Cyclodextrin Nanoparticles Enable Macrophage-Targeted Small Molecule Drug Delivery

Introduction:: Small molecule immunotherapeutics are readily available to treat a myriad of chronic inflammatory diseases, ranging from cancer to heart disease. Their efficacy, however, remains limited by poor solubility, rapid blood clearance, and off-target effects.¹ We have therefore developed cyclodextrin nanoparticles (CDNPs), nanocarriers composed of the macrocyclic host β-cyclodextrin (CD), to improve drug solubility and bioavailability.² Due to its saccharide composition, CDNPs are readily uptaken by macrophages, crucial regulators of the immune microenvironment. Macrophages exist across a spectrum of phenotypes broadly characterized as M1-like (inflammatory) and M2-like (reparatory), the former of which underlie chronic inflammation and failed tissue healing.³ Here, we aim to develop macrophage-targeted therapeutics for M1àM2 re-education that would have a wide range of applications in the treatment of tissue injury or chronic inflammatory diseases.

Materials and Methods:: To reflect the injury environment, we developed a TLR2-driven model of sterile inflammation for drug screening. This two-step screening process was used to examine a library of small-molecule drugs spanning a variety of drug classes to i) identify inhibitors of inflammatory macrophages in RAW-Blue^TM reporter cell lines and ii) identify drugs that promote a reparatory phenotype via large-scale transcriptional analysis (qPCR, nanoString). CDNPs of varying diameter were prepared by EDC/NHS crosslinking of succinylated CD with lysine as previously reported,⁴ and uptake of fluorescently labeled CDNPs was monitored over time in RAW264.7 cells. The biological mechanism (i.e., receptor-mediated endocytosis, phagocytosis) through which CDNPs are uptaken was investigated by pharmacological inhibition. A reporter assay was used to confirm the anti-inflammatory efficacy of the drug loaded nanoparticle in vitro, subsequent to confirming celastrol interaction with CD by surface plasmon resonance (SPR).

Results, Conclusions, and Discussions:: Of 50 small molecule drugs examined, 6 robustly suppressed M1-like activation in initial screens ( >50% suppression, >95% cell viability). Further scrutiny of these hits revealed that only celastrol robustly suppressed the M1-like phenotype while concurrently upregulating M2-associated genes by as much as 10-fold (Fig. 1A). These findings were further confirmed by nanoString analysis in primary murine and human cells (data not shown).⁵ CDNPs of varying diameters were prepared by altering the ratio of EDC/NHS catalyst relative to succinyl reactive sites for amine crosslinking (Fig. 1B). All CDNPs exhibited rapid uptake ( >50% in 6 hrs); though, increasing CDNP size was associated with more rapid uptake kinetics and a greater degree of CDNP accumulation at 24 hrs (Fig 1C). The largest CDNP (z-avg diameter = 123.3±4.3 nm) was therefore selected for continued study. Antibody blocking of MRC1 and SR-A1 reduced CDNP uptake, indicating that receptor-mediated endocytosis directly mediates macrophage-targeting. Pharmacological inhibition of phagocytosis and macropinocytosis further reduced uptake (Fig. 1D). The ability for CDNPs to act as a reservoir for sequestration of celastrol through supramolecular guest-host interactions was confirmed by SPR (K_eq=0.474 mM). Finally, celastrol loading did not inhibit CDNP uptake (Fig . 1E), but drug-loaded CDNP (Cel-CDNP) treatment in vitro showed dose-dependent knockdown of NF-κB and AP-1 mediated inflammatory signaling that was moderately improved relative to soluble drug alone (Fig. 1F).

Drug screens identified celastrol as a potent immunomodulatory agent, which can inhibit the damaging M1-like macrophage phenotype and promote a reparatory M2-like phenotype. CDNPs of larger size accelerate intracellular nanoparticle accumulation, through both receptor-mediated endocytosis and phagocytosis/macropinocytosis. Cel-CDNP demonstrates significant knockdown of inflammatory signaling in vitro and is a promising macrophage-targeted therapeutic to modulate the post-injury immune environment or treat chronic inflammation.


Acknowledgements (Optional): :

References (Optional): : 1) Geldenhuys, Res Rev Drug Deliv (2017). 2) Gadade, Adv Pharm Bull (2020). 3) Martin, Acta Biomater (2021). 4) Rodell, Nat Biomed Eng (2018). 5) Soni, Biomat Sci (2022).