Bioconjugated β-Cyclodextrin–Perfluorohexane Nanocone Clusters as Functional Nanoparticles for Nanoparticle-Mediated Histotripsy

Abstract

Nanocone clusters (NCCs) are new-generation agents of nanoparticle-mediated histotripsy (NMH) recently developed to address the limitations of previously designed nanodroplets (NDs). NCCs can be obtained by simply mixing FDA-approved cyclodextrins (CD) and suitable perfluorocarbons (PFCs), which result in smaller size aggregates, detectable PFC amount, and more stable long-term storage since the obtained powder can be stored and redispersed as needed. Previous experimental and computational studies showed that NCCs consist of an organization of inclusion complexes of CD and PFC around free PFC droplets, and their aggregate behavior depends on the localization of PFC in the cavity and the water solubility of CD derivatives. It has been shown that β-cyclodextrin (βCD) and perfluorohexane (PFH) are ideal candidates for NCCs that can be isolated as a powder with high PFC content among various CD and PFC derivatives. This study focuses on the further development of the selected NCC composition to enhance the potential of NMH therapy while also enabling more detailed future experiments in vitro and in vivo. It is aimed to show the bioconjugation potential of NCCs through the example of the most commonly used functionalization methods such as targeting, PEGylation, and fluorescent labeling. For this purpose, βCD as a building block was monofunctionalized with groups such as azide, alkyne, and amine groups that allow for effective coupling reactions such as the "click" reaction and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling. These monofunctional βCDs were used as building blocks of NCCs in the presence of PFH to obtain functional NCCs as precursors of bioconjugation. EPPT1 as a synthetic peptide specific to uMUC1 and folic acid (FA) as the most commonly used targeting agent along with PEGylation were successfully shown as bioconjugation examples. Lastly, fluorescently labeled NCCs were obtained via fluorescein isothiocyanate (FITC) and alkyne functional NCC reaction through propargyl amine and isothiocyanate group reaction. The obtained bioconjugates were tested in vitro to validate the conjugation, and the ability to lower the histotripsy cavitation threshold, which is necessary for NMH, was demonstrated for all bioconjugates. Overall, the results showed that all obtained bioconjugates successfully lowered the cavitation threshold pressure while also fulfilling the desired bioconjugation metrics to serve as improved tools to enhance NMH as a targeted noninvasive ablation method.

Investigation of Optimum Production Conditions and the Stability of β-Cyclodextrin–Perfluorocarbon Nanocone Clusters for Histotripsy Applications

“…Moreover, βCD is the most used CD derivative that can still preserve the functionalization potential for future applications. Thus, we recently showed that the functional βCD-based NCC can be an effective agent for bioconjugation for various needs such as targeting, PEGylation, and fluorescence labeling . Herein, this favorable NCC structure was further investigated to understand its stability under conditions that might be faced by NCCs during application, such as physiological conditions and circulation.…”

“…Using these varying conditions, the cavitation threshold and bubble cloud behavior at increasing negative pressures for each formulation was investigated. Mechanically tunable agarose gel tissue phantoms , were used as tissue mimicking phantoms, and protocols were followed as in previous histotripsy studies. , NCC-embedded tissue phantoms with different βCD-PFC molar ratios, 1:1 and 1:5 as samples, were used, and empty phantoms (agarose only) were used as histotripsy controls. Agarose powder (Type VII-A, Sigma-Aldrich, St. Louis, MO, USA) was added into saline solutions and boiled until complete dissolution of agarose was achieved followed by a minimum of 20 cycles of flash boiling.…”