PROJECT DESCRIPTION

A Global Problem

The term "cancer" comes from the Latin word for "crab", first used by the ancient Greek physician Hippocrates (460-370 BC) to describe tumors, owing to the finger-like spreading projections that resemble a crab's claws [1]. Cancer is characterized by the uncontrolled growth and spread of abnormal cells leading to death. This abnormal cell proliferation can occur in nearly any tissue or organ in the body, manifesting in various forms. A significant feature of cancer is its ability to invade surrounding tissues and metastasize to distant sites via the bloodstream or lymphatic system [2].

Figure 1: Different Characterizations of Cancer

Despite the diversity in cancer types and behaviors, the common thread among them all is their significant impact on global health. Cancer remains a major public health challenge, exerting a heavy toll on individuals, families, and healthcare systems worldwide. The growing burden reflects population aging, growth, and increased exposure to risk factors like tobacco, alcohol, obesity, and air pollution. High incidence and prevalence rates across populations represent the persistent challenge it poses which outlines the urgent need for a comprehensive approach to cancer control. Increasing prevention efforts, improving early detection and diagnosis, developing advanced treatments, and investing in cancer research are crucial strategies to combat the escalating burden of cancer [3].

Figure 2: Growing Burden of Cancer

Current Treatment Options

Figure 3: Different ways that currently exist to treat cancer

Cancer treatments have advanced significantly, providing various options that target the disease in different ways. However, each treatment has its own limitations. Common treatments like chemotherapy and radiation can also damage healthy cells, leading to adverse side effects due to their non-specificity. As the cancer cells can mutate, they develop resistance to treatments over time, which impedes the effectiveness of these therapies [4]. The genetic diversity of the tumors makes it difficult to eliminate them with a unilateral approach. Furthermore, advanced treatments are often expensive and not easily accessible, resulting in unequal patient outcomes. Given the complexity and diversity of cancer, it is essential to develop more precise treatments and to overcome these limitations [5].

Promising Prospect

Targeted drug therapy offers an encouraging approach to treat cancer by minimizing damage to healthy tissues whilst maximizing efficacy against malignant cells. Over the years, various methods have been developed for this purpose.

Figure 4: The different kinds of Targeted Therapies that are currently being tested [6]

These methods offer several advantages, such as specificity, effectiveness, personalized treatment, and reduced toxicity [7].

Among these methods, antibody-drug conjugates (ADCs) provide an advanced targeted drug delivery method which utilize monoclonal antibodies and allow for the delivery of cytotoxic drugs to the tumor site with the least side effects providing unmatched specificity and precision. Upon binding to the cancer cell surface, the ADC is internalized, leading to the release of the cytotoxic payload within the cell. This targeted approach minimizes damage to healthy tissues, enhances the efficacy of chemotherapy, and reduces systemic toxicity by selective delivery [8].

Challenges associated with Targeted Therapy

Although ADCs show promise, they face several challenges that must be addressed to ensure optimal efficacy and safety. One issue is with extravasation, which is the leakage of the drug from blood vessels into surrounding tissues, affecting how the drug is distributed within the body. Additionally, ADCs can cause immune reactions, making the treatment less effective or causing side effects. There are also limitations in the versatility of linkers and payloads with current site-specific conjugation methods, which restrict the ability to design more effective drugs. Furthermore, there is the risk of immunogenicity, where the body’s immune system reacts against the treatment, potentially leading to adverse reactions. Finally, the extensive bioengineering steps required to develop ADCs lead to higher costs, making them less accessible. [9] Addressing these challenges is crucial for improving the effectiveness and safety of ADCs [10].

Inspiration: Liquid-Liquid Phase Separation

These challenges led us to explore alternative possibilities for targeted drug delivery since its practical applications appeared limited. Through ongoing research at Aalto University, we were introduced to the phenomenon of Liquid-Liquid Phase Separation (LLPS). LLPS occurs when a solution separates into two distinct liquid phases due to specific molecular interactions. In biomaterials and proteins. It is crucial for assembling soluble proteins into complex structures. It drives the formation of dense liquid droplets or coacervates, which concentrate and organize biomolecules, thereby facilitating the development of functional materials and adhesive systems [11].

LLPS: Potential as a Drug Delivery System

Utilizing LLPS as a foundation for a drug delivery system offers a significant advancement in targeted drug delivery. This approach retains all the benefits of targeted delivery while avoiding its associated drawbacks. It effectively compartmentalizes small-molecule drugs, providing protection against harsh conditions and ensuring the encapsulation of active substances. LLPS system allows the enhancement of drug stability and bioavailability, improving drug efficacy by protecting against degradation, and controlling the release of active compounds by delivering it directly to tumors and reducing uptake by healthy tissues. [12] By encapsulating a drug within an LLPS carrier, we aim to achieve targeted delivery with a potentially simpler design compared to ADCs. This could lead to a more cost-effective and efficient approach. Therefore, utilizing our LLPS system with this drug offers an exciting opportunity to develop a more efficacious, targeted, and potentially more affordable treatment option.

Figure 5: How LLPS formation occurs

Doxorubicin

In order to test our LLPS inspired drug delivery system, we opted to choose a well-known chemotherapeutic agent, Doxorubicin as our model drug. It is a potent anthracycline antibiotic with a proven track record against a wide range of cancers [13]. However, its clinical application is hampered by significant dose-dependent side effects, particularly cardiotoxicity, which arise from its non-specific distribution throughout the body [14].

Current attempts to improve Doxorubicin delivery include the well-known targeted delivery methods like using ADCs. In this system, the ADCs link Doxorubicin to tumor-specific antibodies, aiming to deliver the drug directly to cancer cells. While ADCs offer targeted delivery, they have several limitations. Their complex manufacturing processes can significantly increase their cost [15]. Moreover, the large size of ADCs can limit their ability to penetrate deeply into tumors, potentially reducing their efficacy [16]. Furthermore, there can be challenges in ensuring consistent drug-to-antibody ratios within ADC populations, which can affect their therapeutic effectiveness [17].

COALES: Clinical On-Target Adhesive LLPS Engineered System

To address the challenges of existing targeted therapies and improve drug delivery systems, we introduce COALES, a liquid-liquid phase separation (LLPS)-based approach. LLPS is formed by combining two bio-adhesive proteins—MFP-1 (Mussel Foot Protein-1) and ADF-3 (Araneus diadematus fibroin-3)—in a 2:1 stoichiometry via the SpyTag/SpyCatcher system. This process results in two distinct phases: MFP-1 forms the continuous phase, providing structural stability, while ADF-3 forms the dispersed phase, which carries the therapeutic payload. [18]

To begin, a tumor-homing peptide (p160) is integrated into the mussel foot protein (MFP-1) component, which ensures that the system specifically targets and binds to cancer cells.[19,20] The addition of the homing peptide allows us to target cancer cells, which means the necessary dosage of the drug can be reduced and its targeting efficacy can be increased, thereby improving treatment outcomes and minimizing harm to healthy tissues.

ADF-3, on the other hand, is modified with the CfaN intein to serve as the protein scaffold for the drug component. Inteins are self-catalyzing protein sequences that facilitate a controlled splicing event, joining two protein components (CfaC and CfaN in this case) in a highly specific manner. By employing inteins, we ensure a more consistent drug-to-carrier ratio, which provides better control over dosing and enhances the reliability of the drug release. [21] In this system, to further enhance control doxorubicin is conjugated to a separate payload component, CfaC-CGG8, via a pH-sensitive linker, EMCH (N-ε-maleimidocaproic acid hydrazide).[22] Intein sequences are utilized to create a specific and controlled linkage between CfaC and CfaN, allowing for precise conjugation and control over the ratio of drug to protein, which would have been difficult to achieve with traditional chemical conjugation methods.

Additionally, the system incorporates pH-dependent membrane-active peptides (PMAPs) to facilitate endosomal escape.[23] Upon targeting cancer cells and undergoing endocytosis, the LLPS system uses the PMAPs and endosomal protease cleavage sites to prevent degradation within the endosome. In the acidic tumor microenvironment, the pH-sensitive EMCH linker triggers the release of doxorubicin, ensuring the drug is only released at the desired site. This design enhances the drug's therapeutic efficacy while reducing systemic toxicity, particularly in the acidic conditions (pH 6.5-6.8) of the tumor compared to normal physiological pH.[24]

Conclusion

As we explore the complexities and challenges of cancer, solutions like COALES offer promising advancements in drug delivery. The key strength of our drug delivery system is its flexibility to adjust component ratios and integrate multiple drugs into one system. This capability allows us to enhance combination therapies effectively. By targeting different aspects of disease biology simultaneously, this approach offers a promising way to improve treatment outcomes.

While initially, we are focusing on doxorubicin for cancer treatment, the adaptability of our system positions it for broader applications across various drugs and diseases. It can be used as a personalized and effective therapeutic approach for a wide spectrum of medical conditions, not just cancer. Hence, as we continue to our advancement, COALES represents a promising avenue in enhancing targeted drug delivery, offering hope for more effective, safer, and accessible therapies in the fight against cancer and beyond.