Periodic Reporting for period 1 - DynaPIC (Dynamic bonds and polyion complex (PIC) nanoparticles for targeted intracellular peptide delivery)
Reporting period: 2021-01-01 to 2022-12-31
Cytotoxic drugs (i.e. molecules that damage and kill cells) are therapeutic agents with a long history in clinical application. These compounds cause structural damage to cell membranes, being useful as antitumoral and antimicrobial agents due to their tumor-lytic and antimicrobial effects, respectively. However, cytotoxic agents are generally unspecific in action, and they can cause severe toxicity to patients due to indiscriminate killing of host cells. Thus, to unlock the full therapeutic potential of cytotoxic drugs, strategies to control and direct their activity are needed.
Nanomaterials have been widely explored as carriers for the targeted delivery of cytotoxic agents. Capitalising on certain pathological markers, these nanovehicles can release in situ the active cell-damaging molecules they carry, thus minimising off-target interactions and preventing toxicity to the host. Despite decades of development, poor reproducibility in nanomaterial formulation and activity still hampers their widespread application in clinic. Alternatives to these nanocarriers are required as innovative mechanisms to control the selectivity of cytotoxic drugs.
This project aims to tune the cytotoxic effects of a model drug in response to acidic microenvironments, which are characteristic of certain pathologies like cancer and infection. In this design, a cytotoxic peptide has been chemically modified to mask the chemical groups responsible for its membrane-lytic effects. At acidic pH, the masking groups are removed, causing the peptide to aggregate into polyion complexes -PIC for short- and thus accumulate at acidic environments. Thus, PIC peptide aggregates surrounding the tumour or bacteria niches are activated and can exert their toxic effects locally without indiscriminate damage to healthy host tissues buffered at neutral pH.
The innovative aspect of this project is the activation mechanism of the cytotoxic drug, which occurs at a molecular level, not requiring of any nanocarrier to adapt its activity. Addressing this serious healthcare challenge is key to society’s welfare and global economy. The development of new advanced strategies to tackle infection and cancer is critical to address the associated biomedical and economic burden. The concepts developed during this project will resonate across other biomedical fields, making our discoveries translatable to other conditions and target cells based on their modular design, tailoring molecular structure and pH-responsive masking for each application.
Nanomaterials have been widely explored as carriers for the targeted delivery of cytotoxic agents. Capitalising on certain pathological markers, these nanovehicles can release in situ the active cell-damaging molecules they carry, thus minimising off-target interactions and preventing toxicity to the host. Despite decades of development, poor reproducibility in nanomaterial formulation and activity still hampers their widespread application in clinic. Alternatives to these nanocarriers are required as innovative mechanisms to control the selectivity of cytotoxic drugs.
This project aims to tune the cytotoxic effects of a model drug in response to acidic microenvironments, which are characteristic of certain pathologies like cancer and infection. In this design, a cytotoxic peptide has been chemically modified to mask the chemical groups responsible for its membrane-lytic effects. At acidic pH, the masking groups are removed, causing the peptide to aggregate into polyion complexes -PIC for short- and thus accumulate at acidic environments. Thus, PIC peptide aggregates surrounding the tumour or bacteria niches are activated and can exert their toxic effects locally without indiscriminate damage to healthy host tissues buffered at neutral pH.
The innovative aspect of this project is the activation mechanism of the cytotoxic drug, which occurs at a molecular level, not requiring of any nanocarrier to adapt its activity. Addressing this serious healthcare challenge is key to society’s welfare and global economy. The development of new advanced strategies to tackle infection and cancer is critical to address the associated biomedical and economic burden. The concepts developed during this project will resonate across other biomedical fields, making our discoveries translatable to other conditions and target cells based on their modular design, tailoring molecular structure and pH-responsive masking for each application.
The first stages of the project involved the development of a new cytotoxic peptide structure. These molecules are strong lytic compounds due to their surface groups. By chemical conjugation of masking units, we managed to adjust the cytotoxic activity of peptides on a pH-responsive manner. Triggered by pH, cytotoxic groups on the surface of these peptides can be reversibly masked or revealed, as demonstrated in model membrane assays and in vitro cell cultures across the whole biological pH range.
Structural exploration of these peptides allowed a feedback optimisation loop, revealing certain masking groups and positions along the peptide sequence more effective in controlling the pH-dependent activation of these compounds. Libraries of masked peptides were screened in vitro to select for more pH-sensitive and lytic compounds, also allowing for estimations of their effective cytotoxic concentrations. These results set a starting point for in vivo evaluation on animal models, using tumour implants in mice as acidic model environment. As expected, the test compounds showed excellent biocompatibility in mice, being perfectly tolerated at physiological pH (ca. 7.4). Fluorescently labelled compounds allowed biodistribution tracking, showing a clear accumulation at tumour environments due to peptide acidification and accumulation as PIC aggregates. Time-course studies demonstrated a statistically significant reduction in tumour growth as compared to a placebo group without treatment.
Overall, this conceptually new technology has been here proven to be effective in tuning the cytotoxic effects of model compounds based on pH. A manuscript in under preparation to report details on this technology, while the Technology Transfer department at the host university is already studying a potential patent. Results from this specific project has not been disseminated yet in the interest of IPR protection. However, lessons from this design have been applied to other peptide designs and applications by the fellow, resulting in high impact papers (Nat. Commun. 2021, Chem. Sci. 2022, etc.) and communications at international conferences.
Structural exploration of these peptides allowed a feedback optimisation loop, revealing certain masking groups and positions along the peptide sequence more effective in controlling the pH-dependent activation of these compounds. Libraries of masked peptides were screened in vitro to select for more pH-sensitive and lytic compounds, also allowing for estimations of their effective cytotoxic concentrations. These results set a starting point for in vivo evaluation on animal models, using tumour implants in mice as acidic model environment. As expected, the test compounds showed excellent biocompatibility in mice, being perfectly tolerated at physiological pH (ca. 7.4). Fluorescently labelled compounds allowed biodistribution tracking, showing a clear accumulation at tumour environments due to peptide acidification and accumulation as PIC aggregates. Time-course studies demonstrated a statistically significant reduction in tumour growth as compared to a placebo group without treatment.
Overall, this conceptually new technology has been here proven to be effective in tuning the cytotoxic effects of model compounds based on pH. A manuscript in under preparation to report details on this technology, while the Technology Transfer department at the host university is already studying a potential patent. Results from this specific project has not been disseminated yet in the interest of IPR protection. However, lessons from this design have been applied to other peptide designs and applications by the fellow, resulting in high impact papers (Nat. Commun. 2021, Chem. Sci. 2022, etc.) and communications at international conferences.
This is the first pH-masking strategy applied to the targeted aggregation and accumulation of a cytotoxic agent to direct its action. Not only we have demonstrated the pH-controlled response in proof-of-concept experiments with model vesicles and cells lines, but also proved in a real biological setting (i.e. animal models) its efficacy and biocompatibility. This technology is highly adaptable and it could be tailored to other drugs and pH responses, making these concepts extremely valuable for the development of new advanced materials and new-generation therapeutics.
We expect our design to be patented due to its innovative mechanism of action and commercial interest, and publication of these results in scientific journals will be addressed once intellectual property rights have been secured. Dissemination of these advances will also include scientific conferences and outreach activities adapted to the specifics of the audience.
Overall, the pharmaceutical interest of this new biomedical strategy is supported by the promising preclinical results obtained, and further steps towards clinical evaluation may eventually bring this new-generation therapeutic agents to an application for antitumoral/antimicrobial drug approval.
We expect our design to be patented due to its innovative mechanism of action and commercial interest, and publication of these results in scientific journals will be addressed once intellectual property rights have been secured. Dissemination of these advances will also include scientific conferences and outreach activities adapted to the specifics of the audience.
Overall, the pharmaceutical interest of this new biomedical strategy is supported by the promising preclinical results obtained, and further steps towards clinical evaluation may eventually bring this new-generation therapeutic agents to an application for antitumoral/antimicrobial drug approval.