Fabrication of antibody functionalized silk fibroin micro-well arrays using reactive inkjet printing for circulating tumour cell capture

Lead Research Organisation: University of Sheffield
Department Name: Chemical & Biological Engineering

Abstract

Cancer is a group of diseases caused by the abnormal growth of cells and can potentially spread to other parts of the body. It has been the major threat to human health worldwide, counting for 8.2 million deaths (2012). In UK, there are 0.3 million new cancer patients every year with more than 200 types of cancers of which breast, lung, colon and prostate cancers are the most common types. Some of the cancers such as prostate cancer have no early symptoms making the prognosis extremely difficult. Therefore, effective tools for early detection of cancer are highly desired and can greatly increase the chances for successful treatment or extend the lifetime.

Circulating tumour cells (CTCs) are cell seeds that have shed from a primary tumour and circulate in the blood stream and can subsequent growth into additional tumours. Isolation and characterization of CTCs have been proved to be effective in cancer prognosis. However, this task is extremely challenging due to the significant low abundance of CTCs in blood (a few to tens in a millilitre of blood which contains millions of white and red blood cells). Currently, CellSearch is the only FDA approved CTC capture device that require specific equipment to perform the analysis and are not able to discriminate from different types of cancers. Furthermore, the captured CTCs on the filter membrane need to be transferred to a cell culture plate for enumeration/characterization. Hence, there is an urgent need to develop efficient high throughput screen devices/methods that can screen multiple cancers in one test with excellent sensitivity.

The proposed research, envisaging this challenging task and urgent needs, seeks to harness the emerging additive manufacturing technology (reactive inkjet printing, RIJ) and the FDA approved biomaterial (regenerated silk fibroin, RSF) for the fabrication of antibody functionalized micro-well arrays for the capture and sorting of CTCs. The advantages of RIJ are not only the computer assisted design (CAD) that offers the precise delivery of pico-litres (1e-12 litre) of ink at predetermined locations allowing the fabrication of complex micro arrays but also the alternate delivery of different antibody inks (through different print heads) that allows the functionalization of different micro-wells with different cancer biomarker antibodies. The fabricated devices will not only allow us to efficiently capture the rare CTCs but also allow us to discriminate different types of cancer cells, providing a promising tool for efficient cancer diagnosis. The project also extends the knowledge and skills of employing emerging additive manufacturing technology for cost-effective manufacturing of high value products. Therefore, this project firmly aligns with EPSRC priority themes of Healthcare technologies (novel diagnostic tools) and Manufacturing the future and will have significant impact to cancer diagnosis and medical device manufacturing industry.

Planned Impact

There is a global pressure in developing efficient high throughput screen devices/methods for cancer diagnosis. Funding and delivery of this project will have significant input to healthcare technology and provide better cancer diagnosis device/methods for both user industries (medical device companies) and end users (clinicians and patients). This project also explores the use of emerging technology (RIJ) for bio scaffolds and medical device fabrication and takes a step forward in transferring lab research to industrial and clinical applications. Therefore, this project has a number of economic and societal Impacts and will facilitate the UK wealth creation, economic prosperity/global competitiveness & knowledge exploitation. The project is also firmly aligning with EPSRC priority themes of Healthcare Technologies and Manufacturing the Future.

Who are the beneficiaries and how they will benefit from this research?
This research has a wide range of impacts cover knowledge, people, society and economy.

Academics and knowledge: see previous session on academic beneficiaries.

Skilled People: The research project will train skilled PDRA, PhDs and project students and will have direct economic impact via providing skilled people for industry or beneficiary organizations. Specifically, the PDRA working on this project is highly likely to be a young academic.

Business and User industries: in specific, the biomedical, biomaterial and pharma industries. The results from this project will provide the user industries with better technology for medical device/ scaffold fabrication and cancer diagnosis in a cost-effective manner. The technique developed in this project can also be used for other applications in pharmaceutical industry. For example, print micro-arrays for drug screening, cell colony selection. It therefore will facilitate clinical applications and will help the user industries to maintain their leadership in the global healthcare market. The biomedical sector is a key area to support the economic recovery; hence, this project has potential economic benefit. It will also help create job opportunity in user industries.

Public communities: This project will have significant impact to different public communities.
The general public communities: such as patients and clinicians. The eventual beneficiaries of this project are those that will benefit from the growing healthcare techniques. Millions of people are suffering from different cancers that will benefit from early detection/prognosis. The outcome of this project will provide better cancer diagnosis tools and will benefit millions of cancer patients. Taxpayers will benefit from reduced cost of R&D and eventually lower NHS costs.
Public sectors: such as universities and colleges, health & wellbeing agencies. The findings of this project are excellent teaching materials for universities, colleges and can be used as examples for health & wellbeing agencies or charities.

Publications

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Cirillo S (2021) Designed Antitumor Peptide for Targeted siRNA Delivery into Cancer Spheroids. in ACS applied materials & interfaces

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Gregory DA (2016) Reactive Inkjet Printing of Biocompatible Enzyme Powered Silk Micro-Rockets. in Small (Weinheim an der Bergstrasse, Germany)

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Hadianamrei R (2022) Rationally designed short cationic a-helical peptides with selective anticancer activity. in Journal of colloid and interface science

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Hadianamrei R (2023) Surfactant like peptides for targeted gene delivery to cancer cells. in Biochemical and biophysical research communications

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Hadianamrei R (2022) Current state of the art in peptide-based gene delivery. in Journal of controlled release : official journal of the Controlled Release Society

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Jayawardane D (2016) Interfacial Adsorption of Silk Fibroin Peptides and Their Interaction with Surfactants at the Solid-Water Interface. in Langmuir : the ACS journal of surfaces and colloids

 
Description We have been able to utilise a novel printing method that allows us to make chemical reactions in situ on a sample surfaces avoiding the use of large quantities of expensive chemicals to generate patterned surfaces with biologically active species. The Produced samples allow us to quickly and effectively capture specific cancer cells in a flow which will have useful impact in the development of rapid diagnostic devices for hospitals. They also have the potential to be used during dialysis and give rapid cancer cell diagnosis without any further invasive procedures.

In specific, we have successfully coated the surface of the glass for stopping non-specific cell attachment, optimised the printing of silk/ silk with PEG inks onto glass slides. we have successfully printed the patterns on the coated surface with functional groups available for antibody binding and verified through the attachment of fluorescent biotin beads. Cancer cell capture on the printed microarrays has been done both in a designed flow cell system as well as with agitated drips of cancer cells in serum buffer.
For this study four different immobilisation techniques were tested on their ability for printing and cancer cell capture. We are currently in the process of drafting a manuscript on reactive inkjet printing of dot arrays of antibodies on silk films that have successfully captured specific cancer cells.

In addition, reactive inkjet printed silk rockets have been produced that have shown to capture specific cancer cells dependent on immobilised antibodies on their surfaces. This data is also in progress of being written up and will follow once the first paper has been finished.
Exploitation Route The publication we are currently in the process of drafting will give others a chance to use our developed reactive inkjet printing protocol for the immobilisation of other moieties for patterned devices in their pursuit of future targeted species capture such as other specific cancer cells.
Sectors Chemicals,Education,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description 3D printed silk scaffold channels for the guidance and repair of nerve tissues
Amount £15,000 (GBP)
Funding ID RG160662 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2017 
End 04/2018
 
Description Departmental studentship: inkjet printing of silk materials
Amount £60,000 (GBP)
Organisation University of Sheffield 
Sector Academic/University
Country United Kingdom
Start 04/2017 
End 04/2021
 
Description Printable Micro-rockets for Rapid Medical Diagnosis and Biomarker Detection
Amount £483,709 (GBP)
Funding ID EP/N033736/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 12/2016 
End 11/2018
 
Description Syngenta Crop Protection, LLC (silk particles fro DNA extraction)
Amount £115,000 (GBP)
Organisation Syngenta International AG 
Department Syngenta Crop Protection
Sector Private
Country United Kingdom
Start 12/2017 
End 12/2018
 
Title 3D reactive inkjet printer 
Description we assembled a 3D reactive inkjet printer 
Type Of Material Improvements to research infrastructure 
Year Produced 2016 
Provided To Others? No  
Impact will significantly benifit our research group and extend our research in the area of 3D printing 
 
Title Reactive Inkjet Printing and Propulsion Analysis of Silk-Based Self-Propelled Micro-stirrers 
Description a detailed method of using silk solution as bioink for inkjet printing of 3D structure 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2019 
Provided To Others? Yes  
Impact researchers or the general public can watch the video to learn how to make silk solution and ink for 3D inkjet printing. 
 
Description Silk scaffolds swimming devices (Ebbens) 
Organisation University of Sheffield
Department Department of Mechanical Engineering
Country United Kingdom 
Sector Academic/University 
PI Contribution printed silk scaffold micro swimming devices, data analysis, drafting papers
Collaborator Contribution provide experimental methods for swimming devices
Impact publication: Reactive inkjet printing of biocompatible enzyme powered silk micro-rockets. Small 2016, 12, 4048-4055.(Journal Inside Front Cover) news on more than 10 scientific websites: https://www.sciencedaily.com/releases/2016/06/160628113746.htm http://sciencenewsjournal.com/rocketing-towards-safer-drug-delivery/ http://www.popsci.com/tiny-rocket-designed-to-blast-off-inside-human-body http://phys.org/news/2016-06-major-breakthrough-silk-micro-rockets-safely.html#nRlv http://www.americanlaboratory.com/188450-Safe-Biodegradable-Drug-delivery-System-is-Made-From-Silk-Swimming-Devices/ http://www.in-pharmatechnologist.com/Drug-Delivery/Silk-rockets-and-3D-printing-Engineers-work-on-drug-delivery-system http://www.medgadget.com/2016/06/micro-rockets-developed-deliver-drugs-inside-body.html http://www.3ders.org/articles/20160701-3d-printed-silk-micro-rockets-could-swim-drugs-into-your-body.html https://3dprint.com/140790/3d-printed-micro-rockets/ http://healthmedicinet.com/i/new-microscopic-silk-swimming-devices-can-be-used-safely-in-biological-environments/ http://www.eurekalert.org/pub_releases/2016-06/uos--ogl062816.php http://www.gereports.com/5-coolest-things-on-earth-this-week-20/ http://www.hospimedica.com/critical-care/articles/294765053/silk-microrockets-provide-safe-drug-delivery.html http://3dprintingfromscratch.com/2016/07/3d-printed-micro-rocket-drug-delivery-system/ https://www.sheffield.ac.uk/faculty/engineering/news/microrockets-1.586767 http://nextbigfuture.com/2016/07/3d-printed-silk-micro-rockets-are-major.html
Start Year 2016
 
Description silk materials Southwest University China 
Organisation Southwest University
Country China 
Sector Academic/University 
PI Contribution silk printing for cell culture
Collaborator Contribution provide modified silk samples for printing
Impact not yet
Start Year 2017
 
Description silk peptides with manchester 
Organisation University of Manchester
Department Manchester Medical School
Country United Kingdom 
Sector Academic/University 
PI Contribution making silk peptide materials; perform experiments and data analysis
Collaborator Contribution provide equipment and beam time, provide peptide samples
Impact Publication: Interfacial Adsorption of Silk Fibroin Peptides and Their Interaction with Surfactants at the Solid-Water Interface. Langmuir 2016, 32, 8202-8211.
Start Year 2016
 
Description silk printing with Sheffield (Smith) 
Organisation University of Sheffield
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution making silk ink materials and perform printing experiment and data analysis, drafting papers
Collaborator Contribution provide expertise and equipment for printing
Impact Publications Biocompatible silk fibroin scaffold prepared by reactive inkjet printing. Journal of Materials Science 2016, 51, 8625-8630. (Nominated by the Editors for the 2016 Journal of Materials Science Robert W. Cahn Best Paper Prize) Reactive inkjet printing of biocompatible enzyme powered silk micro-rockets. Small 2016, 12, 4048-4055. news on scientific websites: https://www.sciencedaily.com/releases/2016/06/160628113746.htm http://sciencenewsjournal.com/rocketing-towards-safer-drug-delivery/ http://www.popsci.com/tiny-rocket-designed-to-blast-off-inside-human-body http://phys.org/news/2016-06-major-breakthrough-silk-micro-rockets-safely.html#nRlv http://www.americanlaboratory.com/188450-Safe-Biodegradable-Drug-delivery-System-is-Made-From-Silk-Swimming-Devices/ http://www.in-pharmatechnologist.com/Drug-Delivery/Silk-rockets-and-3D-printing-Engineers-work-on-drug-delivery-system http://www.medgadget.com/2016/06/micro-rockets-developed-deliver-drugs-inside-body.html http://www.3ders.org/articles/20160701-3d-printed-silk-micro-rockets-could-swim-drugs-into-your-body.html https://3dprint.com/140790/3d-printed-micro-rockets/ http://healthmedicinet.com/i/new-microscopic-silk-swimming-devices-can-be-used-safely-in-biological-environments/ http://www.eurekalert.org/pub_releases/2016-06/uos--ogl062816.php http://www.gereports.com/5-coolest-things-on-earth-this-week-20/ http://www.hospimedica.com/critical-care/articles/294765053/silk-microrockets-provide-safe-drug-delivery.html http://3dprintingfromscratch.com/2016/07/3d-printed-micro-rocket-drug-delivery-system/ https://www.sheffield.ac.uk/faculty/engineering/news/microrockets-1.586767 http://nextbigfuture.com/2016/07/3d-printed-silk-micro-rockets-are-major.html
Start Year 2016
 
Description tissue engineering (Miller) 
Organisation University of Sheffield
Department Department of Chemical and Biological Engineering
Country United Kingdom 
Sector Academic/University 
PI Contribution contribute on research ideals, making printing inks, printing silk scaffolds for tissue engineering,draft papers, attending conferences
Collaborator Contribution bring expertise of tissue engineering
Impact resulted a publication (Biocompatible silk fibroin scaffold prepared by reactive inkjet printing. Journal of Materials Science 2016, 51, 8625-8630.) (Nominated by the Editors for the 2016 Journal of Materials Science Robert W. Cahn Best Paper Prize) multi-disciplinary: Engineering, biomaterials, tissue engineering
Start Year 2016
 
Description 3D Medicine Printing Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact a talk introducing the 3D printing for tissue culture scaffolds and drug delivery applications. lots of discussions.
Year(s) Of Engagement Activity 2017
 
Description CPI visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact A talk was given to the staff of peintable eclectronics and resulted in further collaboration
Year(s) Of Engagement Activity 2016
 
Description E-MRS 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Poster presentation on the conference. won the best poster price. received lots of attention and discussion.
Year(s) Of Engagement Activity 2016
 
Description Printing for Fabrication (Manchester) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact 2 talks was give on the conferences
Year(s) Of Engagement Activity 2016
URL http://www.imaging.org/Site/IST/Conferences/NIP/Printing_For_Fabrication_NIP.aspx?WebsiteKey=6d978a6...
 
Description The 23 rd Joint Annual Conference of CSCST-SCI (Nottingham) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact more than 100 academics and PDRAs/PhD students attend the conference. result in new collaboration
Year(s) Of Engagement Activity 2016
URL http://cscst.org/Conference2016/index.html
 
Description University of Sheffield, SURE showcase event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact presenting the research ideals and results on the event and resulted in winning of 3 undergraduate research projects relevant to the grant.
Year(s) Of Engagement Activity 2020
 
Description a talk on Sheffield silk conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact a talk on Sheffield silk conference resulted in collaboration.
Year(s) Of Engagement Activity 2017
 
Description invited talk at 3D printing Medical Expo / Conference Maastrict 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact invited talk at the 3D bioprinting conference. generated a lot discussion and networking.
Year(s) Of Engagement Activity 2019
 
Description invited talk at China Pharmaceutical University 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact invited talk at China Pharmaceutical University. generated a lot of discussions and networks.
Year(s) Of Engagement Activity 2018
 
Description invited talk at Loughborough University 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A invitated talk at Loughborough University. generated a lot of discussions.
Year(s) Of Engagement Activity 2018
 
Description invited talk in TBIS 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact more than 100 academics, researchers, PGR students attended the international conference. generated discussions and network.
Year(s) Of Engagement Activity 2018
 
Description invited talk on TBIS conference 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Gave a presentation on 12th Textile bioengineering and informatics symposium
Year(s) Of Engagement Activity 2019
 
Description invited talk on The 2nd International Symposium for Chinese Supramolecular Chemists 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact gave an invited talk on The 2nd International Symposium for Chinese Supramolecular Chemists
Year(s) Of Engagement Activity 2019
 
Description media interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interviewed by Chemistry & Industry magazine (a publication of the Society of Chemical Industry); CEP Magazine (the magazine of the American Institute of Chemical Engineers); Popular Science magazine in the US. and a press release with news on more than 10 scientific websites.
https://www.sciencedaily.com/releases/2016/06/160628113746.htm
http://sciencenewsjournal.com/rocketing-towards-safer-drug-delivery/
http://www.popsci.com/tiny-rocket-designed-to-blast-off-inside-human-body
http://phys.org/news/2016-06-major-breakthrough-silk-micro-rockets-safely.html#nRlv
http://www.americanlaboratory.com/188450-Safe-Biodegradable-Drug-delivery-System-is-Made-From-Silk-Swimming-Devices/
http://www.in-pharmatechnologist.com/Drug-Delivery/Silk-rockets-and-3D-printing-Engineers-work-on-drug-delivery-system
http://www.medgadget.com/2016/06/micro-rockets-developed-deliver-drugs-inside-body.html
http://www.3ders.org/articles/20160701-3d-printed-silk-micro-rockets-could-swim-drugs-into-your-body.html
https://3dprint.com/140790/3d-printed-micro-rockets/
http://healthmedicinet.com/i/new-microscopic-silk-swimming-devices-can-be-used-safely-in-biological-environments/
http://www.eurekalert.org/pub_releases/2016-06/uos--ogl062816.php
http://www.gereports.com/5-coolest-things-on-earth-this-week-20/
http://www.hospimedica.com/critical-care/articles/294765053/silk-microrockets-provide-safe-drug-delivery.html
http://3dprintingfromscratch.com/2016/07/3d-printed-micro-rocket-drug-delivery-system/
https://www.sheffield.ac.uk/faculty/engineering/news/microrockets-1.586767
http://nextbigfuture.com/2016/07/3d-printed-silk-micro-rockets-are-major.html
Year(s) Of Engagement Activity 2016
URL https://www.sciencedaily.com/releases/2016/06/160628113746.htm
 
Description poster presentation on 3D Bionet conference at Sheffield University 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Study participants or study members
Results and Impact around 100 academics/researchers attend the workshop to report/discuss 3D bioprinting related work. A poster of our work was presented and generated lots of discussions and also initiated a collaboration within the university.
Year(s) Of Engagement Activity 2019
 
Description talks on silk printing at southwest university, shanghai jiaotong university, Changzhou university 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact talks at the southwest university, shanghai jiaotong university, Changzhou university, resulted in collaboration with southwest university.
Year(s) Of Engagement Activity 2017