A solvent-free thermosponge nanoparticle platform for efficient delivery of labile proteins

Abstract

Protein therapeutics have gained attention recently for treatment of a myriad of human diseases due to their high potency and unique mechanisms of action. We present the development of a novel polymeric thermosponge nanoparticle for efficient delivery of labile proteins using a solvent-free polymer thermo-expansion mechanism with clinical potential, capable of effectively delivering a range of therapeutic proteins in a sustained manner with no loss of bioactivity, with improved biological half-lives and efficacy in vivo.

Keywords: Nanoparticles; biologics; solvent-free; therapeutic proteins; thermosponge.

Figure 1

Schematic illustration of a thermosponge nanoparticle (TNP) platform. (a) TNP preparation by a one-step nanoprecipitation method. (b) Solvent-free method of protein-loading into TNPs for efficient delivery of labile therapeutic protein drugs. TNPs can be efficiently loaded with desired proteins in a thermoresponsive manner without organic solvents due to the combination of the thermoresponsive swelling behavior of the Pluronic shell of TNPs at 4 °C and the electrostatic interactions between the absorbed proteins and the PLA core of TNPs. The positively charged and negatively charged PLA cores of TNPs were synthesized using PLA-NH₂ and PLA-COOH, respectively, and were tested for loading of relevant therapeutic proteins such as slightly positively charged proteins [IL-10 (isoelectric point (pI) 7.9) EPO (pI 8.3)] and negatively charged proteins [Insulin (pI 5.3) and hGH (pI 5.2)] in deionized water.

Figure 2

Characterization of TNPs. (a) Hydrodynamic diameters and (b) surface charges of TNPs and therapeutic protein-loaded TNPs. (c) Representative TEM image of TNPs. The scale bar is 500 nm. Inset is a high-magnification image with the scale bar representing 50 nm. (d) Swelling and deswelling behavior of TNPs in response to temperature changes. (e) Loading contents (wt %) of therapeutic proteins (IL-10, EPO, insulin, and hGH) into negatively charged or positively charged TNPs. (f) In vitro cumulative release patterns of therapeutic proteins from TNPs in PBS buffer at 100 rpm and 37 °C, analyzed by ELISA (mean ± SD, n = 3).

Figure 3

Bioactivity of proteins released from TNPs. (a) Inhibitory effects on reactive oxygen species (ROS) production by IL-10 at various concentrations (1–100 ng/mL). Intracellular ROS generated from RAW 264.7 macrophage cells by LPS stimulation was measured using a ROS detection reagent. Bioactivity analysis of the inhibitory effects of native IL-10, released IL-10, and loaded IL-10 on ROS production (b) by pretreatment and (c) by post-treatment of IL-10 (n = 3, * p < 0.05, # p > 0.05). (d) Relative mRNA expression of TNF-α, IL-12, and sIL-1Ra after LPS treatment (500 ng/mL) for 4 h, followed by treatment with IL-10 (native IL-10 or released IL-10 at 20 ng/mL) for 2 h at 37 °C (n = 3, # p > 0.05). (e) Western blots were performed to analyze the bioactivity of IL-10 released from TNPs after treatment with IL-10 (native IL-10 or released IL-10 at 20 ng/mL) for 24 h at 37 °C. #1, control; #2, native IL-10; and #3, released IL-10. (f) Bioactivity analysis of native insulin and released insulin (10 nM) on the improved proliferation effect of insulin-dose-dependent human breast cancer cell line MCF-7 (n = 3, # p > 0.05).

Figure 4

Pharmacokinetics of protein-loaded TNPs. Changes in serum protein levels in mice after intravenous administration of (a) IL-10 and IL-10–loaded TNP, and (b) insulin and insulin–loaded TNP. The serum concentrations of proteins were measured at several time points using ELISA kits (mean ± SEM, n = 3).

Figure 5

In vivo anti-inflammatory efficacy of IL-10-loaded TNPs. (a) Therapeutic efficacy of IL-10 and TNPs on ear swelling in a mouse model of allergic contact dermatitis (ACD) at 100 μg IL-10/kg dose via iv administration. (b) Representative histological images of DNFB-treated ears from IL-10 and IL-10-loaded TNP groups. (c) Total neutrophils (CD11b⁺, Ly-6G^high) in skin at 36 h upon acetone or DNFB challenge. All data are expressed as mean ± SEM of n = 4 to 7 per group. * p < 0.05 for saline vs treatment.