BACKGROUND OF INVENTION

The design and synthesis of novel nanomaterials that enhance or assist the passage of biologics, such as proteins, enzymes, and antibodies, across cellular membranes is of significant importance. Cell membranes are impermeable to most macromolecules. Many drug candidates fail to advance clinically because they do not have the properties needed to cross biological membranes and reach their intracellular target. Additionally, poor pharmacokinetics, stability, and off-target effects lead to undesirable biological responses.

The cytosolic delivery of functional proteins could lead to an efficient and specific control of cellular processes for various disease treatments. Despite the success and promise of the first protein-based drugs, current protein-based therapeutics are mainly designed to target the extracellular receptors or secretory proteins due to the cell membrane impermeability of most proteins.

Moreover, many standard laboratory techniques, such as immunostaining and gene editing, may require the intracellular accumulation of proteins inside the cell. A major bottleneck in the advancement of biologics development is due to the membrane impermeability of most proteins, and a lack of effective approaches to delivering proteins to intracellular targets. Current commercially available materials face severe limitations for use, prompting the need for improved protein delivery systems.

The covalent modification of proteins with membrane translocating moieties has been used, however, the risk of altered structure and function has led to significant focus on non-covalent methods. Lipids, peptides, and polymers all rely on relatively weak, non-covalent interactions between the carrier and cargo. The synergistic inter-macromolecular ionic, hydrophobic, and hydrogen bond interactions are essential to achieve proper complexation between the carrier and cargo, interaction with the cellular membrane, and release of the protein inside the cell.

Numerous synthetic polymeric carriers have been developed for the transport of proteins across cell membranes. Specifically, guanidine-rich polymers, relying on the key functional group of cell-penetrating peptides, have gained attention. Due to the unique delocalized positive charge of guanidine, strong ionic and hydrophobic interactions exist with the biomacromolecule cargo. Additionally, the capability of bidentate hydrogen bond interactions assists in the assembly of complexes and cellular entry, especially when multiple guanidine units collectively interact with biomacromolecules in a relatively hydrophobic environment.

Despite the significant advantages, the high positive charge density of guanidine-rich carriers, often results in poor complex stability due to non-specific interactions with other macromolecules in physiological relevant environments, resulting in poor outcomes. For example, the same positive charge is often responsible for nonspecific binding with serum proteins, resulting in destabilization of protein/carrier complexes, alteration of cellular entry pathways, and diminishing overall intracellular entry efficiency. The hydrogen bond interactions in aqueous environments are also dramatically weakened as a large excess of water molecules strongly interacts with guanidine. Therefore, guanidine-based protein delivery systems perform poorly in serum-containing media.

Thus, there is a need to develop novel delivery materials, such as guanidine-rich compounds, carriers or polymers with improved efficacy and balanced hydrophobicity, which overcome the biological barrier. There is also a need to develop methods and approaches for modification and synthesis of guanidine derivatives with improved efficacy.

BRIEF SUMMARY

The subject invention provides materials and methods for intracellular delivery of molecules or therapeutic agents such as drugs, nucleic acids, and proteins. In one embodiment, the subject invention provides nanomaterials as molecule transporters or carriers for targeted delivery of therapeutic agents into cells, preferably, cancer cells for inhibiting the growth of cancer cells and altering gene expression in these cells.

In one embodiment, the nanomaterial of the subject invention comprises a polymer or nanoparticle that comprises a modified guanidine moiety in a plurality of repeating units of a polymer, or on the surface of a nanoparticle.

In one embodiment, the modified guanidine moiety comprises a direct conjugation of a planar carbamoyl group to guanidine, which decreases the pKa, leading to a decreased positive charge environment. Additionally, the hydrogen bonding moiety is extended by the carbamoyl group. This reduced positive charge density, together with new hydrogen bond site, can lead to enhanced interactions between the carrier and cargo, and ultimately, improved protein delivery efficacy.

In a specific embodiment, the nanomaterial or polymer of the subject invention is a guanidylcarbamoyl or carbamoylguanidine derivative. Advantageously, the nanomaterials or polymers having such novel functional groups dramatically enhance intracellular biologics delivery efficiency by improving serum stability, intracellular entry, and release of payloads. Thus, the nanomaterials and polymers of the subject invention function well in the absence or presence of serum, which is superior compared to the existing cargo delivery materials.

For example, the coplanar phenyl group connected carbamoylguanidine derivative exhibits efficient delivery of proteins with various sizes and isoelectric points. The carbamoylguanidine derivative successfully delivers apoptosis-inducing proteins even in a serum containing medium, as compared with most protein delivery systems that show a sharp efficiency decrease in the presence of serum.

In one embodiment, the subject invention provides a guanidine modification to substantially improve functional protein delivery efficiency. The decreased pKa of planar carbamoylguanidine increases local hydrophobicity and hydrogen bond interactions, allowing the formation of stable protein complexes with improved complex stability in a serum-containing medium. The coplanarity of the phenyl group directly introduced to carbamoylguanidine plays a significant role in the cellular entry. The developed functional groups can be introduced to many existing biologic delivery platforms to tackle the issues associated with therapeutic protein delivery.

