Bioconjugation for Tumor Targeting

Bioconjugation involves the chemical linkage of biomolecules, such as antibodies, peptides, or drugs, to form a composite molecule with enhanced functional properties. An ideal therapeutic bioconjugate may consist of a targeted component coupled to a drug or effector molecule, which can deliver an active payload directly to tumor cells, attack invading pathogens, or target other parts of the body for treatment with virtually no binding or toxicity toward non-target cells and organs.

Targeted bioconjugates are also used in in vivo diagnostic procedures to detect and image tumor sites, and in vitro assays to quantify disease markers or drug concentrations in a patient's serum. The specificity and sensitivity of many diagnostic procedures depend on the development of the best biological conjugate that can interact with a specific target and provide sensitive detection capabilities.

Advantages of bioconjugates for tumor targeting

Compared to traditional drugs, bioconjugated drugs have considerable advantages in addition to targeted delivery. Proper bioconjugate design can improve chemical stability, prevent proteolysis or degradation in vivo, increase the half-life of the drug in vivo, reduce off-target effects, reduce unwanted immunogenicity, increase water solubility, reduce drug removal from the kidney or liver, increase specificity of interaction with the desired target, and increase potential cell penetration through endocytosis

Polymer scaffolds and nanoparticles for drug delivery

Polymers in drug delivery systems (PDDS)

Polymer-based drug delivery systems (PDDS) are designed to encapsulate chemotherapeutic agents within biocompatible and biodegradable matrices, which protect the drug from premature degradation in the bloodstream. The structure and composition of these polymers are critical for determining their behavior in vivo. Broadly, PDDS can be classified into four categories based on their structure: linear polymers, branched polymers, nanoparticles, and dendrimers. Each structure offers unique advantages and challenges.

Linear polymers offer simplicity in design and predictable degradation patterns. However, branched and dendritic polymers provide higher drug-loading capacities due to their increased surface area and multiple functional sites. These architectures allow for the conjugation of targeting ligands, such as antibodies or peptides, which enable selective binding to tumor cells. Dendrimers, with their highly branched, tree-like structures, also facilitate multivalent binding, which can significantly enhance the strength of interactions with tumor-specific biomarkers.

Natural polymers, such as hyaluronic acid and chitosan, are often selected for their biocompatibility and inherent biological activity, which can further support drug delivery. Alternatively, synthetic polymers like poly(ethylene glycol) (PEG) and poly(lactic-co-glycolic acid) (PLGA) are favored for their tunable properties, including controlled degradation rates and ease of functionalization.

Advantages of nanoparticles for tumor targeting

Nanoparticles, typically ranging from 10 to 200 nanometers in diameter, have emerged as highly effective drug carriers due to their ability to penetrate biological barriers and accumulate in tumor tissues through the enhanced permeability and retention (EPR) effect. This phenomenon occurs because tumors often have leaky vasculature and poor lymphatic drainage, allowing nanoparticles to passively accumulate in the tumor microenvironment, where they release their therapeutic payloads.

Nanoparticles also provide a platform for active targeting through surface modification. By attaching ligands such as antibodies, peptides, or small molecules, nanoparticles can be directed to specific tumor-associated receptors, thereby enhancing the precision of drug delivery. In addition, nanoparticles can be engineered to respond to specific stimuli in the tumor microenvironment, such as pH, temperature, or enzymatic activity. This capability allows for controlled, site-specific drug release, reducing systemic side effects and enhancing the therapeutic index.

Furthermore, nanoparticles improve the solubility and bioavailability of hydrophobic drugs, which are often poorly soluble in aqueous environments. Many conventional chemotherapeutic agents suffer from limited water solubility, necessitating the use of toxic solubilizing agents. Nanoparticle encapsulation circumvents this issue by embedding the hydrophobic drug within a hydrophilic shell, allowing for safer and more efficient drug delivery.

Functionalization of polymers for enhanced targeting

For instance, antibodies or small molecules that recognize tumor-specific antigens can be covalently linked to the polymer scaffold, ensuring that the drug delivery system homes in on cancerous cells. This targeted approach not only increases the drug concentration at the tumor site but also minimizes off-target effects, reducing the toxicity to normal tissues.

Functionalization is not limited to targeting ligands; polymers can also be tailored to improve drug pharmacokinetics. By modifying the polymer with PEG (polyethylene glycol) chains, for example, the resulting "PEGylated" nanoparticles evade immune detection, allowing for extended circulation times and enhanced tumor accumulation. Additionally, functional groups can be added to the polymer backbone to facilitate triggered drug release. These triggers can be environmental stimuli, such as a lower pH in the tumor microenvironment or specific enzymes that are overexpressed by cancer cells. Upon encountering these conditions, the polymer scaffold degrades or changes conformation, releasing the encapsulated drug at the tumor site.

Incorporating multiple drugs into a single nanoparticle or polymer scaffold allows for combination therapies, where different drugs with complementary mechanisms of action are delivered simultaneously to the tumor.

Antibody-drug conjugates (ADCs)

ADCs are composed of three key components: the monoclonal antibody, the cytotoxic drug, and a linker that connects them. The antibody is designed to specifically bind to antigens that are overexpressed on the surface of cancer cells. Once the ADC binds to the target antigen, it is internalized by the cancer cell through endocytosis. Inside the cell, the linker is degraded, releasing the cytotoxic drug, which then exerts its lethal effect by disrupting critical cellular processes, such as DNA replication or protein synthesis, leading to cancer cell death.

Structural characteristics of ADC. (Thurston, D. E., 2018)

The specificity of the antibody ensures that the cytotoxic drug is delivered primarily to cancer cells, reducing the exposure of normal cells to the toxic effects of the drug. This targeted approach is key to the therapeutic potential of ADCs, minimizing systemic toxicity and enhancing the safety profile of cancer treatments.

While ADCs hold great promise, their development presents several technical challenges. One of the main hurdles is achieving an optimal balance between the stability of the ADC in the bloodstream and efficient release of the drug within the target cell. The linker plays a crucial role in this balance. If the linker is too stable, the drug may not be released effectively inside the cancer cell, reducing the ADC's therapeutic efficacy. Conversely, if the linker is too unstable, the drug could be released prematurely in circulation, leading to off-target toxicity. Linkers can be designed to respond to specific intracellular conditions, such as the acidic environment inside lysosomes or the presence of certain enzymes, to ensure the drug is released only within the cancer cells.

Another challenge is the heterogeneity of antigen expression among tumors. The success of ADCs depends on the presence of specific antigens on cancer cells, but not all tumor cells express these antigens at the same levels. This can lead to incomplete targeting, where some cancer cells evade treatment. Additionally, drug resistance mechanisms, such as reduced internalization of ADCs or increased drug efflux, can limit the effectiveness of these therapies.

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Antibody-directed enzyme prodrug therapy (ADEPT)

ADEPT is designed to selectively activate cytotoxic agents within the tumor environment, minimizing damage to healthy tissues. This approach involves the administration of a non-toxic prodrug that is converted into an active drug only at the tumor site, using an antibody-enzyme conjugate as a targeting mechanism. Antibody components can interact specifically with target tumor cells through characteristic biomarkers on the cell surface, but neither the enzyme nor the antibody has any direct therapeutic activity against the tumor. During treatment, an antibody conjugate is administered to the patient, in which the conjugate finds and binds to tumor cells, and an excess of the conjugate is allowed to clear the system before prodrug is given. The prodrug itself also has no initial therapeutic activity or cytotoxicity because chemical modifications make it harmless. However, when the front drug reaches tumor cells containing the bound ADEPT conjugate, the enzyme acts on the compound, eliminating the chemical modification and initiating the anti-tumor activity of the drug. Therefore, the design of the prodrug must take into account the enzyme used in order to successfully catalyze the removal of its modifying group while producing an active therapeutic agent.

Radiolabeled bioconjugates for tumor imaging and therapy

By attaching radioactive isotopes to antibodies or peptides, these bioconjugates can precisely target tumor cells while minimizing damage to healthy tissues. Radiolabeled bioconjugates are particularly valuable for both tumor imaging and targeted radiotherapy, providing a dual functionality that improves the accuracy and effectiveness of cancer treatment.

The preparation of bioconjugants for radioimmunotherapy involves attaching radioisotopes to antibodies or other targeting molecules, such as peptides or ligands. It can be achieved by the following strategies: (1) Directly modifying amino acid side chains in proteins, peptides or ligands with one or more radioactive atoms. (2) Indirect modification of target molecules by the coordination of bifunctional chelate compounds and the carrying of radioisotopes. (3) by using a polymer carrier or nanoparticle attached to the target molecule, where the carrier is modified to contain the radiolabelling. In each case, one or more radionuclides attach to the targeted molecule to produce a therapeutic agent that delivers a highly concentrated dose of radiation to the tumor site.

One major advantage of radiolabeled bioconjugates is their ability to deliver highly concentrated doses of radiation directly to the tumor microenvironment. The antibody or peptide component ensures that the radiation is delivered selectively to cancer cells that overexpress the target antigen, while the radionuclide emits therapeutic radiation that damages the cancer cell's DNA, inducing cell death. This targeted approach minimizes collateral damage to healthy tissues and allows for the treatment of tumors that are not easily reached by other means.

In cases of metastatic cancer, where tumor cells have spread to multiple locations in the body, radiolabeled bioconjugates offer a systemic treatment option that can seek out and destroy cancerous cells wherever they are located. This makes radiolabeled bioconjugates a valuable complement to other cancer therapies, such as chemotherapy and immunotherapy, which may not be as effective in targeting dispersed tumor cells.