The effect of hyperbranched polyglycerol coatings on drug delivery using degradable polymer nanoparticles

Abstract

A key attribute for nanoparticles (NPs) that are used in medicine is the ability to avoid rapid uptake by phagocytic cells in the liver and other tissues. Poly(ethylene glycol) (PEG) coatings has been the gold standard in this regard for several decades. Here, we examined hyperbranched polyglycerols (HPG) as an alternate coating on NPs. In earlier work, HPG was modified with amines and subsequently conjugated to poly(lactic acid) (PLA), but that approach compromised the ability of HPG to resist non-specific adsorption of biomolecules. Instead, we synthesized a copolymer of PLA-HPG by a one-step esterification. NPs were produced from a single emulsion using PLA-HPG: fluorescent dye or the anti-tumor agent camptothecin (CPT) were encapsulated at high efficiency in the NPs. PLA-HPG NPs were quantitatively compared to PLA-PEG NPs, produced using approaches that have been extensively optimized for drug delivery in humans. Despite being similar in size, drug release profile and in vitro cytotoxicity, the PLA-HPG NPs showed significantly longer blood circulation and significantly less liver accumulation than PLA-PEG. CPT-loaded PLA-HPG NPs showed higher stability in suspension and better therapeutic effectiveness against tumors in vivo than CPT-loaded PLA-PEG NPs. Our results suggest that HPG is superior to PEG as a surface coating for NPs in drug delivery.

Keywords: Camptothecin; Hyperbranched polyglycerol; Nanoparticles; Polylactic acid; Surface coating.

Fig. 1

Characterization of NPs using TEM and DLS. The size distribution and TEM image of NPs comprising PLA-HPG (a), PLA-HPG/CPT (b), PLA-PEG (c) and PLA-PEG/CPT (d). The size of NPs was analyzed by image J on lower resolution images with much larger population of NPs and plotted as histograms. The scale bar represents 200nm. (e) Z-average hydrodynamic diameter of NPs measured by DLS.

Fig. 2

Release profile and emulsion stability of PLA-HPG/CPT and PLA-PEG/CPT NPs. (a) Controlled release profile of the PLA-HPG/CPT and PLA-PEG/CPT NPs. Data are shown as mean ± SD. (b) Emulsion appearance of the PLA-HPG, PLA-HPG/CPT and PLA-PEG/CPT NPs (from left to right) at different time points 30min, 1day and 10 days.

Fig. 3

Cellular uptake of PLA-HPG, PLA-PEG and PLA NPs detected by confocal microscopy. Macrophage derived from U937 were incubated with NPs overnight. The colors of the NPs and nuclei are red and blue respectively. The scale bar represents 25 μm.

Fig. 4

In vitro cytotoxicity of CPT loaded NPs. (a) Cell viability of LLC cells incubated with free CPT, PLA-HPG/CPT and PLA-PEG/CPT NPs for 3 days. (b) Cell viability of LLC cells incubated with PLA-HPG and PLA-PEG NPs for 3 days.

Fig. 5

Comparison of PLA-HPG and PLA-PEG NPs in vivo. (a) Blood concentration of DiD loaded PLA-HPG NPs, PLA-PEG NPs recorded as a function of time after intravenous administration. The fluorescence of NPs was converted to dose and the dose was plotted against the time point of the blood collection and fitted with a two-compartment model (a, inset). Data are shown as mean ± SD (n = 7). Biodistribution of DiD loaded PLA-HPG and PLA-PEG NPs after intravenous administration recorded as percent dose per gram tissue at 12h (b) & 24h (c) and as dose per organ at 12h (d) & 24 h (e). Data are shown as mean ± SD (n = 6).

Fig. 6

Histological study of the DiD loaded NPs in tumors. Tumor cryosections 12 hours after injection of PLA-HPG NPs, PLA-PEG NPs, and PBS were visualized in Dapi, GFP and Cy5 channels at 20x (420 μm × 320 μm). The colors of the NPs, blood vessels and nuclei were red, green and blue respectively.

Fig. 7

Tumor growth inhibition and animal body weight change with therapeutic treatments. (a) Tumor growth on LLC bearing C57BL/6 mice treated with PBS, PLA-HPG NPs, free CPT, PLA-HPG/CPT and PLA-PEG/CPT NPs at 7 and 11 day after tumor inoculation. Data are shown as mean ± SD (n = 7). (b)The body weight of the mice after receiving the therapy.