Endocytic mechanisms for targeted drug delivery

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Abstract

Advances in the delivery of targeted drug systems have evolved to enable highly regulated site specific localization to subcellular organelles. Targeting therapeutics to individual intracellular compartments has resulted in benefits to therapies associated with these unique organelles. Endocytosis, a mechanism common to all cells in the body, internalizes macromolecules and retains them in transport vesicles which traffic along the endolysosomal scaffold. An array of vesicular internalization mechanisms exist, therefore understanding the key players specific to each pathway has allowed researchers to bioengineer macromolecular complexes for highly specialized delivery. Membrane specific receptors most frequently enter the cell through endocytosis following the binding of a high affinity ligand. High affinity ligands interact with membrane receptors, internalize in membrane bound vesicles, and traffic through cells in different manners to allow for accumulation in early endosomal fractions or lysosomally associated fractions. Although most drug delivery complexes aim to avoid lysosomal degradation, more recent studies have shown the clinical utility in directed protein delivery to this environment for the enzymatic release of therapeutics. Targeting nanomedicine complexes to the endolysosomal pathway has serious potential for improving drug delivery for the treatment of lysosomal storage diseases, cancer, and Alzheimer's disease. Although several issues remain for receptor specific targeting, current work is investigating a synthetic receptor approach for high affinity binding of targeted macromolecules.

Introduction

Macromolecular therapeutics are rapidly gaining interest in the area of nanomedicine for their ability to serve as alternatives to traditional drug regimens. Macromolecular conjugation has offered improvements in the delivery of low molecular weight drugs by preventing their passive diffusion into highly circulated tissue systems throughout the body resulting in lowered toxicities and volumes of distribution. Drugs that would potentially benefit from such strategies are those exhibiting low bioavailability and limited therapeutic utility. A variety of macromolecules, such as oligonucleotides, peptides, proteins, and polymers, have been investigated for their biocompatibility and stability in vivo for serving as vehicles for drug delivery; however, even these systems still posses drawbacks, especially with regards to bioavailability [1].

The primary advantage in the use of macromolecules as drug delivery vehicles is their mechanism(s) of cellular internalization. The cell membrane is naturally impermeable to complexes larger than 1 kDa; however, cells posses a variety of active internalization mechanisms to accommodate cellular entry of large molecular complexes. Here, the cell membrane will invaginate to engulf molecules and extracellular fluid in an intracellular membrane-bound vesicle, or endosome, that will subsequently traffic through the cell, a process known as endocytosis. Molecules may reside near the membrane or directly interact with membrane proteins to enable their retention in these vesicles. Analogous to the attachment of drug moieties to high molecular weight carriers, agents such as antibodies and high affinity ligands can be incorporated in order to exploit direct membrane interactions and target these complexes to specific cell populations in organ systems. Once inside the cell, the intracellular fate of the endosomal contents is an important determinant of successful drug delivery. Depending on the membrane interaction and components involved in vesicle formation, endosomes will mature into acidic vesicles which may or may not fuse with lysosomes, which can completely metabolize macromolecules using hydrolytic and enzymatic reactions. Targeting macromolecular complexes with high affinity ligands specific to membrane proteins, namely receptors, can aid in regulating not only the cellular recognition of these carriers but also the trafficking pathway and subcellular localization within the cell.

This review will discuss macromolecular drug delivery systems targeted to the endolysosomal systems of cells. Targeting to this pathway offers several advantages, including the ability to exploit upregulated membrane receptors in certain diseased organs and tissues, to control the intracellular fate for localization to acidic endosomes, and to allow for regulated release of therapeutics from bioresponsive linkers on the vehicle. Next, targeting the macromolecules to an intracellular endolysosomal pathway will enable therapeutic delivery to the unique organelles connected with these trafficking pathways, namely the endosomes and lysosomes. Pathological conditions associated with the endosomes and lysosomes would greatly benefit from therapies directed along these pathway. Finally, current delivery systems allowing for endolysosomal targeting will be discussed, with particular attention to the use of nanoscale carriers.

The major objectives for targeted drug delivery are reducing the nondiscriminate uptake of toxic agents as well as enhancing drug accumulation at the target site. In order to target drugs to specific tissue systems within the body, drug molecules can be directly attached to a targeting agent or complexed with a vehicle, or macromolecule, that contains targeting moieties. Macromolecules can be bioengineered to incorporate a variety of synthetic and natural compounds including drugs, ligands, and radionuclides.

Receptor mediated endocytosis (RME) allows for a more rapid means of ligand targeted internalization compared to that of untargeted complexes. Depending on the receptor-dependent or independent endocytic path, the intracellular trafficking path can also be controlled (Table 1). For example, those macromolecules taken up by clathrin-dependent RME are typically destined for lysosomal degradation; whereas, clathrin-independent RME internalization leads to endosomal accumulation and sorting to a nondegradative path. Appropriate selection of targeting agents could therefore allow for controlled delivery to the lysosomes or endosomes to alleviate conditions associated with these individual organelles, including cancer [2], Alzheimer's disease [3], and most importantly lysosomal storage diseases (LSDs).

Previous modalities of treatment for LSDs have resulted in hepatic buildup of replacement enzymes causing rapid clearance and low bioavailability. The use of macromolecular delivery systems that are targeted to endocytic machinery enable both cell specific as well as organelle specific, in this case the lysosomes, interactions resulting in a more efficacious treatment for these disease states. Work with chemically modified lysosomal enzymes has acted as a proof-of-principle for this theory of directed delivery. For example, glucocerebrosidase, the enzyme deficient in Gaucher's disease, was modified to expose mannose residues allowing for recognition by mannose receptors and subsequent receptor-mediated internalization of the enzyme and trafficking to the lysosomes [4].

The majority of targeted deliveries aim to avoid lysosomal trafficking in an effort to protect the drug molecule or biomolecules from enzymatic degradation. For our focus, this issue need only be addressed for those complexes which are expected to release drug molecules prior to endosomal fusion with lysosomal contents. This matter can easily be resolved by selecting a targeting agent that is known to be internalized via a clathrin-independent mechanism where contents are exposed to the acidic environment of endosomes but are typically not destined to the lysosomally degradative pathway. Alternatively, a growing number of polymer-based therapies are targeting therapeutics through a clathrin-dependent endocytic mechanism for drug localization and release in lysosomes. The endolysosomal pathway is of direct relevance to targeted intracellular drug delivery because not only does endocytosis allow for macromolecular internalization but it enables receptor- and lysosome-specific localization. Using the current knowledge of endocytic mechanisms and the key players involved, drug delivery systems can be bioengineered to exploit these pathways for more specialized intracellular delivery.

Another area with recent successes in macromolecular therapeutics is directed delivery of anticancer drugs. Chemotherapeutics are low molecular weight drugs that quickly diffuse into all tissues in the body thereby frequently causing toxicity leading to dose-related side effects. Modifying the delivery of these compounds, which remains the best form of cancer treatment, through macromolecular conjugation has offered promising results and has currently reached Phase 3 clinical trials. Success in this area can be attributed largely to the structural and physical nature of cancer tissue. To begin, solid tumors recruit new vasculature to support the rapidly dividing cells; however, the process of angiogenesis occurs insufficiently resulting in semipermeable blood vessels and allowing for the entry of otherwise impermeable molecules which are retained in the solid tumor tissue. This effect allows for a higher accumulation of drug conjugated complexes, which are taken up by normal cells through an indirect endocytic mechanism at a much slower rate. Furthermore, cancer cells typically contain upregulated amounts of membrane receptors. For example, the folate receptor is upregulated in over one-third of human cancers. With this in mind, drug delivery vehicles have been conjugated to high affinity targeting agents directed at specific cell surface receptors in cancer tissues including liver, breast, and brain [5]. Another advantage of macromolecular therapy targeting RME systems is the containment of therapeutic macromolecules within endocytic vesicles, thereby bypassing potential effects of multi-drug resistance associated efflux transporters (e.g. P-glycoprotein, MRP, BCRP), which are frequently upregulated in tumor tissues [6].

Section snippets

Understanding endocytic mechanisms for directed targeting

All eukaryotic cells absorb macromolecules through endocytosis, where large or polar substances are engulfed within the cell membrane and contained intracellularly in membrane bound vesicles. Materials, such as proteins and carbohydrates, come in close proximity to the cellular membrane where they may directly interact with membrane-embedded receptors or, indirectly by associating with the bilayer. The selective incorporation of materials is governed by cellular requirements and, depending on

Diseases targeted by endocytically delivered therapeutics

Diseases occurring in endosomes and lysosomes have limited exposure to cellular traffic and are highly regulated at the molecular level. Macromolecular drug delivery systems may be targeted along the endolysosomal system to directly associate with diseases occurring in endosomes and lysosomes, such as Lysosomal Storage Disease (LSD), Alzheimer's, and potentially cancer [2].

Modalities of macromolecular therapeutic delivery

A variety of macromolecular drug delivery systems utilizing endocytic internalization mechanisms have been developed to combat limitations associated with previously established therapies. These systems are very versatile as they are able to incorporate targeting molecules or ligands, imaging agents, and therapeutics moieties. Bioengineering macromolecules or modifying previously existing biomolecules to include targeting molecules enables cell-specific delivery of therapeutics. A variety of

Conclusions and future directions

The advances in cellular biology over the past decade have provided highly useful insight into the translocation and subcellular trafficking of macromolecules using endocytic pathways. Drug delivery scientists have capitalized on this knowledge by using receptors and ligands undergoing endocytosis as targeting moieties for specific cellular organelle targeting. However, our knowledge of endosomal regulation and vesicular trafficking is not complete, leading to several issues that remain to be

Acknowledgements

This work was supported in part by a grant from the Susan G. Komen Foundation.

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