Metabolite Responsive Nanoparticle-Protein Complex

Abstract

Stimuli-responsive polymers are an efficient means of targeted therapy. Compared to conventional agents, they increase bioavailability and efficacy. In particular, polymer hydrogel nanoparticles (NPs) can be designed to respond when exposed to a specific environmental stimulus such as pH or temperature. However, targeting a specific metabolite as the trigger for stimuli response could further elevate selectivity and create a new class of bioresponsive materials. In this work we describe an N-isopropylacrylamide (NIPAm) NP that responds to a specific metabolite, characteristic of a hypoxic environment found in cancerous tumors. NIPAm NPs were synthesized by copolymerization with an oxamate derivative, a known inhibitor of lactate dehydrogenase (LDH). The oxamate-functionalized NPs (OxNP) efficiently sequestered LDH to produce an OxNP-protein complex. When exposed to elevated concentrations of lactic acid, a substrate of LDH and a metabolite characteristic of hypoxic tumor microenvironments, OxNP-LDH complexes swelled (65%). The OxNP-LDH complexes were not responsive to structurally related small molecules. This work demonstrates a proof of concept for tuning NP responsiveness by conjugation with a key protein to target a specific metabolite of disease.

Figure 1.

Lactate responsive nanoparticle. An inhibitor for LDH is incorporated into the NP. When LDH is introduced, the inhibitor acts as a cross-linker between the particle and protein leading to a decrease in NP size. Once the complex is introduced to lactate, the inhibitor is displaced and the cross-linking is eliminated. This leads to a swelling of the particle.

Figure 2.

Quantification of LDH uptake by NPs. Centrifugation was performed to pull down NP–LDH complexes leaving the unbound protein free in supernatant. Bars represent amount of LDH leftover in supernatant. LDH Control corresponds to the maximum amount of LDH added to solutions. NIPAm NP Control represents NIPAm NPs without oxamate inhibitor incubated with LDH. OxNP represents NIPAm NPs containing the oxamate inhibitor. The NIPAm NP Control showed no affinity for LDH, while the OxNP demonstrated affinity for LDH.

Figure 3.

CD Measurements of OxNPs and LDH. CD was performed on OxNP alone and showed no signal. LDH was measured alone as a baseline. OxNP–LDH complexes showed no significant difference from the baseline LDH. After one heat cycle corresponds to measurements of OxNP–LDH complexes after being heated at 37 °C for 10 min followed by cooling and measurement. Each set of conditions demonstrated no denaturation occurring.

Figure 4.

Change in volume of OxNP exposed to small molecules at 25 and 37 °C. All small molecules had a concentration of 10 mM. Pyruvic acid and oxalic acid not shown.

Figure 5.

Change in volume of OxNP–LDH complexes exposed to small molecules at 25 and 37 °C. Low lactic acid concentrations mirroring those found in healthy tissues (1 mM) do not cause a change in volume. High lactic acid concentrations (10 mM) cause a significant change in volume of OxNP–LDH complexes. The box highlights the data point correlated to a large change in volume at 37 °C. Other small molecules (10 mM) elicited no effect on OxNP–LDH complex volume.

Figure 6.

SEM images of OxNPs. OxNPs alone (a); OxNPs with LDH (b); OxNPs with LDH and lactic acid (c).

Figure 7.

CryoTEM images of OxNPs and OxNP–LDH complexes. Image of OxNPs cluster (a); Image of OxNPs with protein bound (b); Magnification of blue box from (b), showing discrete LDH particles (c); Plot profile of selected protein particles (d).

Scheme 1.

Synthetic Pathway to Polymerizable Oxamate Derivative 6

Scheme 2.

NP Synthesis Utilizing 2 mol % Polymerizable Oxamate Inhibitor, 96 mol % NIPAm, and 2 mol % Bis