Folate Binding Protein as a Therapeutic Natural Nanotechnolog

Serum proteins interact with small molecules and nanoparticles in blood, resulting in protein coronas. Protein coronas influence the bioidentity of the molecules and nanoparticles, playing critical roles in biotransport, uptake, and fate. Targeted therapeutics are often "tagged" for removal or sequestration before reaching their intended tissues.;The research presented here is focused on characterizing and taking advantage of a particular protein corona -- the self-aggregation of serum folate binding protein (FBP). FBP is derived from cellular folate receptors, and both bind strongly to folic acid (FA), as well as the antifolate drug methotrexate (MTX). FA has been explored as a targeting agent because folate receptors are overexpressed on many human cancers. Translation to the clinic of FA-targeted therapeutics has been challenging because the interaction of FBP with these materials has not been fully understood or appreciated: therapies tested in vivo are likely to operate by different mechanisms than those predicted by in vitro experiments in the absence of soluble FBP.;Using atomic force microscopy (AFM), we characterized the self-aggregation of FBP on a particle-by-particle basis at physiological concentrations. FBP self-aggregates into nanoparticles (forming FBPNP) at blood serum concentrations. We further explored the effect of concentration and ligand on the aggregation process. The introduction, and subsequent binding to FBP, of FA, MTX, or leucovorin (LEUC, a FA rescue agent) disrupted existing FBPNP, in most cases inducing reaggregation into new FBPNP. Healthy concentrations of FA and therapeutically relevant concentrations of LEUC produced FBPNP distributions that were not statistically different. This provided a new hypothesis for the perplexing phenomenon that LEUC must be used for FA rescue because high-dose FA itself provides no therapeutic benefit. FBPNP with therapeutic levels of FA or MTX had similar distributions, both of which were significantly different from LEUC-FBPNP. We postulated the degree of FBP aggregation acts as a signaling mechanism and dictates uptake of ligated species.;We studied FBP aggregation with two FA-poly(amidoamine) (PAMAM) dendrimers and two FA-poly(ethylene glycol) (PEG) conjugates. Fluorescence spectroscopy experiments showed that FA and FA-conjugates induced conformational changes throughout the protein population, even with an excess of FBP. Using AFM, we demonstrated the PAMAM conjugates produced large aggregates at sub-stoichiometric concentrations. PAMAM-FA-FBPNP and FA-FBPNP have different distributions, suggesting that translation of targeted conjugates has been challenging because FBP does not traffic the conjugates like free FA. PEG should be used with caution because it disrupted healthy FBP aggregation, potentially inducing artificial folate deficiency.;Given the challenges associated with conjugated therapies, we sought to leverage FBP itself as a targeted vector. We hypothesized that pre-binding FBP to MTX would decrease toxicity and increase therapeutic efficacy. We tested this hypothesis in a KB xenograft tumor model in mice. Surprisingly, FBP alone inhibited tumor growth as compared to saline control and free MTX. This is the first time this therapeutic effect of FBP has been reported. We postulate the excess unbound FBP resulted in folate starvation of the tumors. The groups treated with MTX and FBP also showed inhibition of tumor growth, but toxicity increased with FBP concentration. It is likely that instead of specifically targeting cancer cells, FBP facilitated widespread uptake of MTX, resulting in systemic toxicity. The substantial reduction observed in tumor volume suggests that FBP alone could be employed as a chemotherapeutic. Future work should be focused on exploring this exciting possibility.