Surface plasmon resonance as a high throughput method to evaluate specific and non-specific binding of nanotherapeutics

Abstract

Surface plasmon resonance (SPR) is a powerful analytical technique used to quantitatively examine the interactions between various biomolecules, such as proteins and nucleic acids. The technique has been particularly useful in screening and evaluating binding affinity of novel small molecule and biomolecule-derived therapeutics for various diseases and applications including lupus medications, thrombin inhibitors, HIV protease inhibitors, DNA gyrase inhibitors and many others. Recently, there has been increasing interest in nanotherapeutics (nanoRx), due to their unique properties and potential for controlled release of encapsulated drugs and structure-specific targeting to diseased tissues. NanoRx offer the potential to solve many drug delivery challenges by enabling, specific interactions between molecules on the surface of the nanoparticle and molecules in the diseased tissue, while minimizing off-target interactions toward non-diseased tissues. These properties are largely dependent upon careful control and balance of nanoRx interactions and binding properties with tissues in vivo. Given the great promise of nanoRx with regard to engineering specific molecular interactions, SPR can rapidly quantify small aliquots of nanoRx formulations for desired and undesired molecular interactions. Moving forward, we believe that utilization of SPR in the screening and design of nanoRx has the potential to greatly improve the development of targeted nanoRx formulations and eventually lead to improved therapeutic efficacy. In this review, we discuss (1) the fundamental principles of SPR and basic quantitative analysis of SPR data, (2) previous applications of SPR in the study of non-particulate therapeutics and nanoRx, and (3) future opportunities for the use of SPR in the evaluation of nanoRx.

Keywords: Biacore; Molecular interactions; Nanotechnology; Surface plasmon resonance (SPR); Targeted therapy.

Figure 1. Schematic of a typical SPR instrument

Arrows indicate the direction of liquid flow through the system. IFC represents the integrated microfluidic cartridge.

Figure 2. Schematic of an SPR sensor chip and chip surface

Schematic shows a typical CM5 sensor chip that is surface modified with a carboxymethyl-dextran layer.

Figure 3. Example SPR sensorgram data displaying the main parts of the sensorgram

Data shown was obtained by running serial dilutions of a single chain variable fragment anti-Fn14 antibody over the surface of a CM5 chip with immobilized recombinant Fn14 extracellular domain.. “A” marks the pre-injection baseline, “B” represents the beginning of the injection, “C” represents the association phase of the sensorgram, “D” represents the end of the injection, and “E” represents the dissociation phase of the sensorgram.

Figure 4. Use of SPR to optimize formulation chemistry of a dendrimeric nanotherapeutic

SPR is used to confirm the proposed advantages (A) of a methotrexate-dendrimer, G5-MTX_n compared to free methotrexate. SPR binding measurements (B, C) revealed that conjugation of Methotrexate to the G5 dendrimer yielded targeted dendrimers with greatly enhanced binding to a folate binding protein immobilized sensor chip compared to a control dendrimer lacking methotrexate (C). The enhanced binding seen in SPR experiments was confirmed in cell-uptake studies (D). Left panel shows cells not expressing folate receptor and the right panel shows cells expressing the folate receptor. Methotrexate-dendrimer conjugate is localized to only the cells positive for the folate receptor. Figure adapted from ref. [64]

Figure 5. SPR evaluation of specific and nonspecific interactions of a targeted nanoparticles

(A) SPR study of specific binding of PEG-coated polystyrene NP with no ITEM4 antibody (CNP, black), a low density of ITEM4 (CNP-ITEM4 (low), red), and a high density of ITEM4 (CNP-ITEM4 (high), blue) to a CM5 chip with immobilized extracellular domain of Fn14 protein. (B) SPR study of non-specific binding of uncoated NP (no PEG, UNP, black), CNP, CNP-ITEM4 (low), and CNP-ITEM4 (high) to a CM5 chip with immobilized mouse brain extracellular matrix proteins. (C) Zoomed view of dotted box in (B). Figure adapted from ref. [9]