Apoferritin nanocage as drug reservoir: is it a reliable drug delivery system?

KEYWORDS:

• Apoferritin
• protein
• drug carries
• drug delivery
• translatability

1. Introduction

In order to reach the ambitious aim of Ehrlich’s ‘magic bullet’ of developing drug delivery systems (DDS) selectively binding the site of diseases or even the diseased cells, without further affecting patients’ health with side effects, a number of promising approaches were developed in the last decades.

To fit with the ideal design, the ‘perfect’ DDS should be characterized by good properties in:

1. pharmaceutics (i.e. ability to efficiently load different kinds of therapeutic molecules, drug-release profiles adequate to pathologies treatment, and high stability);

2. safety (i.e. biocompatibility); and

3. selectivity (i.e. specific recognition on targeted site and lowering side effects).

The need to find biocompatible materials for the development of DDS strongly drove the research to a concrete tentative of replacing synthetic materials, as porous hollow silica or not-fully biocompatible polymers, with natural materials, endogenously present within the body, thus more acceptable and considered as self by the immune defense system.

Looking at this scope, endogenous self-assembling proteins could be a strategic choice, leading to obtain DDS able to efficiently load molecules, considered as nontoxic for the organism and therefore safe.

More recently, the attention of research in DDSs focused on apoferritin (APO), from ferritin (Ft) family, a protein able to self-assembly in uniform regular nano-sized structures. The overall good biocompatibility of APO and its unique architecture was therefore of interest in order to develop nanocarries able to stabilize small active molecules in its inner core. The low dimension, spherical shape, and high homogeneity [1] are among the key aspects that support the wide interest in APO cage. This novel DDS could lead to longer circulation half-life and eventually to better accumulation rates, in comparison with synthetic DDS (i.e. polymeric nanoparticles, and liposomes), characterized by higher size (50–200 nm) and less homogeneity. Moreover, APO is fully biocompatible and a-toxic, which is a ‘needed’ feature required for its use as nano-DDS.

Besides this aspect on safety, a major interest in APO research lies in ‘targeting’ abilities, classically addressed by surface conjugation of nano-DDS with ligands (peptide, antibodies, etc.) able to target the diseased sites.

The ‘targeting’ approach inevitably lead to the ‘over-crowding’ of the surface of carriers, with a number of issues to be solved from both technological (i.e. production reproducibility and surface characterization) and biological points of view (i.e. interaction with bloodstream proteins, protein corona composition, safety, and immune reaction), and overall the biocompatibility of the final structure

On the contrary, APO possesses site-specific targeting potential, as can be recognized and internalized by Ft-binding receptors, such as the transferrin receptor 1 (TfR1). The overexpression of these receptors, especially on surface of malignant cells, is one of the most important reasons for the growing interest of APO in the application of field of cancer treatment and diagnosis. This overexpression should be considered as an increased presence of TfR in malignant cells, but not as a unique expression of cancer cells. In fact, TfRs are present onto many other cells, greatly varying among cells depending on tissues, as TfR could be easily found in basal epidermis, endocrine pancreas, hepatocytes, Kupfer cells, testis, and pituitary gland [2]. Thus, on the basis of these evidences, the potentiality of APO widens to the application in other fields of nanomedicine as gene therapy, immunology, or liver pathology.

2. APO: from the dream to the reality

Notwithstanding these good premises, some limits emerged in the application of APO as DDS, mainly connected to pharmaceutical formulation, characterization, and standardization of process, but also to its biosafety. In particular, a couple of aspects is up-to-date unclear and poorly analyzed and therefore to be strongly investigated and ameliorated to allow APO to play a major role in DDS, namely:

1. pharmaceutical issues and

2. biosafety issues.

2.1. The pharmaceutical issues

Due to the stringent requirements of loading protocols, a limited number of drugs could be efficiently encapsulated and not always the efficacy of drug/APO complex is higher respect to free drug. Actually, the plethora of experiments on APO formulation did not produce a standardized protocol, able to clearly furnish a reproducible unfolding/refolding of the protein, and especially when the drug is present and ready to be loaded. Chemico-physical properties of drugs as molecular weight, pKa and charge should be taken into great account in planning their encapsulation in the APO core.

In this view, a particular attention should be given to pH values; as a matter of fact, the choice of pH values of the starting solution strongly impact on the type and distribution of charges onto the surface of the protein and therefore could determine rearrangement of the protein conformation. Besides, also pH values applied during the disassembly-reassembly protocol become critical to obtain an effective loading.

Not only pH values, but also many other variables can affect encapsulation efficiency into APO protein nanocarrier as: i) ionic concentration; ii) interactions between ions in solution and functional groups onto protein surface; iii) temperature, which could limit of stability of the protein by inducing its unfolding and denaturation; iv) protein concentration, which can increase the frequency of molecular collisions and can promote aggregation; and v) mechanical stress caused by processes such as mixing, stirring, filtration, dialysis, concentration, etc. [3].

From this point of view, synthetic DDS offer a larger versatility of formulation as, by simply changing the composition of the carriers or the formulation technique, a greater number of molecules could be encapsulated or even adsorbed. Besides, a plethora of polymers could be applied, with different features in terms of charge, hydrophilicity and hydrophobicity, degradation time, and biocompatibility. Moreover, synthetic DDS are studied to reach the goal of achieving controlled and targeted drug release, thus limiting well-known side effects of high systemic or off-target exposure. Thus, synthetic DDS could be really applied to a major number of drugs for encapsulation. In the case of APO, it is very hard to modulate drug release from the inner cage, especially depending on their chemico-physical properties. That way, ions, allowed to enter/exit through pores, could be modulated, but larger molecules could be released from the inner core only as consequence of protein denaturation once in cellular acidic compartment, thus strongly impacting and often decreasing the possibility for release modulation [4]. Moreover, not all the drugs are suitable for efficient encapsulation into APO DDS, as, up-to-today, few technologies are really available as protocols for drug loading.

Thus, a rationale planning of APO as DDS should be based on the ‘prediction/evaluation’ of the combination of the formulative variables, which will govern both the loading efficiency and drug-release kinetics. In fact, the possibility for the drug to penetrate and to escape through the APO inner channels is a strong function of drug/protein electrostatic interactions. These interactions also impact on structural rearrangement of APO, as, depending on its charges, any electrostatic interaction with the drug will strongly affect protein correct reassembly. This electrostatic interaction between charged drug and charged protein also play an important role both on the stability of the encapsulation and on the unwanted absorption of the drug on the protein surface.

Finally, a major issue is surely related to the chemico-physical characterization of APO during all the processes of formulation along with the characterization of the final loaded APO-based DDS. This particular aspect is fully addressed in the review we are proposing in this issue.

2.2. The biosafety issues

It is a common idea that proteins are generally safe and nontoxic; however, the reaction after administration of heterologous APO could lead to (i) activation of immune system against foreign proteins, similar to immune response against pathogens or vaccines; (ii) breach of B- and T-cell tolerance to autologous proteins [5,6], finally resulting in the production of anti-therapeutic protein antibodies (known as antidrug antibodies) able to neutralize or otherwise to compromise the clinical effect of therapeutics [7].

A number of studies use Ft of animal source, mainly derived from horse [8] and pig [9], or in other cases by means of exploitation of recombinant proteins [10,11] and often, the choice of the APO derived from economic aspects. In fact, horse spleen Ft is cheaper with respect to human Ft, but frequently the researchers underestimated the impact on biosafety and in vivo response. Thus, protein source, its purity and its final rearrangement could deeply affect the safety profiles of the protein and the final applicability of the DDS.

Moreover, the advantage of use a natural molecule as starting material for DDS is drastically affected when ex vivo engineering processes are performed in order to link targeting moieties onto the DDS surface. The use of solvent, chemical reagents, and ligands obviously impact on the biocompatibility of formulation; in the same way, protein conformation deeply suffers in terms of stability and maintenance of the tertiary/quaternary structures when a surface modification is applied to proteins. Moreover, the presence of ligand could interfere with the surface integrity, governing or altering the overall biodistribution and body-distribution.

3. Expert opinion

Several evidences provided for the suitability of APO as protein-based DDS, featured by a number of advantages regarding the biocompatibility and application in pathologies, especially in the case of treatment and imaging of cancer. As any new ‘application,’ the starting point consists of the direct evidence of efficacy in treatment, but, as rule, immediately after the first proofs-of-concept, a complete study on formulation, scale-up, translatability, biocompatibility, and biodistribution is strongly required. It is evident that APO-based nano-DDS are up-to-date in the middle of this path.

Good experimental data are present in literature, mainly in the application of APO-nanocarriers for the delivery of anticancer drugs, but deep understanding of the architecture of APO/drugs complexes is still lacking.

This limit is the main drawback in the development of APO as DDSs: from a deep understating of the mechanism and dynamics of complexes formation (between APO and drugs), we will be able to ‘configure’ formulation protocols suitable to a wider plethora of drugs, not only closed up to anticancer drugs or imaging agents.

Moreover, only after a good understanding of the dynamics governing APO/drug complexes formulation, a ‘modification’ in encapsulation protocols will be designed, thus allowing also to move on to ‘surface’ modification of APO for better targeting abilities.

Therefore, especially a multidisciplinary research effort should be given in order to complete the overall ‘painting’ of APO application and formulation and to give strong impulse to the development and clinical translatability of these novel and promising DDS.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Funding

This paper was not funded.