Pharma Voice

Intracellular drug delivery systems: A new paradigm

Intracellular Drug Delivery Systems (ICDDS) could be the solution for many infections. Manan Desai and Aditya Pattani explain the need and prospects of ICDDS.

Many intracellular infections are notoriously difficult to treat since the antibiotic concentrations inside the cells are below the minimum inhibitory concentrations. Nucleotides are ineffective as drugs since they cannot penetrate cell membranes. For a dilemma like this, Intracellular Drug Delivery Systems (ICDDS) could be the solution.

What is ICDDS?

During the course of evolution, cells have developed various mechanisms to prevent the entry of xenobiotics. Some of these mechanisms include—the presence of a lipophilic cell membrane; existence of P-glycoproteins which efflux the drugs out; the occurrence of degradative enzymes and the development of endosomes which are highly acidic and these degrade xenobiotics which are endocytised into the cells. Thus, in order to deliver drugs to an intracellular target successfully, a very specialised system, capable of evading these barriers, is required. Such systems, designed to deliver drugs to intracellular targets are called intracellular drug delivery systems.

Figure 1:The Pathway for receptor mediated endocytosis.

Developing ICDDS

The current intracellular drug delivery systems mimic natural processes, which are known to breach the cellular barrier and achieve internalisation. The natural processes of cellular internalisation are:

Passive transport: Lipophilic molecules can cross the cell membrane passively. They require no special ICDDS, unless an intracellular sustained release is desired. This can be achieved by using nanoparticles, which are slowly biodegradable.

Endocytosis: Eukaryotic cells take up extracellular material by a variety of different mechanisms that are collectively termed endocytosis. It is a mechanism that is unique to these cells and involves internalisation of macromolecules into endocytic vesicles derived from the plasma membrane. It is a conserved multi-step process, consisting of ligand binding, internalisation, formation of transport vesicle, sorting of cargo and cargo distribution. These vesicles come in several different varieties, ranging from the actin dependent formation of phagosomes involved in particle uptake, to smaller clathrin coated vesicles responsible for the internalisation of extracellular fluid and receptor-bound ligands. The best characterised endocytic pathway is the Clathrin Mediated Endocytosis (CME) or the classical pathway, which results in the formation of clathrin coated invaginations that pinch off to make Clathrin Coated Vesicles (CCVs). CME is involved in the internalisation and recycling of receptors participating in signal transduction, uptake of transferrin, an iron binding protein, nutrient import, as well as, in reformation of synaptic vesicles.

A less defined, non-classical, Clathrin Independent Endocytosis (CIE), includes caveolae-mediated endocytosis. Caveolae are small, flask shaped invaginations, characterised by a special lipid composition, distinct from the plasma membrane, enriched in cholesterol and sphingolipids called lipid rafts. It also consists of a cholesterol binding protein, caveolin. An interesting aspect of CIE or raft endocytosis is that the internalised caveolae and rafts bypass the acidic endosomes and lysosomes unlike CME. They certainly provide safer and flexible route for targeted drug delivery. Although, many details are still unclear, understanding of caveolar transport could help in the rational design of more effective drug carriers.

Several natural ligands are known, which are normally internalised by the cells by one of the above mechanisms. Thus, if one attaches a xenobiotic to such a ligand, the drug will also be internalised.

Cell membrane penetration: Several proteins like HIV-1 Tat, Drosophila Antenna-pedia homeoprotein and HSV-1 VP22 have shown to traverse the cell membrane by a process called protein transduction and reach the nucleus, while retaining their biological activity. This is possible due to short "protein transduction domains". Peptides derived from these domains are commonly known as Cell Penetrating Peptides (CPPs).

While some of these peptides are purely cationic, others are amphipathic with a large fraction of basic residues, and again, others are fully hydrophobic. These vary in length from about nine to more than 30 amino acids. These peptides have been used successfully for intracellular delivery of macromolecules with molecular weights, several times greater than their own.

The mechanism of internalisation of CPP has not yet been resolved. A number of investigations of CPP internalisation in cell lines have been carried out and is found to be a multi-step process. However, a mixed mode of internalisation for these has been suggested. The two mechanisms include:

a) An inverted micelle model:

Here, positively charged peptides interact with negatively charged phospholipids of the membrane to form an inverted micelle structure which, is then internalised.

b) To some extent the CPPs also induce endocytosis:

If we attach a drug molecule to these CPP's, the drug molecules will also be able to tranduse across the cell membrane and thus be available for intracellular targets. A significant advantage of CPPs, such as Tat and Penetratin, is that they show insignificant disturbance or impairment to the cell membrane.

Designing ICDDS

In order to rationally design an ICDDS, it is important to fundamentally understand the cellular co-ordination and communication with the external environment. An ideal ICDDS must have the following characteristics:

• It must have a high efficiency for the internalisation of the drug into the cells
• It must be able to carry a high payload of drugs
• It must protect the drugs from the extracellular degradation
• It should exhibit low toxicity and immunogenicity

Various pathways for endocytosis and cell penetrating peptides have been characterised with respect to their molecular mechanisms. This led to the identification of molecules, which induce endocytosis or cell penetration. These have been further used in drug delivery systems.

Initially, drugs were conjugated to the internalisation signals (endocytic molecules or to the CPPs), but the major disadvantage was a low payload efficiency. Thus, the current trend is to attach these internalisation signals to colloidal carriers, especially liposomes and nanoparticles. A commonly used endocytic molecule is Transferrin. Ligands conjugated to this are efficiently internalised. Transferrin has also been conjugated to liposomes and to nanoparticles and has shown superior internalisation as compared to unconjugated carriers.

Similarly, the most commonly used cell penetrating peptides are Tat and penetratin. This peptide has been conjugated to the colloidal carriers and has significantly enhanced the internalisation. Simple PLGA nanoparticles have also been used for sustained intracellular drug delivery, for up to 14 days.

The prospects

ICDDS is an actively researched field especially by groups working on gene delivery. The advances in molecular biology will greatly improve the knowledge base of endocytosis and cell penetration by CPPs. This will lead to the design of newer and safer agents for induction of internalisation into the cells. Introduction of intracellular drug delivery strategies into the clinic is hampered by a limited understanding of the basic mechanisms of macromolecular membrane transport. Also, the development of strategies to breach biological barriers is a major challenge as science moves to exploit the opportunities arising from genomic studies, proteomics, and rational drug design. To summarise, we say that it is clear that CPPs, such as Tat, and endocytogens, such as transferring, are novel vehicles for the rapid translocation of cargo into the cells. They exhibit the properties that make them potential drug delivery agents, and therefore, interesting for practical use in the future.

(The writers are from the Department of Biochemistry, UICT)