Conjugation Strategies for Nanoparticle Aptamer Conjugates

Covalent conjugation of aptamers to substrates or drug delivery vehicles can be achieved most commonly through succinimidyl ester-amine chemistry that results in a stable amide linkage43,43 or through maleimide-thiol chemistry.23,108,116-120 Potential noncovalent strategies include affinity interactions (i.e., streptavidin-biotin) and metal coordination (i.e., between a polyhistidine tag at the end of the aptamer and Ni+2 chelates with immobilized nitrilotriacetic acid on the surface of the polymer particles). These covalent and noncovalent strategies have been used to immobilize a wide range of biomolecules, including proteins, enzymes, peptides, and nucleic acids to delivery vehicles.

We believe that covalently linked bioconjugates may result in enhanced stability in physiologic salt and pH while avoiding the unnecessary addition of biological components (i.e., streptavidin), thus minimizing immunologic reactions and potential toxicity. For covalent conjugation, the aptamer is typically modified to carry a terminal primary amine or thiol group that is in turn conjugated, respectively, to activated carboxylic acid N-hydroxysuccinimide (NHS) ester or malei-mide functional groups present on the surface of drug delivery vehicles. These reactions are carried out under aqueous conditions with a product yield of 80-90%.121 One potential difficulty with maleimide-thiol chemistry is the oxidation of the thiol group attached to aptamers during storage (formation of S-S bond between two thiol-modified aptamers), resulting in dimers of aptamers that are not able to participate in the conjugation reaction with the malimide group on particles. This problem can be partially alleviated by using a reducing agent, such as tris(2-carboxyethyl)phosphine (TCEP), b-mercaptoethanol, or dithiothreitol (DTT), during the conjugation reaction. A potential advantage of using NHS-amine chemistry is that the unreacted carboxylic acid groups on the particle surface make the particle surface charge (zeta potential) slightly negative, thus reducing nonspecific interaction between the negatively charged aptamers and the negative particle surface. Recently, controlled-release nanoparticles generated from PLA-PEG block copolymer with a terminal carboxylic acid group attached to the PEG were conjugated with primary-amine-terminated aptamers.23,103 In this case, the hydrophilic PEG group facilitated the presentation of the carboxylic acid on the particle surface for conversion to activated carboxylic acid NHS ester and conjugation to the primary-amine-modified aptamers.

The conjugation of aptamers to nanoparticles can be qualitatively confirmed by fluorescent microscopy or flow cytometry through the use of fluorescent probes, such as fluorescein isothio-cyanate (FITC), that are conjugated directly to the aptamers or indirectly to complementary oligonucleotides that hybridize to the aptamers.23 Alternatively, analytical approaches, such as x-ray photoemission spectroscopy (XPS), may be used for characterization of the nanoparticle surface to confirm the extent of conjugation. The presence of a hydrocarbon spacer group between the nanoparticle surface and the aptamer should improve the probability of interaction between the aptamer and its target. Furthermore, a consistent density of the aptamer on the surface of nanoparticles can potentially be achieved by utilizing an excess molar amount of aptamer relative to the reactive group on the nanoparticle surface during the conjugation reactions. However, the optimal density of targeting molecule on nanoparticle surface may need to be experimentally determined.122 We have used the covalent conjugation approach to demonstrate a proof-of-concept for nano-particle-aptamer bioconjugates that target the PSMA on the surface of prostate cancer cells and are taken up by cells that express the PSMA protein specifically and efficiently.23 We have also shown using a microfluidic system that these nanoparticle-aptamer conjugates are capable of binding to their target cells under flow conditions suggesting their suitability for in vivo targeted drug-delivery applications.108 Most recently, we have demonstrated the in vivo efficacy of docetaxel-encapsu-lated nanoparticle-aptamer conjugates using a xenograft prostate cancer nude mouse model.14 These approaches have paved the way for the use of aptamers for targeted delivery of drug encapsulated nanoparticles to a myriad of human cancers.

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