Introduction
Various types of delivery systems, including organic/inorganic nanoparticles, small molecule targeting moieties, and antibodies, have been explored as carriers for PROTACs. These systems play a crucial role in addressing the challenges related to poor water solubility and cell permeability of PROTACs, regulating their competitive properties, and achieving selective localization in tumor tissues through passive or active targeting.
Ⅰ. Passive Targeting PROTAC Delivery System Based on Nanoparticles
Over the past few decades, researchers have designed and explored a range of polymer nanoparticles with high biocompatibility, physiological stability, and multifunctionality for drug delivery purposes. Poly(ethylene glycol) block copolymers (PEG) and poly(D-methylene-L-lactide-decoding-glycolide) (PLGA) are among the most representative polymer drug delivery systems, having gained approval from the FDA.
Nanoparticles have long been utilized to modulate the competitive properties of drugs with poor water solubility. Generally, the size of nanoparticles is much larger than that of drug molecules, thus making the solubility of the drug dependent entirely on the solubility of the nanoparticles once loaded. Furthermore, nanoparticles offer prolonged blood circulation for drugs due to their increased stability compared to small drug molecules under physiological conditions. Representative examples of nanoparticles that regulate the physical and chemical properties of drugs include Genexol®-PM (paclitaxel prepared in polymeric micelles) and Doxil® (liposomal doxorubicin).
The abnormal porous vascular structure and insufficient lymphatic drainage in tumor tissues enhance the accumulation of nanoparticles within a specific size range, known as the enhanced permeability and retention (EPR) effect or passive targeting effect. Once located within tumor tissue, nanoparticles can be endocytosed by cancer cells through various uptake pathways, such as microendocytosis, grid protein dependent endocytosis, small hole protein-dependent endocytosis, and grid/small hole protein-independent endocytosis. Subsequently, active drugs are released into the cells. Inspired by the success of nanoparticle-based drug delivery systems, researchers have also started investigating the application of nanoparticles to improve the unfavorable physical and chemical properties of PROTACs.
- Lipid nanoparticles
In 2021, the team led by Xiao Bing Xu at Tufts University employed lipid nanoparticles to deliver BRD4 PROTACs (ARV-771) that were pre-fused with E3 ligase in cells. ARV-771 comprises a BRD4 ligand and a VHL ligase ligand. Initially, the authors fused the pre-fused PROTACs with VHL protein in vitro and then encapsulated them in 80-O14B lipid nanoparticles. Upon entering the cell, the pre-fused PROTACs directly bind to the target protein, leading to the degradation of BRD4 protein. The authors propose that the pre-fused PROTACs have the potential to enhance the degradation efficiency of BRD4 by transforming the UPS system required by ternary complexes into a simpler binary complex system. Additionally, lipid nanoparticles facilitate the internalization of pre-fused PROTACs into cancer cells, enabling their escape from lysosomes and efficient functioning within the cytoplasm.
- Inorganic nanoparticles
Inorganic nanoparticles, including silica, gold, ferric oxide, and quantum dots, present alternative options for PROTAC delivery and demonstrate unique properties compared to organic nanoparticles. In contrast to organic nanoparticles, inorganic nanoparticles allow for more accurate control over their morphology and size distribution. Moreover, their rigid structure reduces the risk of unintended drug leakage at off-target sites. Gold nanoparticles (GNPs) have garnered considerable attention as drug carriers due to their biological inertness, excellent surface modification capabilities, and versatility in providing additional functionalities.
Ⅱ. Active targeting PROTAC delivery Strategy
The active targeting strategy primarily relies on the specific binding affinity between targeted ligands and membrane receptors overexpressed in cancer cells. To address the poor cellular permeability of PROTAC, active targeting components are used for PROTAC delivery as they promote receptor-mediated endocytosis rather than simple transmembrane diffusion. Endocytosis of PROTAC leads to its distribution in the cytoplasm, catalyzing the degradation of target proteins of interest (POIs). Furthermore, active targeting partially facilitates the selective accumulation of conjugated PROTACs in cancer cells, reducing systemic toxicity caused by off-target delivery. Currently, several active targeting components have been reported for PROTAC delivery, categorized as small molecules, antibodies, and aptamers based on their molecular structure.
- Small molecule-targeted PROTAC
Small molecule-targeted PROTAC conjugates represent one approach to PROTAC delivery systems. In 2021, the Jinjian Research Group at Mount Sinai Medical College employed small molecule folic acid groups as targeted ligands for cancer-selective PROTAC delivery. Folic acid groups bind to PROTAC molecules through ester bonds that can be cleaved by intracellular hydrolases. Folate receptors are highly expressed in various cancers, such as ovarian, breast, renal, and colorectal cancers, making folate-caged PROTACs selectively absorbed by cancer cells in a receptor-dependent manner, effectively mediating POI degradation.
Although small molecular ligands confer cancer-specific binding ability to PROTAC molecules, their small size limits their ability to regulate the poor water solubility of PROTACs effectively. Antibodies, on the other hand, possess larger structures that not only address the poor solubility issue but also enhance competitive properties. In addition to active tumor targeting, antibodies typically have longer blood half-lives compared to small molecular ligands, making them more suitable for drug delivery. Antibody-drug conjugates (ADCs) represent a successful case in this regard.
- Antibody-targeted PROTAC
In 2020, Edward W. Tate team BRD4 put forward the concept of Ab-PROTAC for the first time, combining BRD4 PROTACs with the anti-HER2 monoclonal antibody trastuzumab to construct Ab-PROTAC targeting HER2-positive breast cancer. This conjugate successfully degraded BRD4 protein in breast cancer cells. Due to the specific targeting of trastuzumab, Ab-PROTAC conjugates exhibited strong tumor uptake and minimal internalization into HER2-negative normal cells. Following endocytosis by cancer cells, the ester bond between PROTACs and trastuzumab was hydrolyzed, releasing free PROTACs and inducing irreversible BRD4 degradation.
- Aptamer-PROTAC
Nucleic acid aptamer-PROTAC conjugates are composed of short, single-stranded primary structures based on ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). These aptamers have been extensively studied in molecular imaging and drug delivery due to their high binding affinity, stability, and low immunogenicity. In 2021, the Sheng Chunquan research group of Naval military Medical University reported the use of aptamer-PROTAC conjugates, combining BRD4 PROTACs with nucleolar targeting aptamers, for the treatment of breast cancer. The PROTACs can be cleaved in the presence of glutathione (GSH) when connected to aptamers through short disulfide-containing chains. The aptamer-PROTAC conjugate exhibits significant BRD4 degradation activity exclusively in cancer cells with an overexpressed nucleolus, which is advantageous for addressing the poor solubility and competitive properties of PROTACs. Moreover, aptamer-PROTAC conjugates offer several advantages over antibody-PROTAC conjugates, including higher physiological stability and the ability to avoid antibody immunogenicity.
- Actively targeted semi-functional nanoparticles-PROTAC
Direct coupling of PROTAC with antibodies or aptamers has been shown to increase tumor selective accumulation of PROTACs and successfully regulate its adverse competitive properties. However, Ab-PROTAC has limitations in terms of loading capacity. Studies have demonstrated that actively targeted semi-functional nanoparticles can significantly improve the loading problem of PROTACs. Unlike antibodies, nanoparticles are less affected by competition in terms of loading, enabling them to accommodate relatively larger amounts of PROTACs. Furthermore, surface modification of nanoparticles with active targeting moieties can enhance the tumor enrichment of PROTACs through both passive and active targeting mechanisms.