24 h after injection, possibly due to the stealth properties conveyed by PEGylation of the MFLs. Huh and Haam developed multifunctional magneto-polymeric nanohybrids (MNPNs) using polymeric micelles, and they evaluated the in vivo
uses of the particles for simultaneous diagnosis and therapy 199
. Water solubility was achieved by embedding the hydrophobic magnetic nanocrystals and Dox within amphiphilic poly(ethylene glycol)-block-poly(D,L-lactic-co-glycolic acid) (PEG-PLGA) using nanoemulsion methods, and Herceptin was conjugated to the terminal PEG-PLGA carboxyl groups for selective targeting. Manganese ferrite (MnFe2
) and Dox were present in the MNPNs at 41.7 wt% and 3.3 wt%, respectively. In vivo
MRI and therapeutic tests in a mouse model bearing NIH3T6.7 cells demonstrated that the Herceptin-conjugated multifunctional polymeric micelles were site-specifically delivered to the tumor tissues that overexpressed HER2/neu receptors. The particles retarded the rapid growth of the tumors. Gao et al
. used polymeric micelles to prepare αvβ3 integrin targeting theranostic nanoparticles 200
. The amphiphilic block copolymers of maleimide-terminated poly(ethylene glycol)-block-poly(D,L-lactide) (MAL-PEG-PLA) and methoxy-terminated poly(ethylene glycol)-block-poly(D,L-lactide) (MPEG-PLA) were used to form micelles. Oleic acid- and oleylamine-stabilized SPIONs and Dox were encapsulated into polymeric micelles via a solvent evaporation method, and thiol-containing cRGD was attached to the surfaces of the micelles. In vitro
MRI and cytotoxicity studies confirmed the ultrasensitivity, enhanced MRI contrast properties, and αvβ3 integrin-specific therapeutic response to the multifunctional nanoplatform. The presence of ionizable ammonium groups on Dox (PKa
=7) suggested that the drug release rate should be pH-dependent.
In addition to organic coatings, core-shell structures, such as biocompatible silica- or gold-covered magnetic nanoparticles, have provided an attractive approach to developing stealth nanoparticles. Silica shells serve as protective stable nanoparticle coatings under aqueous conditions. The ability to encapsulate functional molecules within the nanoparticle matrix is a unique feature of these nanostructures. Hyeon and Moon developed Fe3
nanocrystal-embedded, core-shell mesoporous silica nanoparticles, and they demonstrated their multifunctional application to simultaneous MR/optical imaging and drug delivery 201
. This study suggested a precise method for controlling the size of the silica nanoparticles smaller than 100 nm. The surfactant cetyltrimethylammonium bromide (CTAB) provided an organic template for the formation of a mesoporous silica shell and stabilized the hydrophobic Fe3
nanocrystals in an aqueous solution. The sol-gel process occurred through the template by using tetraethylorthosilicate (TEOS) and rhodamine B isothiocyanate (RITC)-labeled aminopropyltriethoxysilane (APS), and generated amine groups containing silica shell, to which PEG was covalently conjugated via succinimidyl end group to render further biocompatibility. Dox molecules loaded onto the as-synthesized Fe3
(R)-PEG NPs to convey therapeutic properties. The core-shell structure exhibited magnetic and fluorescent properties, as well as a therapeutic index, suggesting the utility of the nanostructure in biomedical theranostic applications. On the other hand, gold provides several advantages as a coating material due to its inertness and its unique ability to absorb near-IR radiation. Hyeon and Cho et al.
described magnetic gold nanoshells (Mag-GNS) consisting of gold nanoshells encapsulating magnetic Fe3
nanoparticles as a novel nanomedical platform for simultaneous diagnostic imaging and thermal therapy 202
. Monodisperse 7 nm Fe3
nanoparticles stabilized with 2-bromo-2-methylpropionic acid (BMPA) were covalently attached to amino-modified silica spheres through a direct nucleophilic substitution reaction between the bromo groups and the amino groups. Gold seed nanoparticles were then attached to the residual amino groups of the silica spheres. Finally, a complete 15 nm thick gold shell embedded with Fe3
nanoparticles formed around the silica spheres to generate Mag-GNS. To target breast cancer, an anti-HER2/neu antibody was conjugated onto the surfaces of the Mag-GNS. SKBR3 breast cancer cells treated with Mag-GNS could be detected using a clinical MRI system, followed by selective destruction by near-IR radiation.
A new class of non-biofouling zwitterionic materials was recently developed 203
. The low fouling properties with respect to blood serum or plasma were attributed to strong interactions between the zwitterions and the neighboring water molecules, thereby offering good colloidal stability. The zwitterionic state was macroscopically neutral with a net zero charge that provided a non-fouling surface 204-206
. In vivo
tests of the zwitterion-coated QDs demonstrated that the surfaces were protein-resistant, which made it possible for the QDs to remain small relative to the PEG-decorated particles 207,208
. Jiang et al.
developed poly(carboxybetaine acrylamide) (polyCBAA)-functionalized surfaces that were used to stabilize gold nanoparticles using the ATRP method 209
. This surface platform was highly resistant to nonspecific protein adsorption, and it presented abundant carboxyl groups for biomolecule immobilization. However, ATRP reactions require surface-grafted initiators and oxygen-free conditions, which limit their practical application. This problem was addressed by another strategy, the 'graft-to-surface' method 210
, in which a zwitterionic poly(carboxybetaine methacrylate) (pCBMA) polymer was grafted onto the surface via two 3,4-dihydroxyphenyl-L-alanine (DOPA) adhesive moieties, and iron oxide nanoparticles were fabricated from the as-synthesized pCBMA-DOPA2
. Amine-containing cRGD peptides were immobilized onto the nanoparticles via the EDC/NHS chemistry. pCBMA-DOPA2
-decorated magnetic nanoparticles exhibited a lower macrophage uptake than the dextran-coated magnetic nanoparticles. Uptake by human umbilical vein endothelial cells (HUVEC) was considerably higher due to cRGD-mediated targeting, as demonstrated by MRI studies.
Optimal density of the non-biofouling moieties and targeting ligands
Non-biofouling moieties, present as shielding materials, not only provide a steric barrier to prevent nonspecific protein absorption, they can tailor the surface properties of the nanoparticles to avoid recognition by the RES. The successful in vivo
performance of a nanoparticle system relies critically on the non-biofouling properties, the molecular weight, the surface structural conformation, and the surface coverage ratio 211,212
. The surface density and conformation are important features for improving the stealth and targeting efficiency. To this end, PEG-linked liposomal nanoparticle surfaces have been engineered. As shown in Figure , individual PEG chains on a liposomal surface exhibited a Flory dimension, Rf, which represents the volume occupied by each flexible PEG molecule 213
Figure 17 Representations of different PEG conformations, formed through their incorporation onto surfaces at different densities. (A) Low surface coverage levels of PEG lead to the “mushroom” configuration (D>Rf). (B) High surface coverage (more ...)
A high surface coverage and a high concentration of the PEG-lipids within a liposomal formulation decrease the distance, D, between each PEG molecule on the nanoparticle surface. If D>Rf, the PEG chains self-assemble into a random-coil like 'mushroom' configuration. On the other hand, if D<Rf, the lateral pressure between the overcrowded PEG extends the PEG chains to a semi-linear 'brush' configuration 214
. In general, the brush configuration of surface PEGs conveys greater protein repulsion and enhanced nanoparticle lifetime in the blood stream 215-217
. This surface configuration can also decrease the mobility of the PEG chains, thereby diminishing the stealth functionalities of the PEG layer 218
, and hinder binding between the targeting ligands of the nanoparticles and the target cancer cells 213
. Therefore, the surface coverage of the nanoparticle must be optimized during the design of any theranostic system.
Despite the critical role of the targeting ligand density on the nanoparticle surfaces, few studies have reported maximization of a nanoparticle system targeting efficacy. Higher concentrations of the targeting ligands on the nanoparticles often increase cellular uptake 219,220
. Targeting ligands must be present on the nanoparticle surfaces at concentrations that exceed a minimum threshold for binding 221
. However, some studies also demonstrated that high ligand densities do not improve binding to the target cancer cells and can even promote nonspecific interactions with endothelial and other non-cancerous cells, which increases immunogenicity, thereby causing opsonization-mediated clearance of the nanoparticles 222
. Gao et al
. prepared 0%, 5%, or 16% RGD ligand-immobilized Dox-loaded polymeric micelles to investigate their in vitro
targeting and therapeutic properties 200
. The 16% RGD ligand-bearing nanoparticles displayed the highest cellular uptake and cytotoxicity in αvβ3 integrin-overexpressing SLK cells. Berkland et al.
modulated the reactive sites on the nanoparticle surfaces for peptide conjugation by controlling the mixture ratio of the two surfactants during nanoparticle fabrication 223
. They prepared PLGA nanoparticles with several ratios of the carboxyl and hydroxyl groups in the Pluronic®
surfactant (100:0 75:25, 50:50, 25:75, and 0:100 (v/v)), and cLABL peptides were conjugated to the surfaces to achieve intercellular cell adhesion molecule-1 (ICAM-1) targeting. Incubation of the cLABL-PLGA nanoparticles with the target A549 cancer cells revealed a maximum uptake for surfactant ratios of 50:50 or 25:75. Interestingly, the minimum uptake occurred for the surfactant ratios of 100:0 and 75:25. The enhanced receptor binding at moderate ligand densities may have been due to receptor behavior during the binding process. ICAM-1 mobility on the cell surface leads to clustering during ligand binding 224
, and a high density of the targeting ligands can disturb the clustering. This result suggests that the ligand spacing should be considered during surface engineering of targeted nanoparticle systems. Recently, Ashley et al
. developed targeted silica nanoporous particle-supported lipid bilayers that provided enhanced surface fluidity, which recruited multiple peptides to the target cancer cells to promote multivalent effects 6
. The unique long-range fluidity of the nanoparticle surface promoted a high affinity between the targeting peptides on the nanoparticles and the target cancer cells at low peptide densities (6 peptides per particle). This specific targeting is crucial for reducing nonspecific interactions and enhancing specific affinity, which maximize the selective delivery of a cargo.
It has become increasingly clear that the tumor-targeting properties of the nanoparticles optimized in vitro
are not predictive of the in vivo
performance. Farokhzad and Langer identified maximally targeted and maximally stealth surface engineering conditions for in vitro
and in vivo
performance using PSMA targeted aptamer-conjugated Dtxl-loaded self-assembled nanoparticles 225
. Nanoparticles were prepared with different compositions of the self-assembled diblock copolymers and aptamers, and the optimal aptamer density on the nanoparticle surface was initially determined in vitro
. Increasing the ligand density to 5% significantly increased the nanoparticle uptake by the target cells (LNCaP), whereas further increase in aptamer density modestly increased the nanoparticle uptake. These results indicated that the optimum ligand density for PSMA-specific endocytosis in vitro
was 10-80 nmol aptamer per μmol nanoparticle. LNCaP xenograft mouse models injected with the targeted nanoparticles showed that increasing the aptamer density from 0% to 5% significantly increased nanoparticle retention in tumors, but the retention decreased for aptamer densities beyond 10%. The authors suggested that higher aptamer densities may have reduced the nanoparticle stealth properties, resulting in rapid clearance by the liver. Gabizon et al
. optimized the ligand density in the Her2-targeted PEGylated liposomal Dox system (HT-PLD) in vivo
for ligand ratios of 7.5, 15, or 30 per liposome 226
. The best safety margin and in vivo
performance resulted from a ligand density of 15 ligands per liposome in the HT-PLD formulation. A 30 ligand ratio accelerated plasma clearance in the tumor-bearing mice, and the 7.5 ligand ratio reduced cytotoxicity after in vivo
The role of nanoparticle geometry in tumor targeting has received relatively little attention, although it is important for determining the binding affinity for a target cell. Sailor et al
. systemically optimized in vivo
tumor targeting by varying the nanomaterial shape (elongated versus spherical), targeting ligand type (cell surface targeting versus extracellular matrix targeting), ligand surface coverage, and attachment chemistry (Figure ) 227
. They prepared two types of tumor-targeting peptides (F3 or CREKA) and conjugated the peptides to magnetic nanoworms (NWs) or magnetic nanospheres (NSs) at varying numbers of targeting peptides and for varying PEG lengths. Intravenous injection of the magnetic nanostructures in the tumor xenograft mice models revealed that the in vivo
tumor-targeting properties of the NWs were superior to those of the NSs due to multivalent interactions between the elongated NWs and the receptors on the tumor cell surfaces. The smaller neutral CREKA targeting moiety was more effective than the larger positively charged F3 targeting moiety, presumably because multiple copies of the highly cationic F3 caused a large increase in the surface charge on the particles, which facilitated clearance by the MPS-related organs. The most effective number of CREKA peptides was 60 per NW. Above 60 peptides per NW, the blood circulation time decreased. For a given number of peptides bound to the NWs, the presence of a PEG linker facilitated peptide targeting by reducing conformational restriction as well as increasing the residence time of the nanostructures in the blood stream. The short SMCC linker restricted the targeting peptide conformation. These results suggest some design guidelines for the development of targeted multifunctional nanoparticle systems for cancer imaging and therapy.
Figure 18 Three parameters were varied to optimize in vivo tumor targeting: the shape of the nanoparticle, the type of the targeting ligand, and the nature of the molecular linker. Two types of surface linkers were used to attach the targeting groups to the magnetic (more ...)