Engineering Human Epidermal Growth Receptor 2-Targeting Hepatitis B Virus Core Nanoparticles for siRNA Delivery in Vitro and in Vivo

Hepatitis B virus core (HBc) particles acquire the capacity to disassemble and reassemble in a controlled manner, allowing entrapment and delivery of drugs and macromolecules to cells. HBc particles are made of 180–240 copies of 21 kDa protein monomers, assembled into 30–34 nm diameter icosahedral particles. In this study, we aimed at formulating HBc particles for the delivery of siRNA for gene silencing in vitro and in vivo. We have previously reported recombinant HBc particles expressing ZHER2 affibodies, specifically targeting human epidermal growth receptor 2 (HER2)-expressing cancer cells (ZHER2-ΔHBc). siRNA was encapsulated within the ZHER2-ΔHBc particles following disassembly and reassembly. The ZHER2-ΔHBc–siRNA hybrids were able to secure the encapsulated siRNA from serum and nucleases in vitro. Enhanced siRNA uptake in HER2-expressing cancer cells treated with ZHER2-ΔHBc–siRNA hybrids was observed compared to the nontargeted HBc–siRNA hybrids in a time- and dose-dependent manner. A successful in vitro polo-like kinase 1 (PLK1) gene knockdown was demonstrated in cancer cells treated with ZHER2-ΔHBc–siPLK1 hybrids, to levels comparable to commercial transfecting reagents. Interestingly, ZHER2-ΔHBc particles exhibit intrinsic capability of reducing the solid tumor mass, independent of siPLK1 therapy, in an intraperitoneal tumor model following intraperitoneal injection.


siRNA encapsulation efficiencies by aldehyde/sulfate latex beads assay
To reassure the siRNA encapsulation into Z HER2-ΔHBc particles, we performed a beads coupling study with HBc-siRNA hybrid particles. siNEG Att0655 was encapsulated into Z HER2 -ΔHBc AF480 particles at 1:1 HBc:siRNA molar ratio and coupled with aldehyde/sulfate latex beads ( Figure S2). The aldehyde/sulfate latex contains high density of aldehyde groups grafted to the surface of the polymer particle, which enables facile coupling of proteins. This will allow the nano-sized HBc particles to be detected using flow cytometry. According to flow cytometry analysis (Figure S2), only beads coupled with Z HER2 -ΔHBc AF480 particles showed enhanced in median fluorescent intensity (MFI) in FL-1 channel (green bars), whereas S-3 the untreated beads, which acted as a negative control, did not show any significant increase. These results indicated that the aldehyde/sulfate latex beads were successfully coupled with Z HER2 -ΔHBc AF480 particles. Meanwhile, 1.4-and 1.5-fold increases (blue dots) in MFI in FL-4 channel of was observed when siNEG Att0655 was encapsulated into Z HER2 -ΔHBc AF480 particles, using Method I or Method II, respectively. However, as shown in Figure 1B, a 2.7-fold increase in MFI (blue dot) was observed for HBc-siRNA particles prepared by Method III. Taken together, these results indicate that the HBc particles could encapsulate siRNA into their interior. Moreover, the siRNA encapsulated using Method III showed the highest amount of siRNA encapsulated into HBc particles, compared to the other HBc-siRNA hybrid particles prepared by Method I and Method II. This result is in agreement with the results obtained by gel retardation assay (Figure 1), which also show that approximately 1 mol of siRNA is encapsulated in 1 mol HBc particles when encapsulated using Method III.
Method III will be used later to encapsulate siRNA into HBc particles for further studies.

In vitro cellular uptake HBc-siRNA hybrids subjected to serum competition
The intracellular uptake of HBc-siRNA hybrid particles in vitro in cancer cells were further studied subjected to serum competition. Cell uptake was evaluated using flow cytometry detected at FL-1 and FL-4 channels, and expressed as the fold increase in median fluorescent intensity (MFI). MDA-MB-468 (HER2 -) and SKBR-3 (HER2 +++ ) cells were treated with Lipofectamine®-siNEG Atto655 complexes or Z HER2 -ΔHBc AF488 -siNEG Atto655 hybrids at 1 or 24 h post incubation in the media ± 10% serum. As shown in Figure S3, by FL-1 detector, a significantly decreased cellular uptake was observed when MDA-MB-468 (light blue bars) or SKBR-3 (light green bars) cells were treated with Z HER2 -ΔHBc AF488 -siNEG Atto655 hybrids in serum-containing media, compared to those treated in serum-free media. Furthermore, uptake of siNEG Atto655 detected by FL-4 channel also showed significant reduction (dark bars) when both cell lines were treated in serum condition.
The same result was observed when cells were treated with Lipofectamine®-siNEG Atto655 complexes. These results suggested that the presence of serum did affect and reduce the intracellular uptake in cancer cells; however, the uptake of siRNA could be still observed.
S-4 Figure S1. A scheme summarizing methods used for siRNA encapsulation into HBc particles. siRNA was encapsulated into Z HER2 -ΔHBc using one of the three different methods, dis-assembly/re-assembly by urea, osmotic shock or dis-assembly/re-assembly by DTT/CaCl 2 , namely as Method I, Method II and Method III.