To be useful as nanoparticle formulations, however, they must be stable Three key observations emerge from this study. and false-negatives in cell-based assays.5C7 The formation of colloidal particles has been reported for thousands of compounds (http://advisor.bkslab.org),8 including those from screening libraries and from clinically used drugs, such as anticancer, cardiovascular, and antiretroviral therapeutics.6,9C13 While these aggregates are a serious nuisance for early drug SSR 69071 discovery, they have potentially interesting properties as drug formulations. Colloidal aggregates have many aspects desired for delivery as the self-assembly of these compounds leads to defined nanoparticles composed entirely of the active molecule. To be used for drug delivery, the aggregates must be stable found that many biopharmaceutics classification system (BCS) class II and IV drugs form colloidal aggregates in simulated intestinal fluid, suggesting colloid formation could play a role in drug formulation and bioavailability.14 Recently, Wilson demonstrated that colloids formed from amorphous solid dispersions can act as reservoirs and enhance plasma drug exposure after oral delivery.15 Frenkel found that orally administered colloid-forming non-nucleoside reverse transcriptase inhibitors were directed to the lymphatic circulation.16 The presence of proteins can further impact colloidal drug transport. For example, Owen demonstrated that colloids do form in standard cell culture conditions (10% serum) and observed that colloidal chemotherapeutics did not cross into cells, resulting in an apparent loss in drug cytotoxicity.17 Efforts to exploit and study colloidal aggregates in high protein milieus have been hindered by their transient stability. Even in biochemical buffers, most small molecule aggregates are only transiently stable, often flocculating and precipitating over several hours. Recently, strategies to stabilize colloidal particles under physiologically relevant conditions have been developed. Co-aggregation with polymeric surfactants, azodyes, or protein coronas all stabilized drug colloids over many days in buffered and serum-containing media.18C20 Colloids of the estrogen receptor antagonist, fulvestrant, and the investigational anthracycline prodrug, pentyloxycarbonyl-(conditions, their stability in biomimetic, high serum conditions remains unknown, largely because the methods to even measure such stability have been unavailable. We describe a new method to measure the critical aggregation concentration in high-serum content media and demonstrate that fundamental drug colloid properties, such as those that dictate the onset of aggregation, are significantly changed under drug circulation properties and increase the plasma half-life compared to monomeric formulations. RESULTS AND DISCUSSION Few techniques are available to probe the integrity of amorphous nanostructures in complex media. In biochemical buffers, drug colloids can readily be defined by dynamic light scattering (DLS); however these techniques are ineffective in the presence of serum due to scattering from serum proteins themselves, which is only further complicated as serum content is increased.21 Alternatively, FRET pairs can be SSR 69071 absorbed into the self-assembled colloids, where they can report on their gross structural integrity.20,22,23 Accordingly, we designed such a strategy to study colloidal drug aggregate stability in serum-containing media Cholesterol-modified BODIPY dyes can be readily incorporated during colloid formation due to the hydrophobic and amorphous nature of drug aggregates.4,20 These dyes have substantial fluorescence intensity within drug colloids but have very low intensity when not associated with a drug aggregate or when colloids are disrupted with detergents (Figure S2). Thus, we investigated the presence and stability of colloidal aggregates of fulvestrant, in high-serum conditions exploiting the fluorescence intensity changes of a BODIPY FRET pair. We first investigated the effects of dilution and media composition on the critical aggregation concentration of fulvestrant. In protein-free media, many colloid-forming compounds, including fulvestrant, aggregate at low micromolar concentrations, as measured by light scattering (Figure S3).9 To measure the CAC of fulvestrant in serum-containing media, colloids were formulated with 10 0.0001 between all groups by one-way ANOVA with Tukeys posthoc (= 3, mean SD). Drug colloids, which will ordinarily flocculate and precipitate Rabbit Polyclonal to RPL3 over several hours, require a stabilizing excipient to remain in buffer and serum-containing media for longer times.9 We investigated the role of excipients in stabilizing fulvestrant colloids in high-serum conditions, which mimic the environment. Here, using the same hydrophobic dyes, which lose fluorescence intensity as they become released when the colloids disassemble or precipitate,20,25 we measured the stability of colloids in complex protein media. Fulvestrant colloids were formulated at 500 = 3, mean SD). We hypothesized that serum-stable fulvestrant-UP80 colloids would increase the circulation SSR 69071 of fulvestrant compared to a solubilized monomeric form of the drug (Figure 3). As colloid formation can be disrupted by detergents, we used 5% UP80 to.