A Versatile Brij-Linker for One-Step Preparation of Targeted Nanoparticles

Abstract

Background: Most frequently the functionalization of nanoparticles is hampered by time-consuming, sometimes harsh conjugation and purification procedures causing premature drug release and/or degradation. A strategy to circumvent multi-step protocols is to synthesize building blocks with different functionalities and to use mixtures thereof for nanoparticle preparation in one step. Methods: BrijS20 was converted into an amine derivative via a carbamate linkage. The Brij-amine readily reacts with pre-activated carboxyl-containing ligands such as folic acid. The structures of the building blocks were confirmed by different spectroscopic methods and their utility was assessed by one-step preparation and characterization of nanoparticles applying PLGA as a matrix polymer. Results: Nanoparticles were about 200 nm in diameter independent of the composition. Experiments with human folate expressing single cells and monolayer revealed that the nanoparticle building block Brij mediates a "stealth" effect and the Brij-amine-folate a "targeting" effect. As compared to plain nanoparticles, the stealth effect decreased the cell interaction by 13%, but the targeting effect increased the cell interaction by 45% in the monolayer. Moreover, the targeting ligand density and thus the cell association of the nanoparticles is easily fine-tuned by selection of the initial ratio of the building blocks. Conclusions: This strategy might be a first step towards the one-step preparation of nanoparticles with tailored functionalities. Relying on a non-ionic surfactant is a versatile approach as it might be extended to other hydrophobic matrix polymers and promising targeting ligands from the biotech pipeline.

Keywords: PEGylation; folic acid; nanoparticles; non-ionic surfactant; targeted delivery.

Figure 1

Carbonyldiimidazole-mediated activation of the hydroxyl group of BrijS20, coupling of PEG-diamine and acylation with folic acid NHS-ester.

Figure 2

Suggested particle morphology: The hydrophobic stearyl moiety of BrijS20 is inserted into the PLGA particle matrix; the hydrophilic PEG moiety covers the particle surface. The hydroxyl groups can be modified with an amine group (blue square) as an anchor for further molecules and further with folic acid (yellow dot) as a targeting ligand.

Figure 3

(a) Particle size and PdI before and after the lyophilisation process of all particle suspensions prepared; A: plain PLGA-NPs; B: PLGA/BrijS20-NPs; C: PLGA/BrijS20-amine-NPs, D: PLGA/BrijS20-amine-folic acid conjugate-nanoparticles (one-step particle preparation); E: PLGA/BrijS20-amine + folic acid—NPs (post-preparation particle functionalization); (b) particle size and PdI after dispersion in water or isotone HEPES buffer pH 7.4. The size and size distribution of particle suspensions in HEPES—buffer stored at 4 °C were determined after 24 h and after one week.

Figure 4

(a) Zeta potential and amine or folate density of PLGA nanoparticles prepared in one step (PLGA, PLGA/Brij-amine and PLGA/Brij-amine-FA) or stepwise (PLGA/Brij-amine + FA). Poly-d,l-lactide-co-glycolide (PLGA), BrijS20 (Brij), 1,8-diamino-3,6-dioxa-octane (DA), folic acid (FA). Nanoparticle suspensions were prepared with 13 mg Brij-amine or Brij-amine-FA. (b) The density of folic acid on the particle surface correlates with the amount of BrijS20-amine-folate used for nanoparticle preparation.

Figure 5

Cell binding and internalization of targeted and non-targeted nanoparticles in cell suspension assays (a) after incubation for two hours at 4 °C (hatched area) and 37 °C (plain area) and monolayer experiments (b) (KB cells). Cell binding was examined at 4 °C whereas uptake was analyzed at 37 °C. Results of monolayer and cell suspension assays using flow cytometry (FACS) are depicted in relation to the cell-associated fluorescence intensity of unmodified PLGA particles (at 4 °C). ** p < 0.01.

Figure 6

Visualization of the cellular uptake of BODIPY labeled nanoparticles (green); KB-cells were incubated with DMEM (A) or the respective nanoparticle formulation ((B): PLGA-nanoparticles; (C): PLGA/Brij-amine-FA-nanoparticles) for two hours at 37 °C and after fixation in paraformaldehyde with the nucleus stain HOECHST 33342 (blue) and the membrane stain alexa-fluor 488 WGA (red). Scale bar indicates 20 µm.