Abstract
Conjugation of small molecule agonists of Toll-like receptor 7 (TLR7) to proteins, lipids, or polymers is known to modulate potency, and the physical form or formulation of these conjugates is likely to have a major effect on their immunostimulatory activity. Here, we studied the effect of formulation on potency of a 1,2‑di‑(9Z‑octadecenoyl)‑sn‑glycero‑3‑phosphoethanolamine (DOPE) conjugated TLR7 agonist (DOPE-TLR7a) alongside assessing physical form using Dynamic Light Scattering (DLS), Nanosight Particle Tracking (NTA) analysis and Small Angle X-ray Scattering (SAXS). A very high potency of DOPE-TLR7a conjugate (EC50 around 9 nM) was observed either when prepared by direct dilution from DMSO or when formulated into 400–700 nm large multilamella liposomes containing dimethyldioctadecylammonium bromide salt (DDA) and DOPE. When prepared by dissolution in DMSO followed by dilution in aqueous culture medium, 93 ± 5 nm nanoparticles were formed. Without dilution from solution in DMSO, no nanoparticles were observed and no immunostimulatory activity could be detected without this formulation step. SAXS analysis of the conjugate after DMSO dissolution/water dilution revealed a lamellar order with a layer spacing of 68.7 Å, which correlates with arrangement in groups of 3 bilayers. The addition of another immunostimulatory glycolipid, trehalose‑6,6‑dibehenate (TDB), to DOPE:DDA liposomes gave no further increase in immunostimulatory activity beyond that provided by incorporating DOPE-TLR7a. Given the importance of nanoparticle or liposomal formulation for activity, we conclude that the major mechanism for increased potency when TLR7 agonists are conjugated to macromolecules is through alteration of physical form.
Highlights
The molecular pathways of immune activation both by pathogen associated molecular patterns (PAMP) recognition via pattern recognition receptors (PRR) such as TLR (Medzhitov and Janeway, 1998; Medzhitov et al, 1997; Takeda and Akira, 2004) and by particulate adjuvants for example by nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3)/inflammasome mediated pathways (Eisenbarth et al, 2008) have been elucidated
The Toll-like receptor 7 (TLR7) agonist UC-1V150 was conjugated to the phospholipid DOPE as previously described and noted for their remarkable potency compared to the unconjugated TLR7 agonist (Chan et al, 2009)
Our data shows that the formation of TLR7 agonist containing nanoparticles distinctly modulates pro-inflammatory activity
Summary
The molecular pathways of immune activation both by pathogen associated molecular patterns (PAMP) recognition via pattern recognition receptors (PRR) such as TLR (Medzhitov and Janeway, 1998; Medzhitov et al, 1997; Takeda and Akira, 2004) and by particulate adjuvants for example by nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3)/inflammasome mediated pathways (Eisenbarth et al, 2008) have been elucidated These advances include insight into the immunomodulatory properties of alum, which was thought to rely on a deposition effect to enhance the persistence of presented antigens to antigen presenting cells (APC) (Verdier et al, 2005). The physical form and self-assembly properties of TLR2 stimulatory lipopeptides have recently been studied in much greater detail, revealing that aggregation into micellular or fibrillar structures, has a profound effect on biological activity (Castelletto et al, 2016; Hamley et al, 2014)
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