(M0930-07-38) Immune Cells Activating Biotin-Decorated PLGA Protein Carrier

Purpose: Nanoparticle formulations have long been proposed as protein carriers and immune modulators owing to their ability to entrap proteins as well as possessing immunostimulatory or immunosuppressant properties. Poly(lactic-co-glycolic acid) (PLGA) remains one of the most studied polymers for controlled release and nanoparticle drug delivery, and numerous studies exist proposing PLGA particles as protein carriers. In this work we report using PLGA nanoparticles modified with biotin (bNPs) to deliver proteins via adsorption, and stimulate professional antigen-presenting cells (APCs).

Methods: Biotin was incorporated into PLGA microparticles using DSPE-PEG-biotin at a ratio of 10:1 PLGA: DSPE-PEG-biotin. bMPs were manufactured using solvent evaporation emulsification with dichloromethane as the organic phase containing PLGA and DSPE-PEG-biotin and 1% polyvinyl alcohol in water as the aqueous phase. Fluorescently tagged bMPs were manufactured in the same manner with the addition of Nile Red in the organic phase. Particle physical properties were evaluated by scanning electron microscopy (SEM) for particle shape, and dynamic light scattering (DLS) for diameter, dispersity index and zeta potential. Surface accessible biotin on the particles was quantified by a pulldown assay using 800CW streptavidin by the ratio of fluorescent protein removed from the supernatant to that of blank PLGA particles. Binding of bCAT to bMP with an avidin linker was confirmed by incubation of bCAT with avidin-coated bMPs. The incubated particles were centrifuged at 11k rpm, and the supernatants analyzed for CAT/ bCAT activity using UV-Vis as described above. bMPs coated with avidin and bCATs were characterized by DLS for diameter and zeta potential. Rat alveolar macrophages (RAMs) and mouse splenic cells (harvested from adult BALB/c mice) were incubated for 1 hour with bNPs. The supernatant was analyzed for cytokine concentration using ELISA. To determine the impact of biotin on cytokine response, bNPs were pre-incubated with avidin, bovine serum albumin (BSA) or dissolved using 1N NaOH. bNPs containing a near infrared dye (NIR815) were coated with near infrared streptavidin (Streptavidin 680RD) were injected subcutaneously into C57 mice and 28 days later the popliteal draining lymph nodes and spleens were collected from each mouse and imaged at 700 nm and 800 nm using an Odyssey Imager.

Results: Surface accessible biotin bound both biotinylated catalase (bCAT) through avidin as well as streptavidin-horse radish peroxidase (HRP). Analysis of the HRP found that activity on the bNPs was preserved once captured on the surface of bNP (Figure 1). Further, bNPs were found to have self-adjuvant properties, evidenced by bNP induced IL-1β, IL-18, and IL-12 production in vitro in APCs, thereby licensing the cells to generate T helper type 1 (Th1)-type T cell responses. Th1 responses have favorable indications in immunization, infectious disease, cancer and in early wound healing. In fact, mixed lymphocytes from mice primed with ovalbumin (OVA) and bNPs ex vivo responded to restimulation better than OVA alone or plain PLGA nanoparticles+ OVA (Figure 2). For bNPs, we found that we can modulate cytokine production by blocking biotin on the particle surface. Indeed, cytokine production was reduced in avidin pre-coated bNPs (but not with other proteins) suggesting that the pro-inflammatory response is specific to exposed biotin on the surface of bNPs. bNPs and avidin were also shown to colocalize to the spleen and draining lymph nodes in mice for up to 28 days when injected subcutaneously into footpads (Figure 3).

Conclusion: These data provide evidence that bNPs should be explored further as potential adjuvanting carriers for proteins across a wide variety of conditions in which a biased Th1 type response is desired.


Physicochemical properties of bNP. (A) Intensity vs size plots of control NP (○), 100:1 bNP (●) and 10:1 bNP (●). (B) Zeta-potential plots of 10:1 bNP (●), bNP +avidin (●), and bNP +avidin +bCAT (●). (C) SEM micrograph of 10:1 bNP. (D) Streptavidin 800 CW remaining in supernatant following incubation with 10:1 bNPs (■), 100:1 bNPs (■) and control NPs (□), as determined by fluorescence (774/795 ex/em). (E) Intensity vs size plots of bNP (●), bNP +avidin (●), and bNP +avidin +bCAT (●). (F) Activity of bCAT (●) as compared to CAT (○) remaining in supernatant following incubation with bNPs. (G) Activity of streptavidin-HRP determined by TMB consumption. (H) SDS-PAGE electrogram of pellet (-) and supernatant of avidin-coated 10:1 bNP vs control NP +avidin, and PBS +avidin following trypsin incubation. *, **, ***, ****, ns, p < 0.05, < 0.01, < 0.001, < 0.0001 and not significant, respectively, by 1-way ANOVA.


bNP Activation of Antigen Presenting Cells (APC). IL-1β (A) and IL-18 (B) release from RAMs incubated with bNPs for 1 hour. IL-1β (C), IL-12 (D), IL-4 (E) and IFN-g (F) release from BALB/c mice splenic cells following bNP treatment with and without avidin. IFN-g release from mouse splenic cells 24 hours after priming with OVA treatment, with or without bNP (G) and 48 hours after restimulation with OVA (H). There was no significant difference between the two doses and so both doses were pooled in the final analysis. *, **, ***, ****, ns, p < 0.05, < 0.01, < 0.001, < 0.0001 and not significant, respectively, by 1-way ANOVA.


Colocalization of bNP and Avidin in vivo. NIR images of spleens from mice injected with bNPs coated with streptavidin, bNPs or streptavidin (A). NIR images from popliteal DLNs from injection side and non-injection side after treatment with streptavidin bNPs, bNPs or streptavidin (B). The 700 nm and 800 nm panels were from the same lymph nodes or spleen.