Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Apr 25;14(5):408.
doi: 10.3390/ph14050408.

Polymerized Albumin Receptor of Hepatitis B Virus for Evading the Reticuloendothelial System

Kurumi Takagi  1 , Masaharu Somiya  2 , Joohee Jung  3 , Masumi Iijima  1   4 , Shun'ichi Kuroda  1   2
Affiliations

Polymerized Albumin Receptor of Hepatitis B Virus for Evading the Reticuloendothelial System

Kurumi Takagi et al. Pharmaceuticals (Basel). .

Abstract

Various strategies, such as optimization of surface chemistry, size, shape, and charge, have been undertaken to develop nanoparticles (NPs) as DDS (drug delivery system) nanocarriers for evading the reticuloendothelial system (RES) in vivo. We previously developed a hollow NP composed of hepatitis B virus (HBV) surface antigen L proteins and lipid bilayers, hereinafter referred to as bio-nanocapsule (BNC), as a nonviral DDS nanocarrier. Such a BNC harbors the HBV-derived human hepatic cell-specific infection mechanism, and intravenously injected BNCs by themselves were shown to avoid clearance by RES-rich organs and accumulate in target tissues. In this study, since the surface modification with albumins is known to prolong the circulation time of nanomedicines, we examined whether the polymerized albumin receptor (PAR) of BNCs contributes to RES evasion in mouse liver. Our results show that NPs conjugated with peptides possessing sufficient PAR activity were captured by Kupffer cells less efficiently in vitro and were able to circulate for a longer period of time in vivo. Comparing with polyethylene glycol, PAR peptides were shown to reduce the recognition by RES to equal content. Taken together, our results strongly suggest that the PAR domain of BNCs, as well as HBV, harbors an innate RES evasion mechanism. Therefore, the surface modification with PAR peptides could be an alternative strategy for improving the pharmacodynamics and pharmacokinetics of forthcoming nanomedicines.

Keywords: albumin; bio-nanocapsule; hepatitis B virus; nanoparticle; polymerized human serum albumin receptor; reticuloendothelial system.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structures of Bio-nanocapsule (BNC) and hepatitis B virus (HBV). HBV is composed of HBsAg embedded in lipid bilayer, HBcAg (HBV core antigen), DNA polymerase, and the HBV genome. Similarly, BNC is composed of HBsAg L proteins embedded in lipid bilayer. The L protein contains three domains, including the pre-S1 region containing a human hepatocyte-recognizing domain, the pre-S2 region with a polymerized albumin receptor (PAR) domain, and the S region with three transmembrane-spanning segments. The number of amino acids in each domain is indicated in parentheses. BNCs could be used for the in vivo pinpoint delivery of genes and drugs in mouse models via intravenous injection.
Figure 2
Figure 2
In vivo imaging analysis of LPs and BNC-LP complexes. Each mouse was injected intravenously with Rh-labeled LPs, either with or without 10 µg of BNCs. After 30, 180, and 300 min, Rh-derived fluorescence was detected from the ventral side of mice using an in vivo imaging system OV-100. The orientation (head and tail) of mice is shown by arrows. White and green arrowheads indicate liver and intestine, respectively.
Figure 3
Figure 3
In vitro albumin-binding activity of BNCs. Polymerized (A) and monomeric (B) forms of human (HSA), bovine (BSA), or mouse (MSA) albumins that were conjugated to resins were incubated with either BNCs or ΔBNCs at 37 °C for 1 h, washed with PBS 4 times, subjected to 12% SDS-PAGE, followed by silver staining. The amounts of precipitated BNCs and ΔBNCs were determined by densitometry. Forty percent input, the loading control. ΔBNCs, PAR-deleted BNCs.
Figure 4
Figure 4
In vitro pHSA-binding activity of BNC-derived peptides. (A) Synthetic peptides containing the putative PAR region. Peptide 1, Leu-12 to Tyr-21 of the pre-S2 region; peptide 2, Thr-7 to Ala-24 of the pre-S2 region; peptide 3, peptide 1 containing a mutation of Tyr-21 to Pro-21 (underlined); and peptide 4, peptide 2 containing a mutation of Tyr-21 to Pro-21 (underlined). (B) Pull-down assays with peptide-conjugated resins (right panel). Left panel, calibration of pHSA. Resins were incubated with pHSA at 37 °C for 1 h, washed with PBS 4 times, subjected to 12% SDS-PAGE, followed by silver staining. The border between stacking and separating gels is indicated by a white arrowhead. (C) Competition assays using pHSA-conjugated resins (right panels). Left panels, calibration of BNCs (0.03125, 0.0625, 0.125, 0.25, 0.5, 1, and 2 μg as protein). Resins (containing 10 μg pHSA) were incubated with BNCs (20 μg) in the presence of specific peptides (0, 1.25, 2.5, 5, 10, 20, and 40 μg) at 37 °C for 1 h, washed with PBS 4 times, subjected to 12% SDS-PAGE, followed by silver staining. The amounts of precipitated BNCs were determined by densitometry.
Figure 5
Figure 5
In vitro uptake of fluorophore-labeled microspheres and BNCs by Kupffer cells. After opsonization of Cy3-labeled BNCs and NYO-labeled microspheres (naked, PEG-modified, HSA-modified, MSA-modified, peptide 2-modified with/without pHSA treatment, or STI-modified), about 5 × 104 Kupffer cells were incubated with the NYO-labeled microspheres (about 5 × 108 particles) or Cy3-labeled BNCs (about 5 × 108 or 5 × 109 particles) at 37 °C for 30 min and subjected to the FACS analysis. The fractions of Kupffer cells were predefined by the forward scatter/side scatter dot plots derived from CD11b+ cells. Distributions of microspheres and BNCs in Kupffer cells were indicated as open histogram. Controls (untreated Kupffer cells) were indicated as shaded histograms. The percentages (%) of NYO-labeled or Cy3-labeled Kupffer cells were presented as numbers.
Figure 6
Figure 6
Blood concentration–time curves of NYO-labeled microspheres. At 10 min, 30 min, and 60 min after intravenous injection with NYO-labeled microspheres (100 μg/mouse), blood samples were collected and processed as described in Materials and Methods. The concentrations of fluorophores were determined by a fluorescence microplate reader (n = 5; mean ± SEM; t-test, * p < 0.05).
Figure 7
Figure 7
Evaluation of hepatotropic properties of microspheres and BNCs. (A) In vivo uptake of fluorophore-labeled microspheres by mouse livers. About 100 μg of NYO-labeled microspheres (naked, PEG-modified, HSA-modified, MSA-modified, peptide 2-modified, or STI-modified) were administrated intravenously into each mouse. After 10 min, livers were isolated and subjected to the in vivo imaging analysis. The color bar shows NYO-derived fluorescence intensity. Bar, 1 cm. (B) After opsonization of Cy3-labeled BNCs and NYO-labeled microspheres (naked, PEG-modified, HSA-modified, MSA-modified, peptide 2-modified, or STI-modified), about 5 × 104 mouse primary hepatocytes were incubated with NYO-labeled microspheres (about 1 × 109 particles) or Cy3-labeled BNCs (about 1 × 109 or 1 × 1010 particles) at 37 °C for 30 min and then subjected to the FACS analysis. Distributions of microspheres and BNCs in hepatocytes were indicated as open histograms. Controls (untreated hepatocytes) were indicated as shaded histograms. The percentages (%) of NYO-labeled or Cy3-labeled hepatocytes were presented as numbers.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Stolnik S., Illum L., Davis S. Long circulating microparticulate drug carriers. Adv. Drug Deliv. Rev. 1995;16:195–214. doi: 10.1016/0169-409X(95)00025-3. - DOI
    1. Vonarbourg A., Passirani C., Saulnier P., Benoit J.-P. Parameters influencing the stealthiness of colloidal drug delivery systems. Biomaterials. 2006;27:4356–4373. doi: 10.1016/j.biomaterials.2006.03.039. - DOI - PubMed
    1. Huang Z.-Y., Hunter S., Chien P., Kim M.-K., Han-Kim T.-H., Indik Z.K., Schreiber A.D. Interaction of Two Phagocytic Host Defense Systems. J. Biol. Chem. 2011;286:160–168. doi: 10.1074/jbc.M110.163030. - DOI - PMC - PubMed
    1. Furumoto K., Nagayama S., Ogawara K.-I., Takakura Y., Hashida M., Higaki K., Kimura T. Hepatic uptake of negatively charged particles in rats: Possible involvement of serum proteins in recognition by scavenger receptor. J. Control. Release. 2004;97:133–141. doi: 10.1016/j.jconrel.2004.03.004. - DOI - PubMed
    1. Owens D.E., Peppas N.A. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int. J. Pharm. 2006;307:93–102. doi: 10.1016/j.ijpharm.2005.10.010. - DOI - PubMed

LinkOut - more resources