Advancing Precision Medicine: Protein Engineering and Nanoparticle Delivery in Modern Therapeutics

Proteins are fundamental in the regulation of cell processes, playing roles that span from directing complex signaling pathways to overseeing critical physiological functions. Any disruption in protein function—whether due to genetic mutations, structural malformations, or imbalances in cellular environments—can cascade into severe diseases, including various cancers. The discovery of this connection between protein function and health has driven research into protein therapy, a highly specific treatment approach. Unlike traditional drugs, which often affect multiple pathways and bring unwanted side effects, protein therapy offers precision. It targets malfunctioning proteins directly, either by replacing defective proteins or by recalibrating cell signaling to restore health. This therapeutic precision could transform treatment options for genetic disorders and conditions that have evaded traditional therapies.

However, for protein therapy to be effective, proteins must be delivered to the exact cellular and subcellular locations where they’re needed, which remains a significant challenge. Proteins are diverse molecules, with each type differing in size, charge, and solubility. These differences influence how proteins interact with cell membranes and determine whether they can penetrate cells. Unfortunately, most proteins are not naturally cell-permeable, making it difficult to get them across the cell membrane. Even when researchers do achieve intracellular delivery, targeting proteins to specific organelles, like the nucleus or mitochondria, adds another layer of complexity. Consequently, researchers are pursuing new methods for efficient, targeted protein delivery, as the efficacy and potential of protein-based treatments hinge on overcoming these barriers.

The rise of CRISPR/Cas9 technology has brought new hope to the field, as it provides a molecular “scalpel” for editing genes within mammalian cells. This technology allows scientists to directly manipulate genetic material, opening up potential treatments for previously untreatable genetic disorders. However, CRISPR’s promise is only partially realized until we can reliably deliver Cas9 and guide RNAs into cells and ensure they reach the right subcellular compartments. The precise delivery of therapeutic proteins is no longer just an aspiration—it is an urgent requirement in the development of next-generation treatments.

Nanoparticle Strategies for Enhanced Protein Delivery

Protein therapy’s potential hinges on our ability to deliver these biomolecules effectively into cells and, even more critically, into targeted subcellular compartments. Over recent years, researchers have explored various approaches to accomplish this. Viral nanoparticles initially attracted attention due to their potential to deliver therapeutic proteins directly into cells, leveraging viruses’ natural ability to invade host cells. However, the use of viral vectors raised significant concerns regarding safety and immune response, as they may trigger immune reactions or insert genetic material in ways that could lead to unintended effects, limiting their use in the clinic.

Consequently, attention has turned to non-viral carriers, such as lipid-based nanoparticles. These carriers are appealing because they are versatile and can encapsulate different therapeutic agents. Liposomes and lipid nanoparticles, traditionally used for gene delivery, have garnered interest for protein delivery as well, given their ability to protect encapsulated biomolecules and release them under controlled conditions. But proteins, with their complex chemical structures, pose unique challenges that gene-based approaches do not encounter. For instance, each protein’s hydrophobic or hydrophilic nature influences its compatibility with lipid-based carriers. Traditional lipid nanoparticles must be fine-tuned for each protein, complicating the design of a single solution for multiple types of proteins.

To address these limitations, researchers have pioneered a combinatorial library approach. By synthesizing a wide array of lipid nanoparticles with varying head groups and tail lengths, they can systematically screen for the best formulations to encapsulate and deliver specific proteins. This strategy not only identifies effective lipid-protein combinations but also provides insights into the interactions between lipid structures and protein stability. By studying these structure-activity relationships, scientists are building a foundation for designing nanoparticles tailored to the unique requirements of each therapeutic protein. This customized approach represents a significant step forward in achieving efficient, stable, and specific protein delivery.

Innovations in Lipid Nanoparticles for Targeted Delivery

The evolution of cationic lipid nanoparticles for gene delivery laid the groundwork for adapting this technology to protein delivery. The recent FDA approval of Patisiran, a cationic lipid-siRNA formulation, for TTR-mediated amyloidosis demonstrates the viability of lipid nanoparticles in treating genetic diseases. Yet, while this success is encouraging, the intricacies of protein delivery present additional challenges. Unlike nucleic acids, proteins vary greatly in their size, shape, and chemical properties, which complicates the task of developing a universal delivery method.

One of the key challenges in protein delivery is maintaining the integrity of these complex molecules during and after encapsulation in lipid nanoparticles. Any alteration in a protein’s three-dimensional structure can compromise its function, potentially nullifying its therapeutic effects. Moreover, proteins often need to be released in specific cellular compartments to perform their functions effectively. For example, some therapeutic proteins must reach the cell nucleus to exert their effects, while others may need to localize to the mitochondria. This level of precision requires nanoparticles that are not only capable of carrying proteins but also engineered to release them in response to cellular cues.

Researchers have addressed these challenges by modifying lipid nanoparticle structures to increase delivery efficiency and targeting precision. By introducing chemical modifications to lipid tails, such as unsaturated or bioreducible hydrophobic chains, scientists have enhanced the nanoparticles’ ability to release encapsulated proteins inside cells. This improvement in intracellular delivery has proven particularly valuable for cancer therapies, where the ability to specifically target tumor cells while sparing healthy cells is essential. Such advances underscore the critical role of lipid design in developing effective protein therapies, as the structural diversity within combinatorial lipid libraries allows researchers to match the right carrier with the right therapeutic molecule.

Engineered Lipids for Efficient Cytotoxic Protein Delivery

The development of combinatorial lipid libraries has unlocked new possibilities for the intracellular delivery of cytotoxic proteins, such as RNase A and saporin. These proteins can induce cell death by interfering with fundamental cellular processes—RNase A by degrading RNA and saporin by inactivating ribosomes. When delivered to the cytosol, these proteins trigger apoptosis, a programmed form of cell death, which is a promising strategy for treating cancer. However, the challenge lies in achieving efficient delivery without affecting non-target cells or tissues.

By screening a combinatorial library of synthetic lipids, researchers identified specific lipid formulations capable of delivering cytotoxic proteins effectively. The screening revealed that variations in lipid head amine structures and tail lengths significantly influence delivery efficiency. Proteins with high isoelectric points, such as RNase A and saporin, interact differently with lipid nanoparticles based on their net charge and hydrophobicity. In tests with cancer cell lines, certain lipids exhibited greater affinity for saporin, indicating that hydrophobic interactions between the protein and lipid tail play a crucial role in successful delivery.

The implications of these findings extend beyond saporin and RNase A. The combinatorial approach allows scientists to investigate how slight alterations in lipid structure affect protein delivery and to optimize nanoparticle formulations for a broad range of therapeutic proteins. This method has already yielded lipid-protein combinations that inhibit tumor growth in animal models, underscoring the therapeutic potential of lipid nanoparticles as vehicles for protein delivery. As the library expands, researchers anticipate developing increasingly precise and effective cancer treatments, pushing the boundaries of protein therapy.

Reversible Chemical Modifications: Enhancing Protein-Lipid Complexation

The diversity in protein structures necessitates strategies that enhance their compatibility with lipid nanoparticles. Chemical modification is one such strategy, allowing researchers to alter a protein’s surface properties to improve interactions with lipid carriers. One approach is to adjust a protein’s charge, which can strengthen electrostatic interactions with cationic lipid nanoparticles. Modifying proteins with cis-aconitic anhydride, for example, converts positively charged lysine residues into negatively charged carboxylates, enhancing the protein’s binding to lipid nanoparticles.

This approach has proven especially effective for proteins that struggle with cellular uptake in their native form. Notably, researchers observed a significant increase in cellular delivery and activity with modified proteins, as the charge alterations enhance endosomal escape once inside cells. Additionally, this modification is pH-sensitive; in the acidic environment of the endosome, the cis-aconitic amide linkage is cleaved, restoring the protein’s native structure and activity. This precision engineering ensures that proteins reach their target in a functional state, maximizing their therapeutic potential.

Moreover, the use of hyaluronic acid (HA) for protein modification has introduced a dual-function approach. HA not only enhances a protein’s electrostatic interaction with lipid nanoparticles but also targets CD44 receptors, which are overexpressed in many solid tumors. This dual modification directs therapeutic proteins specifically to cancer cells, sparing healthy cells. This targeted approach has shown promise in preclinical models, where HA-conjugated proteins demonstrated increased efficacy against tumor cells, highlighting the potential of chemical modification to improve the selectivity and impact of protein-based treatments.

Boronic Acid Conjugation for Cancer-Specific and Nuclear Targeting

Building on the success of chemical modification, researchers have explored boronic acid conjugation to enhance the selectivity and functionality of therapeutic proteins. Boronic acid exhibits unique reactivity to reactive oxygen species (ROS), particularly hydrogen peroxide, which is elevated in many cancer cells. By conjugating therapeutic proteins with boronic acid, researchers have developed a method to activate these proteins specifically within the ROS-rich environment of tumors. This strategy minimizes off-target effects, as the proteins remain inactive in normal cells with lower ROS levels, thus providing a selective approach to cancer treatment.

Beyond cancer cell targeting, boronic acid conjugation offers an innovative solution for targeting proteins to specific subcellular compartments. Nuclear localization is critical for therapies involving gene regulation or genome editing, as proteins must reach the nucleus to perform their intended functions. Boronic acid conjugation has shown potential as a nonpeptide nuclear localization signal, guiding proteins to the nucleus through active transport pathways. In tests with green fluorescent protein (GFP) conjugated with boronic acid, researchers observed efficient nuclear accumulation, which is essential for treatments requiring precise genomic intervention.

This dual functionality of boronic acid—targeting cancer cells based on ROS sensitivity and directing proteins to the nucleus—positions it as a powerful tool in therapeutic protein design. By leveraging boronic acid’s chemical properties, researchers can engineer proteins that are not only highly selective but also precisely localized, paving the way for advanced cancer therapies and gene-editing applications.

Expanding CRISPR/Cas9 Delivery through Engineered Lipid Nanoparticles

The CRISPR/Cas9 genome-editing system has revolutionized molecular biology by enabling precise modifications to the genetic code. This capability holds enormous potential for treating genetic disorders, but the practical application of CRISPR relies on effective delivery methods. Delivering Cas9 protein and guide RNA (gRNA) into cells and targeting them to the nucleus is challenging due to the size and charge of these molecules. Researchers have turned to engineered lipid nanoparticles as a solution, adapting the combinatorial lipid strategy for CRISPR delivery.

By designing bioreducible lipids that degrade within the cell, scientists have created nanoparticles that can encapsulate and release Cas9 and gRNA upon cellular entry. These lipids feature disulfide bonds that break down in the presence of cellular reducing agents, facilitating Cas9 release from the nanoparticle complex. The screening of various bioreducible lipids has yielded formulations capable of delivering Cas9 and gRNA with high efficiency, comparable to commercial transfection agents.

In addition to Cas9, researchers have demonstrated the efficacy of these nanoparticles for other genome-editing proteins, such as Cre recombinase. The ability to target and edit genes with high precision in vivo has been a major achievement, demonstrating the feasibility of using lipid nanoparticles for CRISPR delivery. This technology promises to expand the therapeutic applications of CRISPR/Cas9, offering a non-viral, biocompatible solution that could be adapted for a range of genetic interventions.

Looking Forward: Integrating Chemistry and Nanotechnology for Precision Medicine

The convergence of lipid nanoparticle engineering and protein modification strategies is reshaping the landscape of protein therapy and gene editing. Future advancements are likely to focus on refining nanoparticle designs to improve targeting specificity and delivery efficiency. Advanced imaging techniques, such as quantitative fluorescence and electron microscopy, will provide insights into protein localization within cells, guiding further optimization of lipid structures for specific therapeutic applications.

In addition to refining lipid nanoparticles, expanding the chemical toolkit for protein modification will unlock new therapeutic possibilities. Techniques that allow for site-specific modifications, such as the incorporation of non-natural amino acids, will enable researchers to precisely control protein structure and function in situ. This precision will be essential for developing therapies that are both highly effective and minimally invasive.

Ultimately, the integration of combinatorial lipid nanoparticle synthesis with advanced protein modification techniques promises to bring a new generation of protein therapeutics to the clinic. By leveraging the strengths of chemistry, nanotechnology, and molecular biology, researchers are developing tools to treat diseases with a level of specificity and efficacy previously thought unattainable. As these technologies continue to evolve, they hold the potential to transform the way we approach medicine, offering hope for targeted treatments that address the root causes of disease.

Study DOI: https://doi.org/10.1021/acs.accounts.8b00493

Engr. Dex Marco Tiu Guibelondo, B.Sc. Pharm, R.Ph., B.Sc. CpE

Editor-in-Chief, PharmaFEATURES

Register your interest [here] at Proventa International’s Clinical Operations and Clinical Trials Supply Chain Strategy Meeting this 14th of November 2024 at Le Meridien Boston Cambridge, Massachusetts, USA to engage with thought leaders and like-minded peers on the latest developments in the clinical space and regulatory affairs.