**1. Introduction**

### **1.1 Noninvasive vaccine delivery by nanocarriers**

Currently, most vaccines, drugs, and diagnostic/therapeutic agents are administered through invasive routes such as injection. There has been vast interest in the development of noninvasive, targeted, stable, and convenient drug delivery platforms that obviate the drawbacks of invasive delivery methods (reviewed in [1–6]). Systemic drug delivery through noninvasive routes requires that the delivery platform protects the drug compound while it traverses physiological barriers. Noninvasive delivery platforms, as in the case of traditional delivery platforms, should also distribute the drug effectively and selectively so that only the targeted cells receive the therapeutic agent. The rapid evolution of nanotechnology has shed light on the huge potential of nanocarrier platforms for targeting and drug delivery. Recent developments in the optimization of drug nanocarriers in terms of packaging, delivery, and targeting have the potential to revolutionize noninvasive administration and delivery of therapeutics and diagnostics through the mucosa. Several nanocarrier systems have been developed that take advantage of these

developments and additionally show diminished toxicity in nontargeted cells and tissues. Despite these early successes, instability under physiological conditions, inefficient targeting, toxicity, and lack of bioavailability impose serious limitations for the development of an effective mucosal delivery platform.

While there are several routes of mucosal drug delivery, the oral and nasal routes are among the most safe and preferred by patients. The inherent characteristics of a nanocarrier such as structural composition, size, and natural stability play major roles in the potential success of a drug delivery system (reviewed in [1–4, 7–9]). For many nanocarrier platforms, problems with enzymatic degradation, limited penetration of the thick mucosal layer, and subsequently transportation of drugs through transcellular or paracellular routes are major shortcomings. The majority of currently available nanocarriers consist of simple structures that are on the nanometer to micrometer scale. Theranostic delivery vehicles that are currently used or considered for use fall into a handful of categories including polymers, lipids, solid-lipid carriers, gold carriers, nanotubes, immunostimulant complexes, magnetic carriers, and virus-like particles (VLPs)

[3, 8, 9]. The size and exact composition of these nanocarriers are commonly altered and optimized based on their intended application. The key factors required for entry and distribution of theranostics include high degrees of bioavailability, the ability to withstand physiological conditions without degradation or premature exposure of the drug, and efficient distribution by overcoming the physical and enzymatic barriers through noninvasive routes.

#### **1.2 Nanocarrier platform based on viral capsids**

Evolutionarily, viruses adapt and coevolve with their host. Genetic engineering techniques and elucidation of viral structures have enabled virologist to generate empty capsids, called VLPs, which retain the physical characteristics of the capsid structure but lack the viral genome. VLPs thus exhibit the structural characteristics of the authentic virus but are incapable of replicating. In addition to being noninfectious, VLPs are generally nontoxic, biodegradable, and highly biocompatible. Structurally, the symmetrical configuration of VLPs allows them to be developed as nanocarrier systems that can entrap not only foreign nucleic acids but also peptides and imaging agents within their internal cavity. The exterior surface of VLPs, in some cases, can be tagged with targeting ligands without disruption of the VLP structure. The VLP assembles spontaneously and forms highly ordered structures following recombinant expression of the capsid protein (CP) in prokaryotic, eukaryotic, and cell-free protein expression systems. Currently, there are numerous ongoing VLP-based clinical trials worldwide [10–13]. From these clinical trials, a handful of VLP-based vaccines have been approved by the US FDA and other governmental regulatory agencies. For example, VLPs of hepatitis B virus (HBV), human papillomavirus (HPV), influenza virus, human parvovirus, and Norwalk virus have shown success in clinical trials or have been commercially developed as vaccines. The effectiveness of the delivery of therapeutic and/or diagnostic payloads using VLPs, as well as VLP surface modulation by attachment of ligands and tracking molecules, has been recently reviewed [10, 11, 14]. Here, a description of the key advantages and application of hepatitis E virus nanoparticles for use in vaccine development will be discussed.
