Revisiting 30 years of biofunctionalization and surface chemistry of inorganic nanoparticles for nanomedicine

Abstract

In the last 30 years we have assisted to a massive advance of nanomaterials in material science. Nanomaterials and structures, in addition to their small size, have properties that differ from those of larger bulk materials, making them ideal for a host of novel applications. The spread of nanotechnology in the last years has been due to the improvement of synthesis and characterization methods on the nanoscale, a field rich in new physical phenomena and synthetic opportunities. In fact, the development of functional nanoparticles has progressed exponentially over the past two decades. This work aims to extensively review 30 years of different strategies of surface modification and functionalization of noble metal (gold) nanoparticles, magnetic nanocrystals and semiconductor nanoparticles, such as quantum dots. The aim of this review is not only to provide in-depth insights into the different biofunctionalization and characterization methods, but also to give an overview of possibilities and limitations of the available nanoparticles.

Keywords: biofunctionalization; chemistry surface; gold nanoparticles; magnetic nanoparticles; quantum dots.

Figure 1

Schematic representation of a multifunctional nanocarrier. These innovative NPs comprise nucleic acids such as RNA and DNA used for gene silencing approaches and in colorimetric assays, respectively. Aptamers and anticancer drug molecules are also used for delivery to the target tissue. Carbohydrates may be useful as sensitive colorimetric probes. PEG is used to improve solubility and decrease immunogenicity. Responsive nanocarriers can also trigger reaction upon external stimuli through the functionality of valuable tumor markers, peptides, carbohydrates, polymers and antibodies that can be used to improve nanocarrier circulation, effectiveness, and selectivity. Multifunctional systems can also carry fluorescent dyes that are used as reporter molecules tethered to the particle surface and employed as tracking and/or contrast agents.

Figure 2

Polyvalent PEGylated gold nanoparticles. (A) Bioconjugation of the surface-modified gold nanoparticles with different thiol-PEG layer composition (SH-EG(7)-CH₂-COOH and SH-(CH₂)₃-CONH-EG(6)-CH₂-N₃), TAT peptide and thiol-dsRNA oligonucleotide. (B) Mechanism for the enhancement of the dsRNA loading on AuNPs functionalized with PEG chains and TAT peptide. The azide group has a resonant structure with a positively polarized behavior that can attach the negatively charged thiolated oligonucleotide to the gold surface. The azide-containing chain also encloses an amide group near the gold surface that could play a role in approaching the thiol group of the oligonucleotide to the gold surface. This amide group could form a hydrogen bond with one of the hydroxyl groups of the ribose group near the thiol group on the oligonucleotide. (C) Determination of the number of TAT chains bound to AuNPs by the EDC reaction as a function of the initial peptide concentration in the reaction mixture. Blue bars AuNP@COOH/N₃ and red bars AuNP@COOH. (D) Loading of thiolated oligonucleotide (HS-dsRNA) on AuNPs functionalized with TAT peptide and with both PEG-azide and PEG-COOH and only with PEG-COOH. Blue bars AuNP@COOH/N₃ and red bars AuNP@COOH (Sanz et al., 2012). Reproduced with permission from Sanz et al. (2012), Copyright 2013.

Figure 3

Fluorescent-AuNPs. (A) Chemical structure of a FAM–DNA–AuNP and schematic illustration of its FRET-based operating principles. (B–E) Confocal fluorescence and phase-contrast images of living cells. (B) Fluorescence image of macrophages incubated with the probe for 30 min at 37°C. (C) Fluorescence image of probe-stained macrophages stimulated with PMA for 1 h at 37°C. (D) Bright field image of live macrophages shown in (C), confirming their viability. (E) AO staining of probe-loaded macrophages, confirming their viability (Tang et al., 2008). Reproduced with permission from Tang et al. (2008), Copyright 2013.

Figure 4

Silencing the Silencers with hairpin-DNA-AuNPs—Gold nanobeacons. (A) Specific Au-nanobeacons are capable of intersecting miRNA pathway, leading to recovery of previously downregulated gene expression while simultaneously discriminating cells where silencing is occurring. The fluorescence signal may allow for tracking cell internalization and sub-cellular localization. The Au-nanobeacons' potential for anti-cancer therapeutics via the silencing of the silencers is demonstrated by blocking the endogenous microRNA pathway via an Anti-miR Au-nanobeacon complementary to the mature microRNA-21 (miR-21), commonly upregulated in cancer phenotypes. (B,C) Au-nanobeacons silencing of endogenous silencers—silencing of miR-21. Confocal imaging (scale bar, 10 μm) shows internalization of 50 nM (B) Anti-miR Au-nanobeacon 50 nM and (C) Nonsense Au-nanobeacon. Target (mature miR-21) recognition leads to change of Anti-miR Au-nanobeacon conformation in the cytoplasm with concomitant fluorescence signal (red, Cy3) encircling the cell nuclei (blue, DAPI) (Conde et al., 2013b). Reproduced with permission from Conde et al. (2013b), Copyright 2013.

Figure 5

Gold nanoparticles functionalized with multiple biomolecules: PEG, cell penetration peptide (TAT), ammonium quaternary groups, and siRNA. Two different approaches were employed to conjugate the siRNA to the AuNPs: (A) ionic approach, interaction of the negatively charged siRNA to the modified surface of the AuNPs through ionic interactions; (B) covalent approach, use of thiolated siRNA for gold thiol binding to the NPs.

Figure 6

Quantum dot protein biosensors. (A) Schematic showing the common design, chemical and sensing elements, including FRET-based biosensors: (1) peptide modularity, (2) peptide labeling, (3) attachment to QDs, and (4) FRET-based sensing for both the caspase 3 proteolytic sensor (left) and Ca²⁺ sensor (right). The 4-pendant carboxyl groups that interact with Ca²⁺ ions are shown in red on the CaRbCl structure. Within the Ca²⁺ sensor peptide sequence, Aib is the synthetic amino acid α-aminoisobutyric acid. Reactive dye structures are shown where appropriate along with the chemical linkages attaching them to the peptides. (B) Representative, superimposed spectra collected from 550 nm emitting QD donors surface functionalized with 85:15 DHLAPEG600-COOH/DHLA-PEG750-OMe ligands and covalently conjugated to increasing molar ratios of Texas Red-labeled substrate peptide. Samples excited at 350 nm. (C) Proteolytic assay data from exposing a constant concentration of 550 nm emitting QDs conjugated to 4 Texas Red substrate peptides to a constant concentration of caspase 3 enzyme. Derived Km and Vmax values are given. An R² = 0.98 was obtained for the fitting of the curve. (D) Representative, superimposed, and deconvoluted spectra collected from 580 nm emitting QD donors self-assembled with increasing CaRbCl-acceptor labeled peptides. Samples were excited at 350 nm. (E) Normalized acceptor/donor PL area ratios for 580 nm QDs self-assembled with ~2 CaRbCl-acceptor labeled peptides exposed to selected ionic materials. The ratio from the native unexposed sensor was set to an initial value of 1 for comparison purposes (Prasuhn et al., 2010). Reproduced with permission from Prasuhn et al. (2010), Copyright 2013.

Figure 7

Gold glyconanoparticles. (A) Strategy for studying carbohydrate ± carbohydrate interactions based on 2D and 3D models that mimic carbohydrate presentation at the cell surface. Preparation of (B) glyconanoparticles; (C) hybrid glyconanoparticles; (D) fluorescence glyconanoparticles (Barrientos et al., 2003). Reproduced with permission from Barrientos et al. (2003), Copyright 2013.

Figure 8

EDC coupling reaction. The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is a zero-length crosslinking agent used to couple carboxyl groups to primary amines. In the presence of N-hydroxysulfosuccinimide (Sulfo-NHS), EDC can be used to convert carboxyl groups to amine-reactive Sulfo-NHS esters. The addition of Sulfo-NHS stabilizes the amine-reactive intermediate by converting it to an amine-reactive Sulfo-NHS ester, thus increasing the efficiency of EDC-mediated coupling reactions. Excess reagent and crosslinking by products are easily removed by washing with water. Once EDC is water soluble, the crosslinking can be done under physiologic conditions without adding organic solvent.

Figure 9

Highly active magnetic nanoparticle-antibody conjugates. (A) Two-step immobilization mechanism proposed when using Ab that bind to the MNPs through the most reactive amines—random immobilization. (B) Covalent attachment via the polysaccharide moieties of the antibody to the MNPs—oriented immobilization. (C) Capacity to capture HRP of the anti-HRP anchored to MNPs by different orientations. The protein content of all anti-HRP-functionalized MNPs was similar (2 μg Ab per mg MNP) (Puertas et al., 2011). Reproduced with permission from Puertas et al. (2011), Copyright 2013.

Figure 10

Maleimide coupling reaction. Maleimide reacts with free sulfhydryl group(s), forming stable thioether linkages, at physiological pH. It is useful for bioconjugation of proteins with −SH groups and the coupling of two thiols to form a disulfide linkage.

Figure 11

Click chemistry reaction. The copper-catalyzed cycloaddition of azides and alkynes (CuAAC) developed for click chemistry joins an organic azide (N₃) and alkyne together producing a mixture of 1,4- and 1,5-triazoles.

Figure 12

Ionic coupling. Coupling of negatively charged siRNA (left) and of positively charged proteins (right) to negatively or positively charged AuNPs.

Figure 13

Hydrophobic coupling. Use of amphiphilic polymers as nanoparticle and drug delivery moieties.

Figure 14

Biotin-avidin system. Schematic of inhibition assay method based on the photoluminescence quenching of SA-QDs by Biotin-AuNPs. SA denotes the streptavidin immobilized on the surface of QDs, and Av is the externally added avidin (Feng et al., 2013). Reproduced with permission from Feng et al. (2013), Copyright 2013.