PLGA nanoparticle--peptide conjugate effectively targets intercellular cell-adhesion molecule-1

Abstract

Targeted delivery of therapeutics possesses the potential to localize therapeutic agents to a specific tissue as a mechanism to enhance treatment efficacy and abrogate side effects. Antibodies have been used clinically as therapeutic agents and are currently being explored for targeting drug-loaded nanoparticles. Peptides such as RGD peptides are also being developed as an inexpensive and stable alternative to antibodies. In this study, cyclo(1,12)PenITDGEATDSGC (cLABL) peptide was used to target nanoparticles to human umbilical cord vascular endothelial cell (HUVEC) monolayers that have upregulated intercellular cell-adhesion molecule-1 (ICAM-1) expression. The cLABL peptide has been previously demonstrated to possess high avidity for ICAM-1 receptors on the cell surface. Poly( dl-lactic-coglycolic acid) nanoparticles conjugated with polyethylene glycol and cLABL demonstrated rapid binding to HUVEC with upregulated ICAM-1, which was induced by treating cells with the proinflammatory cytokine, interferon-gamma. Binding of the nanoparticles could be efficiently blocked by preincubating cells with free peptide suggesting that the binding of the nanoparticles is specifically mediated by surface peptide binding to ICAM-1 on HUVEC. The targeted nanoparticles were rapidly endocytosed and trafficked to lysosomes to a greater extent than the untargeted PLGA-PEG nanoparticles. Verification of peptide-mediated nanoparticle targeting to ICAM-1 may ultimately lead to targeting therapeutic agents to inflammatory sites expressing upregulated ICAM-1.

Figure 1

Scanning electron micrograph of PEMA-coated PLGA nanoparticles.

Figure 2

Fluorescent intensity increased in HUVEC as nanoparticle concentration increased. cLABL peptide modified PLGA nanoparticles binding time was one hour, followed by washing and incubation for 2 hours prior to cell lysis.

Figure 3

Fluorescent intensity of nanoparticles associated with HUVEC was significantly higher for targeted cLABL nanoparticles for short binding times (p<0.001 for 10 min and 30 min). Nanoparticle binding time was followed by washing and incubation for 2 hours prior to cell lysis.

Figure 4

Fluorescent micrographs demonstrate that the binding of PLGA nanoparticles mediated by cLABL occurred rapidly. Significant localized fluorescence was noted at 15 minutes for cLABL nanoparticles; however, PLGA-PEG nanoparticles required >2 hours to demonstrate similar localization to cells. No significant differences in fluorescence were observable after 4 hours. Nanoparticle binding time was 10 minutes, followed by washing and incubation for 2 hours prior to cell lysis or imaging.

Figure 5

Nanoparticle binding was studied at 4°C to mitigate energy dependant endocytic pathways. Binding of cLABL nanoparticles was still apparent at low temperature Nanoparticle binding time was one hour, followed by washing and incubation for 2 hours prior to cell imaging.

Figure 6

Fluorescent microscopy was utilized to assess the colocalization of nanoparticles (green) with lysosomes using Lysotracker (red). Images confirm the rapid binding of cLABL nanoparticles. In addition, cLABL nanoparticles rapidly trafficked to the lysosome with colocalization visible within 15 minutes. PLGA-PEG nanoparticles began to appear in lysosomes after two hours. Nanoparticle binding time was 10 minutes, followed by washing and incubation for different time prior to cell imaging.

Figure 7

Lysosomes were labeled with fluorescent dextran (red) to confirm results obtained with Lysotracker using cLABL nanoparticles. Again, nanoparticles (green) rapidly colocalized with lysosomes and remained for about 4–6 hours. For times >4–6 hours, colocalization was no longer as apparent. Nanoparticle binding time was 10 minutes, followed by washing and incubation for different time prior to cell imaging.

Figure 8

Yellow pixels signifying colocalization of nanoparticles (green) with lysosomes (red) were quantified over time demonstrating the rapid and transient localization of cLABL nanoparticles (circles) with lysosomes. Relatively fewer PLGA-PEG nanoparticles transitioned to lysosomes at later times (>2–4 hours).

Figure 9

(A) Fluorescence spectrophotometry was used to quantify ICAM-1 expression in response to INF-γ and normalized to basal ICAM-1 expression (blank). (B) Fluorescent spectrophotometry demonstrated that fluorescent intensity increased in HUVEC as IFN-γconcentration increased (p<0.001 for 1.25 U/μL and 2U/μL). Nanoparticle binding time was one hour followed by washing and incubation for 2 hours prior to cell lysis or imaging.

Figure 10

Fluorescent spectrophotometry demonstrates that binding of cLABL nanoparticles was inhibited by pre-incubating HUVEC with free peptide (p<0.001 for 0.5 U/μL and 2 U/μL). PLGA-PEG nanoparticle binding was unaffected. Nanoparticle binding time was 1 hour, followed by washing and incubation for 2 hours prior to cell lysis or imaging.