Nanocarrier Lipid Composition Modulates the Impact of Pulmonary Surfactant Protein B (SP-B) on Cellular Delivery of siRNA

Abstract

Two decades since the discovery of the RNA interference (RNAi) pathway, we are now witnessing the approval of the first RNAi-based treatments with small interfering RNA (siRNA) drugs. Nevertheless, the widespread use of siRNA is limited by various extra- and intracellular barriers, requiring its encapsulation in a suitable (nanosized) delivery system. On the intracellular level, the endosomal membrane is a major barrier following endocytosis of siRNA-loaded nanoparticles in target cells and innovative materials to promote cytosolic siRNA delivery are highly sought after. We previously identified the endogenous lung surfactant protein B (SP-B) as siRNA delivery enhancer when reconstituted in (proteo) lipid-coated nanogels. It is known that the surface-active function of SP-B in the lung is influenced by the lipid composition of the lung surfactant. Here, we investigated the role of the lipid component on the siRNA delivery-promoting activity of SP-B proteolipid-coated nanogels in more detail. Our results clearly indicate that SP-B prefers fluid membranes with cholesterol not exceeding physiological levels. In addition, SP-B retains its activity in the presence of different classes of anionic lipids. In contrast, comparable fractions of SP-B did not promote the siRNA delivery potential of DOTAP:DOPE cationic liposomes. Finally, we demonstrate that the beneficial effect of lung surfactant on siRNA delivery is not limited to lung-related cell types, providing broader therapeutic opportunities in other tissues as well.

Keywords: nanoparticles; pulmonary surfactant; siRNA delivery.

Figure 1

Visual representation of the core-shell surfactant-coated nanogel structure. SiRNA-loaded dextran nanogels (siNGs) were coated with Curosurf^® (poractant α; porcine derived clinical pulmonary surfactant (PS)) or with a PS-inspired lipid coating containing the surfactant protein B (SP-B) and an anionic lipid mixture. PC = phosphatidylcholine, PG = phosphatidylglycerol.

Figure 2

Biological efficacy of surfactant-coated nanogels on (non-)pulmonary cell lines. (a,c,e,g) Flow cytometric quantification of cellular uptake of siCy5-loaded nanogels (siNGs) with and without Curosurf^® (CS) coating. (b,d,f,h) Gene silencing potential of siNGs and Curosurf^®- coated NGs (siNGs-CS). Despite the strongly reduced cellular uptake of siNGs following Curosurf^® coating, both formulations reach comparable levels of gene knockdown on the different cell lines studied. Experiments were performed with a fixed NG concentration (30 µg/mL) and siRNA concentration (50 nM), except for the MH-S cell line, for which we used a final siRNA concentration of 100 nM. Experiments on H1299_eGFP and silencing of MH-S cell lines are the result of three independent biological repeats (n = 3), other experiments are performed in technical triplicate.

Figure 3

Impact of lipid phase transition temperature on formation and delivery efficiency of SP-B containing proteolipid-coated nanogels. (a) Evaluation of eGFP silencing in H1299_eGFP cells by uncoated or (proteo)lipid-coated siNGs with different lipid mixtures, supplemented with 0.4 wt% SP-B. All experiments were performed with a fixed NG concentration (30 µg/mL) and siRNA concentration (50 nM). The SP-B effect is strongly influenced by the type of lipid with which it is combined, highlighting the importance of a fluid lipid membrane in the formulation of the core-shell nanocomposites. (b) Chemical structures and phase transition temperatures (Tc) of the different PC lipids tested. Statistical analysis was performed via an unpaired t-test. Data are represented as the mean ± SD (n = 2) and statistical significance is indicated (**** p < 0.0001, ns = not significant).

Figure 4

Impact of cholesterol on biological efficacy of proteolipid-coated nanogels. Evaluation of (a) cellular uptake and (c) gene silencing potential in H1299_eGFP cells of siRNA-loaded nanogels (siNGs) coated with lipid mixtures containing physiological cholesterol (CHOL) levels (2.5, 5 to 10 wt%). Data show one representative (technical triplicate) of two independent experiments; formulations with different siRNA concentrations showed the same trend (data not shown). Evaluation of (b) cellular uptake and (d) gene silencing potential of siNGs coated with increased cholesterol fraction in the outer layer (~25 wt%) (n = 3). Cholesterol exceeding physiological levels partially hinder SP-B promoted siRNA delivery. All experiments were performed with a fixed NG concentration (30 µg/mL) and siRNA concentration (50 nM). LIP = DOPC:PG (85:15); LIP SP-B = DOPC:PG (85:15) + SP-B 0.4 wt%; LIP SP-B CHOL = DOPC:CHOL:PG (60:25:15) + SP-B 0.4 wt%. Data are represented as the mean ± SD and statistical significance is indicated (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001, ns = not significant).

Figure 5

Role of the anionic lipid in biological efficacy of proteolipid-coated nanogels. (a) Cellular uptake and (b) gene silencing evaluated on H1299_eGFP cells via flow cytometry. SiRNA-loaded nanogels (siNGs) were coated with a mixture of DOPC:PG, DOPC:PhS or DOPC:PI (weight ratio 85:15). The presence of negatively charged lipids is required to allow the formation of the core-shell structure via electrostatic interactions. The replacement of the anionic phosphatidylglycerol (PG) with phosphatidylserine (PhS) or phosphatidylinositol (PI) does not abrogate SP-B’s beneficial effect on siRNA delivery. All experiments were performed with a fixed NG concentration (30 µg/mL) and siRNA concentration (50 nM). Statistical analysis was performed via an unpaired t-test. Data are represented as the mean ± SD (n = 3) and statistical significance is indicated (* p < 0.05, ** p < 0.01, **** p < 0.0001, ns = not significant).

Figure 6

Impact of dextran nanogel core structure on SP-B mediated siRNA delivery. (a) Gene silencing potential of (proteolipid-coated) siRNA-loaded NGs (siNGs) constructed with the hydrolysable dex-HEMA or the stable dex-MA. Both formulations were coated with a mixture of DOPC:PG (85:15 wt%) here abbreviated as LIP, with or without SP-B. Although the stable dex-MA shows a less pronounced eGFP knockdown, SP-B promotes siRNA delivery equal to the degradable dex-HEMA. Data are a summary of two independent experiments. Data are represented as the mean ± SD (n = 2) and statistical significance is indicated (** p < 0.01, *** p < 0.005, ns = not significant). (b) Gene silencing of (proteo)lipid-coated dex-HEMA NGs with an intact or degraded NG core. All experiments were performed with a fixed NG concentration (30 µg/mL) and siRNA concentration (5 nM). Data show one representative graph of two independent experiments; formulations with increased SP-B fraction showed the same trend (data not shown).

Figure 7

Evaluation of SP-B effect on DOTAP:DOPE liposomes for siRNA delivery in H1299_eGFP cells. (a) Cellular uptake and (b) gene silencing potential of DOTAP:DOPE LPXs (final concentrations siRNA are 0.5, 1 and 5 nM) with and without SP-B (1 wt%). The inclusion of SP-B in the cationic liposomal formulation does not result in any enhanced delivery effect. For uptake data, statistical analysis was performed using a one sample t-test. Data are represented as the mean ± SD (n = 2) and statistical significance is indicated (ns = not significant).