Final Report Summary - BAN-CANCER (Smart Bandage for Cancer Margin Theranostics)
Cancer is the leading cause of death for those under age 85 in the developed world. One in two men and one in three women are affected by cancer during their lifetime. Successful cancer treatment relies on early diagnosis to enable surgical intervention and on the accuracy of such surgical resection providing maximal tumour removal to significantly reduce the chance of recurrence. Both early diagnosis and surgical accuracy benefit from the detection and identification small cluster of tumour cells and the ability to intervene on them. Ultimately, notwithstanding the advancement in molecular based diagnostics, the current gold standard to detect the location of cancer cells is histological assessment of biopsies (for early diagnosis) or resections (for surgery). While histology can provide very accurate assessment of the pathological state of a tissue it is highly operator dependent, requiring making informed decisions on the sampling of analysis sites within the tissue and subjective assessments of pathological state. The time requirements for histological analysis effectively prevent the deployment of therapeutic strategies in combination with the diagnostic intervention.
There is thus a need for a tool capable of screening large areas of tissue and identify the ideal locations of choice for histological assessment and therapeutic intervention, defined as the most likely to display abnormal tissue. With BAN-CANCER we aim to prove the principle that such tool can be realized in the form of a bandage decorated with a regular array of vertical nanoneedles to be applied to an area under investigation, either in the margin region during surgery or a tissue suspected of dysplasia during diagnosis.
BAN-CANCER proposed developing nanoneedles for sensing and drug delivery. In this context, over the course of the two-year project we have realized a sensor for the detection of Cathepsin B activity which was successfully applied to patient-derived samples, as well as a drug delivery platform capable of localized delivery of DNA in vivo. The efficacy of such delivery was validated by the expression of vascular endothelial growth factor (VEGF) from plasmid DNA that resulted in the formation of neovasculature in a murine model.
The appropriateness, timeliness and impact of our approach to developing integrated solutions for sensing and delivery is testified by three publications originated within the context of the project and published in Nature Materials, ACS Nano and Advanced Materials, three of the top four journals in material science according to impact factor metrics. The Nature Materials publication dealt with the development and testing of the drug delivery platform, the Advanced Materials with the development and testing of the sensing platform and the ACS Nano investigated the details of the cell-nanoneedle interface to provide assess the impact of nanoneedle based treatments and to provide novel insight into the mechanisms of nanoneedles interaction with cells.
In the form developed in this project BAN-CANCER consists of a vertical array of porous silicon nanoneedles that were either functionalized with a fluorescently labelled peptide capable of sensing the activity of Cathepsin B protease or loaded with nucleic acids for delivery. The nanoneedles enabled interaction with the cytosol of cells, allowing intracellular drug delivery and discrimination of cells by means of their Cathepsin B activity. The nanoneedles applied to live tissue in murine models showed proficient localized delivery of nucleic acids, capable of inducing neovasculature formation. In the sensing context, the nanoneedles applied to resected tumour tissue discriminated tumour and healthy regions by means of their Cathepsin B activity.
The aims of the project were identified as:
1) The development of a nanoneedle platform for cell interfacing in a pre-clinical setting
2) The synthesis of a sensing element to recognize cancer cells
3) Assembly of the nanoneedle sensor
4) In vitro testing of intracellular delivery and sensing
5) Assessment of sensing and delivery efficacy in pre-clinical models
The project has fulfilled all its aims by the end of the final reporting period.
Nanoneedle geometry for cell interfacing was investigated by generating nanoneedles with different length, tip diameter and pitch. Nanoneedles with 2 micron pitch, 4 micron length and 100nm tip diameter were found to be minimally toxic and proficiently interface with cells. Such nanoneedles were used for all further studies. The nanoneedles developed were biodegradable and completely dissolved within few days. A chip with 16 million nanoneedles could withstand 1N of compressive forces without failure.
The peptide CFKK was identified as the most responsive substrate to Cathepsin B cleavage and was employed in the development of the fluorescence based Cathepsin B sensor. The peptide was conjugated to the nanoneedles and equipped with a fluorescent label for sensing aberrant activation of this cancer biomarker.
The nanoneedles were proven to be non-toxic to cells in culture and were tested to optimize the cell interface for proficient intracellular sensing. When interfaced from the top of a cell culture they were able to both deliver bioactive payloads and to discriminate cells by their Cathepsin B activity. In this setting the delivery platform was able to regulate gene expression and the sensor could discriminate cancer cells from healthy cells.
When applied to live tissue the nanoneedles were able to deliver a payload to a local region without eliciting an observable immune response or causing damage to tissue architecture. The payload delivery was confined to the area of nanoneedle application, and gene regulation was observed through the delivery of plasmid DNA, leading to the formation of new blood vessels. When tested on resected samples of human tumours and matching healthy regions the nanoneedle sensor could discriminate between the two based on Cathepsin B activity.
This project has thus demonstrated the feasibility of developing a nanoneedle-based delivery platform for nucleic acids coupled with a sensor to detect enzymatic activity in cancer and healthy tissue with the aim of discriminating within the two and providing gene therapy support. This knowledge will contribute to the development of more accurate theranostic platforms for early diagnosis and intraoperative margin intervention.
There is thus a need for a tool capable of screening large areas of tissue and identify the ideal locations of choice for histological assessment and therapeutic intervention, defined as the most likely to display abnormal tissue. With BAN-CANCER we aim to prove the principle that such tool can be realized in the form of a bandage decorated with a regular array of vertical nanoneedles to be applied to an area under investigation, either in the margin region during surgery or a tissue suspected of dysplasia during diagnosis.
BAN-CANCER proposed developing nanoneedles for sensing and drug delivery. In this context, over the course of the two-year project we have realized a sensor for the detection of Cathepsin B activity which was successfully applied to patient-derived samples, as well as a drug delivery platform capable of localized delivery of DNA in vivo. The efficacy of such delivery was validated by the expression of vascular endothelial growth factor (VEGF) from plasmid DNA that resulted in the formation of neovasculature in a murine model.
The appropriateness, timeliness and impact of our approach to developing integrated solutions for sensing and delivery is testified by three publications originated within the context of the project and published in Nature Materials, ACS Nano and Advanced Materials, three of the top four journals in material science according to impact factor metrics. The Nature Materials publication dealt with the development and testing of the drug delivery platform, the Advanced Materials with the development and testing of the sensing platform and the ACS Nano investigated the details of the cell-nanoneedle interface to provide assess the impact of nanoneedle based treatments and to provide novel insight into the mechanisms of nanoneedles interaction with cells.
In the form developed in this project BAN-CANCER consists of a vertical array of porous silicon nanoneedles that were either functionalized with a fluorescently labelled peptide capable of sensing the activity of Cathepsin B protease or loaded with nucleic acids for delivery. The nanoneedles enabled interaction with the cytosol of cells, allowing intracellular drug delivery and discrimination of cells by means of their Cathepsin B activity. The nanoneedles applied to live tissue in murine models showed proficient localized delivery of nucleic acids, capable of inducing neovasculature formation. In the sensing context, the nanoneedles applied to resected tumour tissue discriminated tumour and healthy regions by means of their Cathepsin B activity.
The aims of the project were identified as:
1) The development of a nanoneedle platform for cell interfacing in a pre-clinical setting
2) The synthesis of a sensing element to recognize cancer cells
3) Assembly of the nanoneedle sensor
4) In vitro testing of intracellular delivery and sensing
5) Assessment of sensing and delivery efficacy in pre-clinical models
The project has fulfilled all its aims by the end of the final reporting period.
Nanoneedle geometry for cell interfacing was investigated by generating nanoneedles with different length, tip diameter and pitch. Nanoneedles with 2 micron pitch, 4 micron length and 100nm tip diameter were found to be minimally toxic and proficiently interface with cells. Such nanoneedles were used for all further studies. The nanoneedles developed were biodegradable and completely dissolved within few days. A chip with 16 million nanoneedles could withstand 1N of compressive forces without failure.
The peptide CFKK was identified as the most responsive substrate to Cathepsin B cleavage and was employed in the development of the fluorescence based Cathepsin B sensor. The peptide was conjugated to the nanoneedles and equipped with a fluorescent label for sensing aberrant activation of this cancer biomarker.
The nanoneedles were proven to be non-toxic to cells in culture and were tested to optimize the cell interface for proficient intracellular sensing. When interfaced from the top of a cell culture they were able to both deliver bioactive payloads and to discriminate cells by their Cathepsin B activity. In this setting the delivery platform was able to regulate gene expression and the sensor could discriminate cancer cells from healthy cells.
When applied to live tissue the nanoneedles were able to deliver a payload to a local region without eliciting an observable immune response or causing damage to tissue architecture. The payload delivery was confined to the area of nanoneedle application, and gene regulation was observed through the delivery of plasmid DNA, leading to the formation of new blood vessels. When tested on resected samples of human tumours and matching healthy regions the nanoneedle sensor could discriminate between the two based on Cathepsin B activity.
This project has thus demonstrated the feasibility of developing a nanoneedle-based delivery platform for nucleic acids coupled with a sensor to detect enzymatic activity in cancer and healthy tissue with the aim of discriminating within the two and providing gene therapy support. This knowledge will contribute to the development of more accurate theranostic platforms for early diagnosis and intraoperative margin intervention.