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Gold nanoparticles (AuNPs) are a suitable platform for development of efficient delivery systems. AuNPs can be easily synthesized, functionalized, and are biocompatible. The tunability of the AuNP monolayer allows for complete control of surface properties for targeting and stability/release using these nanocarriers. This review will discuss several delivery strategies utilizing AuNPs.
One of the greatest challenges facing chemotherapy today is developing drug delivery systems (DDSs) that are efficacious and have therapeutic selectivity . Both passive and active targeting approaches have been utilized with nanocarriers such as dendrimers [2, 3], liposomes , metal nanoparticles , polymer micelles and vesicles [6, 7]. These DDSs have improved in the targeted delivery of the therapeutics for cancer treatment.
The application of gold nanoparticles (AuNPs) as a DDS is a rapidly expanding field [8, 9]. Their inherent properties make them a very promising vehicle for drug delivery. Controlled fabrication of various sized particles (1–150 nm) with limited size dispersity has been established , and using ligand place exchange reactions , multifunctional monolayers can be fabricated. This structural diversity enables particle surfaces to contain multiple targeting agents and/or chemotherapeutics. In addition the core in essence is non-toxic, biocompatible, and inert [12–14].
The diverse functional possibilities of AuNPs allows for a variety of approaches for DDS design. Hydrophobic drugs can be loaded onto AuNPs through non-covalent interactions, requiring no structural modification to the drug for drug release . Likewise, covalent conjugation to the AuNP through cleavable linkages can be used to deliver prodrugs to the cell and the drug can then be released by external  or internal  stimuli. Regardless of the approach used, the tunability of the AuNP monolayer is crucial for internal or external release mechanisms.
The use of AuNPs for therapy, biosensing, and diagnostics has been reviewed [18–22]. This review will focus on the use of AuNPs as a viable DDS and the influence on the overall function provided by the monolayer.
The fabrication of AuNPs with varying core sizes makes them an appealing platform for DDSs. A variety of synthetic methods exist for the fabrication of core-shell AuNPs, as summarized in Table 1. The one-pot method of Brust and Schiffrin et al. allows for the rapid and scalable synthesis of monolayer protected AuNPs . AuCl4− salts are reduced by NaBH4 in solution with the desired ligands. By changing the ligand to gold stoichiometry core sizes from 1.5 nm to 6 nm can be fabricated. To create larger particles citrate reduction of gold salts[24, 25] or ripening approaches  are widely used. Improved diversity of AuNPs is achieved by synthesizing a mixed monolayer by post-functionalization or directly functionalizing with appropriate ligands. A widely utilized method for the formation of mixed monolayer protected AuNPs is using a place-exchange reaction developed by Murray et al. . In this method, ligands with thiol functionality exchange in equilibrium with the bound ligands of AuNPs. The tunability of the ligand structure allows for enhanced delivery applications, such as the use of poly(ethylene glycol) and oligo(ethylene glycol) moieties to create a more biocompatible particle [27–33].
For a DDS to be effective, the interaction between cells and the delivery particles must be understood. Charge , size and surface functionality  affect the intracellular fate and cellular uptake of AuNPs. Rotello and Vachet have shown charge and hydrophobicity contributed to determining the cellular uptake of functionalized AuNPs . Chan et al. have reported cellular uptake is likewise dependent on the size of nanoparticles . In these studies, AuNPs ranging in size from 2–100 nm were coated with Herceptin and were tested for breast cell internalization mediated by the ErbB2 receptor. The most efficient cellular uptake was observed with particles ranging from 20–50 nm. Apoptosis was also enhanced by 40–50 nm particles. In vivo studies of passive targeting of tumors was also performed with AuNPs ranging from 10–100 nm . The authors found smaller AuNPs rapidly diffused into the tumor matrix, whereas larger AuNPs stayed near the vasculature (Figure 1).
Stellacci et al. found ligand shell morphology affects cell membrane penetration of AuNPs . It was shown that cytotoxic AuNPs with structured ligand shells could directly pass through the plasma membrane of cells without the creation of pores. Surface modifications to the ligand shell allow for targeting of specific organelles. Brust et al. utilized transmission electron microscopy to demonstrate that modifying the AuNP surface with cell penetrating peptides the nucleus and other organelles could be specifically targeted (Figure 2). Feldheim et al. have also shown nuclear targeting of AuNPs by modifying the surface with a nuclear localization sequence .
Drug release and transport are major factors for creating an efficient DDS. Nanocarriers can be loaded with drugs through non-covalent interactions or by covalent conjugation . using a prodrug that is processed by the cell . AuNPs provide an excellent platform for DDS design due to the functional versatility of their monolayers.
Regulation of drug release is crucial for DDSs . One useful approach for controlling the rate and site of release is to use an external stimulus to trigger release [44–49]. Photocleavable “cages” have been used to reduce the activity of a drug, with activation occurring upon uncaging [50, 51]. Nakanishi et al. have used this approach to develop a photoresponsive nanocarrier of amines. Using near-UV irradiation, a carbamate linkage could be dissociated by the photocleavable reaction of the 2-nitrobenzyl group. They found histamine had no biological activity while caged but after irradiation it became active.
Rotello et al. have demonstrated an AuNP platform with a photo-cleavable o-nitrobenzyl ester moiety that upon light irradiation dissociates changing the surface potential to negative from positive, intracellularly releasing adsorbed DNA . In further studies the authors observed light-controlled release of the anticancer drug 5-fluorouracil from AuNPs (Figure 3) . The mixed monolayer of the AuNPs was composed of photocleavable moieties and zwitterionic ligands. Improved solubility and limited cellular uptake was achieved by the use of the zwitterionic ligand. Using near-UV irradiation (365 nm) the photocleavable orthonitrobenzyl group could efficiently cleave the 5-fluorouracil from the AuNP. The observed IC50 value on a per particle basis for the AuNP was 0.7 μM. No significant cell death was observed in cells treated with only light AuNPs or only light.
DDSs have been created utilizing glutathione (GSH) as an endogenous agent for drug release. These systems rely on the drastic difference between intracellular GSH concentrations (1–10 mM) [54, 55] and extracellular thiol concentrations (cysteine 8 μM, GSH 2 μM) [56, 57]. Disulfide exchange or place-exchange reactions with the intracellular GSH can release prodrugs bound to the AuNP. Also the monolayer of the AuNP limits reactivity with cysteine residues of proteins in the bloodstream [58–60].
A DDS utilizing glutathione (GSH) for payload release was developed by Rotello et al. . A mixed monolayer of fluorogenic ligands (HSBDP) and cationic ligands (TTMA) was attached to AuNPs with a core diameter of 2 nm (Figure 4). Cellular penetration was assisted by the TTMA ligands on the surface of the nanoparticle. When attached, the Au core quenches the fluorescence of the BODIPY moiety of the HSDBP [61, 62]. Fluorogenesis occurred after cellular uptake through the intracellular GSH in human liver cells (Hep G2) or GSH treatment in cuvette. Treatment of mouse embryonic fibroblast cells (containing greater than 50% lower intracellular GSH levels than Hep G2) with varying levels of glutathione monoester (GSH-OEt) verified GSH-mediated release of the dye. Increasing concentrations of GSH-OEt showed a dose dependent relationship in fluorescence signal. Regulation of intracellular GSH levels likewise controlled the effectiveness of AuNP-mediated DNA transfection .
The surface charge and monolayer structure of AuNPs heavily influence the rate of ligand-displacement by intracellular thiols (e.g. GSH), giving rise to a controlled release of payload [64, 65]. Kotov et al. observed the anti-proliferative effect against K-562 leukemia cells of 6-mercaptopurine-9-β-D-ribofuranoside was notably increased when attached to an AuNP in comparison to the free form drug . Improved intracellular transport of the AuNPs preceding the GSH-mediated release in the lysosomes and cytoplasm was used to explain the enhancement.
Enhanced selectivity and potency can be achieved by targeting overexpressed receptors in cancer cells. El-Sayed et al. observed selective delivery and enhanced potency of conjugated tamoxifen to their AuNPs . The increased potency is attributed to an enhancement of cellular uptake of their AuNPs compared to the passive diffusion of the free drug. The binding of the estrogen receptor alpha in the plasma membrane facilitated improved selectivity for drug delivery. Alemzadeh et al. utilized the overexpressed folate-receptor in cancer cells to develop targeted delivery system for doxorubicin . Folate-modified PEG ligands were conjugated to doxorubicin to comprise the monolayer of their AuNP. The AuNPs had increased cytotoxicity to cells with overexpression of folate-receptors and reduced cytotoxicity to healthy cells when compared to free doxorubicin.
Mukherjee et al. also developed a system to deliver the platinum containing drug cisplatin targeting the folate-receptor . The delivery system targets cancer cells preferentially and enhances cytotoxicity. The cisplatin retains its cytotoxic effects while conjugated, and the folic acid protects healthy cells from the cisplatin. Lippard et al. combined DNA functionalized AuNPs with Pt(IV) prodrugs for a novel DDS . After the Pt-DNA-AuNPs enters the cell the Pt(IV) is reduced to release cytotoxic cisplatin. The Pt(IV) species exhibit greater biological stability when compared to the more reactive Pt(II) species leading to less side effects. Wheate et. al observed enhanced cytotoxicity of oxaliplatin when tethered to AuNPs . The tumor cell concentrations of oxaliplatin were greater for the AuNP delivered drug than when compared to the free drug.
Encapsulating unmodified drugs into the monolayer of the AuNP non-covalently avoids potential issues associated with using a prodrug strategy. Using appropriate ligands, a hydrophobic environment is created to incorporate the drugs into the monolayer. The further one goes from the gold core, the smaller the ligand density becomes [72, 73]. Using EPR spectroscopy, Pasquato et al. observed the partitioning of radical, lipophilic probes between bulk water and the monolayer of an AuNP. As seen in Figure 5, they found that guest encapsulation was favored by the greater radial monolayers of smaller particle cores [74, 75].
Rotello et al. fabricated a biocompatible AuNP to deliver drugs to cancer cells via their incorporation into the monolayer . The 2.5 nm core AuNPs were functionalized with a hydrophobic alkanethiol interior and a tetra(ethylene glycol) (TEG) hydrophilic shell. The ligands possessed zwitterionic headgroup which minimized nonspecific binding with cell surface functionalities and biomacromolecules [77, 78] (Figure 6). Kinetically encapsulated in the monolayer, the hydrophobic payloads were stable in serum and buffer. Fluorescence microscopy, using a hydrophobic fluorophore, and drug efficacy measurements of therapeutics verified membrane-mediated diffusion into cells without cellular uptake of the particles. These DDS are great candidates for passive targeting utilizing the enhanced permeability and retention effect .
Inducing necrosis and apoptosis using reactive oxygen species is a potential strategy for therapeutics. Burda et al. utilized this strategy to develop a photodynamic therapy (PDT) with PEGylated AuNP-Pc4 (Si-phthalocyanine) . The PEG ligands serve multiple purposes. First they inhibit non-specific binging to biomacromolecules and prevent colloidal aggregation. Theligands also, through Van der Waals interactions, encapsulate the phthalocyanine photosensitizing agent (Figure 7a). An efficient delivery process was verified by monitoring the release of Pc4 from the nanocarrier in vitro in a two-phase solution system and in vivo in cancer-bearing mice with enhanced accumulation of Pc4 in tumor sites. The Au NP-Pc4 conjugates decreased the time necessary for PDT from 2 days to 2 hours versus the free drug (Figure 7b).
Kim et al. developed a DDS using non-covalently loaded anti-cancer drugs which are released by glutathione . β-Lapachone, an anti-cancer drug, was encapsulated in the cyclodextrin moieties. Poly(ethylene glycol) was used to prevent degradation before delivery. The AuNPs were also functionalized with an anti-epidermal growth factor receptor antibody as a targeting agent. In addition to this, the targeting antibody increased the extent of apoptosis. Schoenfisch et al. have developed a pH responsive system which at acidic pHs releases nitric oxide from AuNPs . This potentially gives the ability to control cellular processes such as immune response, angiogenesis, and vasodilation [82, 83]. Using a phosphate linker Hwu et al. conjugated paclitaxel to Fe3O4 particles and AuNPs . Phosphodiesterase could then release the paclitaxel.
AuNPs show great potential for the creation of DDSs. Their stability, tunable monolayers, functional flexibility, low toxicity, and ability to control the release of drugs offer many possibilities for further development of DDSs. Additional investigations of these systems will be required to fully understand their pharmacokinetics, interactions with the immune system, and the extent of cytotoxicity due to surface and size of the AuNPs. Continued research into these nanoscale delivery vehicles will expand the understanding of the interactions of these materials with biological systems, and promoting the development of more effective DDSs.
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