Release date:2023/5/27 15:26:07

As an emerging therapeutic strategy for degrading disease-causing target proteins by hijacking the ubiquitin-proteasome system (UPS), PROTACs have the potential to revolutionize trends in the pharmaceutical industry. However, the development of many PROTACs has not been smooth, and most have not made it through the preclinical phase of drug development. There are many reasons for this, and one of the most challenging obstacles is the inability to ensure precise delivery of PROTACs to their targets.

To realize the medical value of PROTRAC for patients, it is important to combine PROTAC with advanced delivery systems to improve its physicochemical properties, enhance its targeting and reduce its off-target side effects. The combination of PROTAC and multifunctional delivery systems will open up new research directions in the field of targeted protein degradation.

Development Challenges of PROTACs

Currently, most traditional therapeutic strategies are based on "occupancy-driven" mode of action to inhibit the function of disease-causing target proteins for disease treatment. Unlike traditional small molecule inhibitors and antagonists, protein degradation technology can address challenges that cannot be addressed by traditional small molecules or biomolecules due to their ability to induce the degradation of disease-causing target proteins. Compared to inhibitors and antibodies, PROTACs have longer-lasting therapeutic effects because they catalyze and irreversibly remove oncogenic proteins.

Despite the outstanding features of PROTACs for cancer treatment, their translation to clinical trials has been stalled due to inherent shortcomings in their structural properties.

1. PROTAC has a large molecular weight ( over 800 Da), thus it is poorly soluble in water, resulting in its low bioavailability.
2. The highly polar surface of PROTAC limits its permeability and hinders its ability to cross physiological barriers and cell membranes.
3. Since E3 ligases are widely expressed at disease sites and in normal tissues, this is likely to lead to side effects such as off-targeting of PROTAC.
4. PROTACs with high intracellular concentrations tend to form binary complexes that produce Hook effects, which can reduce target degradation activity and hinder the design of their in vivo dosing.


Figure 1. Mechanism diagram and limitations of PROTAC-mediated protein degradation, source: reference [2]

Poor solubility, poor permeability, low bioavailability, non-specific biodistribution and unpredictable Hook effect hinder the clinical translation of PROTACs.

In addition to modifying the chemical structure of PROTAC molecules to overcome these drawbacks, drug delivery systems can be an effective alternative strategy because they can conveniently govern the overall PK behaviors of PROTACs toward showing better therapeutic outcomes. 

PROTAC Delivery Systems

Currently, various types of delivery systems including organic/inorganic nanoparticles, small molecule targeting moieties and antibodies have been explored as PROTAC carriers (Figure 2). They can modulate the poor water solubility and cell permeability of PROTACs, modulate their PK profiles, and promote selective localization of PROTACs to tumor tissues through passive or active targeting approaches.


Figure 2. Carriers for PROTAC delivery. Source: reference [1]

Nanoparticle-Based PROTAC Delivery System

Over the past few decades, there has been a proliferation of research on nanoparticle-based designed delivery systems. Clinical results of approved nanomedicines (e.g. Abraxane and Doxil) have shown that the drug carriers can successfully reduce the side effects of the drugs themselves, thus keeping the toxicity of chemotherapeutic drugs that require multiple doses under control. For PROTAC, nanocarriers do overcome the extra-tissue targeting side effects of protein degraders.

Furthermore, the successful combination of COVID-19 vaccine with lipid nanoparticles holds great promise for therapeutic agents with poor physicochemical properties but superior therapeutic efficacy, such as PROTACs. On the other hand, nanoparticles can diversify the route of administration (from a single oral dose to a wider range of delivery options such as intravenous injection) and expand the indications of PROTACs.

Nanoparticles for drug delivery can be broadly classified into organic and inorganic nanoparticles, and organic nanoparticles are further specified as polymeric and lipid nanoparticles.


Figure 3. Nanoparticle-Based PROTAC Delivery System, Source: reference [2]

Lipid Nanoparticles

Liposomes can be composed of a large number of substructures and are multifunctional carriers. Most lipid-based nanoparticles are spherical vesicles that often have consistent multiple water-soluble components encapsulated within their lipid bilayers. Liposomes have many advantages as drug delivery systems, such as simple formulation, good biocompatibility, high bioavailability, excellent loading capacity, and flexible and adjustable physicochemical properties.

Studies on lipid-based PROTAC delivery have been mainly conducted by K. Patel's research group. In their first report on PROTAC delivery, Kolliphor ELP®, an industrial pharmaceutical surfactant, was newly employed in a lipid-based nanoformulation of BRD4 degraded ARV-825 and its application to vemurafenib (BRAF inhibitor) resistant melanoma. The PROTAC nanoformulation was measured to be approximately 45 nm in diameter, effectively alleviating the poor water solubility of ARV-825. The nanoformulation showed higher cellular uptake and cytotoxicity to melanoma cells compared to the free PROTAC solution. Similarly, PEGylated lipid nanoparticles composed of three different lipid molecules (PEG 2.000-conjugated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; PEG2,000-DSPE, PRECIROL® ATO 5, CAPTEX® 300 EP/NF) and two different surfactants (poloxamer 407 and Tween 80) were also evaluated for ARV-825 delivery. The encapsulation of ARV-825 in PEGylated lipid nanoparticles provided stable and durable dispersity in aqueous media with good hemocompatibility.


Figure 4. BRD4 degraded ARV-825, Source: reference [2]

Polymeric nanoparticles

Polymer nanoparticles have a variety of structures and properties, and they are made from monomers or polymers derived from natural or synthetic sources. Due to their high biocompatibility, simple preparation process and adjustable physical and chemical properties, polymeric nanoparticles are excellent delivery carriers. These nanoparticles can also carry a variety of therapeutic agents, both small and large molecules.

For decades, a large number of polymeric nanoparticles with high biocompatibility, physiological stability, and multiple functions have been designed and explored for drug delivery. Among them, block copolymers of polyethylene glycol (PEG) and poly(D,L-lactide-co-glycolide) (PLGA) are among the most representative polymeric drug delivery systems and have been approved by the FDA.

A. Sarawat et al. encapsulated bromodomain 4 (BRD4)-degrading PROTACs into PEG-PLGA nanoparticles for the treatment of pancreatic cancer. The nanoparticles protecting ARV-825 from metabolic degradation in a polymer matrix are called ARV-NP (Figure 5), and the appropriate size (<200 nm) allows passive targeting to solid tumors through EPR effects. The PEG-rich surface facilitates prolonged circulation time. Based on in vitro characterization results, ARV-NP exhibited good physical stability, controlled drug release and negligible hemolysis.

Figure 5. ARV-NP, Source: reference [1]

Inorganic nanoparticles

Nanoparticles synthesized from inorganic materials, including gold, iron and silica, can be used as versatile drug delivery systems because of their ability to form a variety of sizes, structures and geometries. Biocompatible and stable inorganic nanoparticle delivery systems can help reduce the risk of unfavorable drug leakage at off-target sites. In particular, gold nanoparticle (GNP) has been widely studied as a drug carrier due to its bioinertness, well-established surface modification method, and versatility to endow additional functionalities.

Wenyuan liu's group at China Pharmaceutical University designed a GNP-based multi-headed PROTAC delivery system (Cer/Pom-PEG@GNP) for the treatment of non-small cell lung cancer (NSCLC) (Figure 6). Anaplastic lymphoma kinase (ALK)-binding warhead (Cer-PEG-SH) and CRBN E3 ligase receptor (Pom-PEG-SH) were conjugated to GNPs with a diameter of ~32 nm via thiol-gold interactions to make a complex of ALK-degrading PROTACs (Figure 5). According to the author, the multi-targeting feature of Cer/Pom-PEG@GNPs promotes the interaction between ALKs and E3 ligases, thus exhibiting better therapeutic effects than small molecule bifunctional PROTACs. Furthermore, the application of GNPs not only modulates the PK difference of PROTACs, but also regulates the tumor-specific enrichment of PROTACs through EPR effects. However, inorganic nanoparticles other than gold have not been used for PROTAC delivery.


Figure 6. GNP-based multi-headed PROTAC. Source: reference [1]

Targeting Moiety-PROTAC Conjugates

Active targeting strategies primarily exploit the specific binding affinity of targeting ligands to cancer overexpressing membrane receptors. By using the active targeting fraction for PROTAC delivery, the poor cellular permeability of PROTAC can be modulated as they are internalized by promoting receptor-mediated endocytosis rather than by a simple transmembrane diffusion pathway. The endocytosed PROTAC is distributed in the cytoplasm, which in turn catalyzes the degradation of POIs. Moreover, active targeting partially promotes the cancer-selective enrichment of conjugated PROTACs and reduces their off-target delivery-induced systemic toxicity. Currently, several active targeting moieties  have been reported for PROTAC delivery, which can be sequentially classified as small molecules, antibodies and aptamers based on their molecular structure.

Small molecule-PROTAC Conjugates

In 2021, J. Liu et al. adopted small molecular folate groups as targeting ligands for cancer-selective PROTAC delivery. The folate group binds to PROTAC molecules by forming ester groups that can be cleaved by intracellular hydrolases. Folate receptors are highly expressed in various cancers (e.g. ovarian, breast, kidney and colorectal cancers) compared to other normal tissues, and thus the "folate caged" PROTACs are specifically taken up by cancer cells in a receptor-dependent manner and successfully mediate POI degradation.


Figure 7. Folate molecules-PROTACs conjugation. Source: reference [1]

Although small molecule ligands can confer a cancer-specific binding ability to PROTAC molecules, the molecular size of these ligands is too small to effectively modulate problems such as poor water solubility of PROTAC molecules.

Antibody-PROTAC Conjugates

Antibodies, which can specifically target antigens on cancer cell membranes, are proteins with average molecular weights of ~150 kDa, approximately 100 times heavier than usual PROTAC molecules. Due to their relatively bulky structure, antibodies not only compensate for the poor solubility of conjugated PROTACs, but also determine PK profiles. In addition to their aggressive tumor-targeting properties, antibodies typically have a longer blood half-life than small molecule ligands and are therefore better suited for drug delivery. There is no more successful case than that of ADC drugs.

In 2020, Edward W. Tate's team first introduced the mAb-PROTAC concept. By combining BRD4-degrading PROTACs with an anti-HER2 monoclonal antibody (trastuzumab), they successfully constructed mAb-PROTACs targeting HER2-positive breast cancer and successfully degraded the BRD4 protein in breast cancer cells (Figure 8). Due to the specific targeting of trastuzumab, mAb-PROTAC conjugates exhibited strong tumor uptake with little internalization into HER2-negative normal cells. After endocytosis of PROTACs into cancer cells, the ester bond with trastuzumab is hydrolyzed, releasing free PROTACs and inducing irreversible BRD4 degradation.


Figure 8. Antibody-PROTACs conjugation. Source: reference [1]

Aptamer-PROTAC Conjugates

Aptamers are ribonucleic acid (RNA)- or deoxyribonucleic acid (DNA)-based targeting moieties with short and single-stranded primary structures. Due to their high binding affinity, stable reproducibility and low immunogenicity, aptamers have been extensively studied in molecular imaging and drug delivery.

In 2021, Chunquan Sheng's group at Second Military Medical University reported that aptamer-PROTAC conjugates composed of BET-degrading PROTACs and nucleolin-targeting aptamers can be used in the treatment of breast cancer. PROTACs are attached to aptamers through short chains containing disulfide groups linked to the aptamer, which can be cleaved in the presence of glutathione (GSH). This aptamer-PROTAC conjugate has significant BET degradation activity exclusively to cancer cells overexpressing nucleoli, favorably modulating the poor solubility and PK profile of PROTACs. In addition, aptamer-PROTAC conjugates are more advantageous than antibody-PROTAC conjugates because they have higher physiological stability and can circumvent the immunogenicity of antibodies.


Figure 9. Aptamer-PROTAC Conjugates, source: reference [3]


PROTAC is considered to be a novel therapeutic strategy with unlimited potential. However, the poor water solubility and low cell permeability of PROTAC contribute to the poor pharmacokinetic (PK) properties, thus limiting the use of PROTAC in the clinic. The development of delivery systems for PROTAC can accelerate its translation from experimental to clinical use. These delivery systems can optimize the physicochemical properties of PROTAC, enable targeted delivery, promote intracellular accumulation and improve its degradation potency.

However, challenges and opportunities exist, and several challenges remain for the delivery of PROTAC. First, additional carrier materials and pharmaceutical excipients may increase the manufacturing costs associated with PROTAC. Secondly, the safety of PROTAC and the corresponding drug delivery system must be strictly guaranteed to optimize their effects while ensuring that their side effects are within acceptable limits.

Although PROTAC-based delivery systems are still in their infancy, the future is promising and more safe and effective PROTAC systems are expected to be developed to improve the physicochemical properties of PROTAC and bring benefits to patients!

As a professional PEG derivatives supplier, Biopharma PEG has been focusing on the development of a full range of medical applications and technologies for nanocarrier systems, including various types of nanoparticles, liposomes, micelles, etc. We are committed to providing a variety of PEG-liposome derivatives, including mPEG, DSPE lipids with different molecular weights and functional PEG. We can also produce and provide some PEG products used as PROTAC linkers.

[1] Moon, Y.; Jeon, S.I.; Shim, M.K.; Kim, K. Cancer-Specific Delivery of Proteolysis-Targeting Chimeras (PROTACs) and Their Application to Cancer Immunotherapy. Pharmaceutics 2023, 15, 411.
[2] Chen, Y.; Tandon, I.; Heelan, W.; Wang, Y.; Tang, W.; Hu, Q. Proteolysis-Targeting Chimera (PROTAC) Delivery System: Advancing Protein Degraders towards Clinical Translation. Chem. Soc. Rev. 2022, 51, 5330–5350.
[3] He S, Gao F, Ma J, Ma H, Dong G, Sheng C. Aptamer-PROTAC Conjugates (APCs) for Tumor-Specific Targeting in Breast Cancer. Angew Chem Int Ed Engl. 2021;60(43):23299-23305. doi:10.1002/anie.202107347

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