Periodic Reporting for period 4 - TROJANANOHORSE (Hybrid immune-eluding nanocrystals as smart and active theranostic weapons against cancer)
Período documentado: 2020-09-01 hasta 2021-08-31
Cancer, together with circulatory diseases, is one of the primary causes of mortality worldwide and its incidence is expected to rise by about 70% over the next 2 decades. There is a continuous development of new smart and nanosized materials as therapeutic and diagnostic platforms or even theranostic ones. However a huge disproportion between these nanotherapeutic treatments with respect to the conventional ones proposed to patients is present. Indeed the various nanomaterials for theranostic applications developed so far show several smart characteristics (controlled drug delivery, cancer cell targeting ability and biocompatibility), but still their immunogenicity, stability in biological media and off-target delivery - in case of drug-delivery systems - show important challenges. Similarly, little is known to the final destiny of the nanomaterials at the end of their functions in the human body. All these key challenges make the already-developed nanosized systems still incomplete for theranostics in nanomedicine.
The ERC Starting Grant project TrojaNanoHorse (TNH) aimed to cover the gap between the present nanomedicine tools and the clinical requirements, and strived to develop a non-immunogenic hybrid nanosystem. The ERC project was divided in three objectives aiming to: (1) construct the TNH and proof the therapeutic capability and targeting action; (2) test the biodegradation and safety of the nanoconstruct; (3) proof the bio-imaging and diagnostic capabilities in a multimodal way, thus rendering the whole TNH a complete theranostic tool. As a result, the developed TNH nanosystem is equipped by a therapeutically active nanocrystalline core with multifunctional features, such as bio-imaging capabilities and stimuli responsive action for therapy, as well as a biodegradation and safe-by-design behaviour to be not harmful per se for the biological system. The TNH is programmed to kill the tumor cells without the use of potentially toxic drugs, i.e. chemotherapeutics. Furthermore, the TNH have a biomimetic lipid bilayer shell autologously derived from the patients, thus rendering the whole nanoconstruct fully biomimetic, haemocompatible (thus injectable) and naturally non-immunogenic. The TNH is then completed by targeting ligands in order to be safely and precisely directed to the cell objective.
The main focus of the project is anticancer therapy, in particular against leukaemia, having of course a fundamental impact in the fight against this tremendous disease. The high flexibility of the TNH concept with its hybrid core-shell nanostructure can be successfully applied to other nanomaterials of suitable nanodimensions and to solid tumors (upon development of efficient targeting), but also to cardiovascular diseases, kidney infections, gastrointestinal illnesses, wound therapy or even extended to animal care and other domains, as for example antibacterial treatments.
In a Nanomaterial or Nanotechnology era, as we are now, it is still difficult to recognize and standardize the risks associated to the exposure of living systems to nano-objects. Therefore, the safety and biodegradation assessments performed during this project will contribute in defining standardized nanotoxicology essays, new protocols and guidelines which at present are lacking, leading to a strong input on the regulatory aspects and standards of next-generation nanomedicine therapeutics.
The new methods and design of the instrument set up developed under this project were patented so they can be transferable to industry or generate spin-offs for the systematic use of therapeutically stimuli-responsive nanoparticles and diagnostics for clinical cancer therapy. In particular, the project have proposed also the use of already commercially-available instruments in clinical practice for the stimuli application to this novel TNH nanoconstruct, thus establishing new applications of these machines against cancer and speeding up the research in this field.
Concerning the impact of the TrojaNanoHorse project in science, we expect to open new horizons in the nanomedicine field with strong inter- and multi-disciplinary developments.
The ERC Starting Grant project TrojaNanoHorse (TNH) aimed to cover the gap between the present nanomedicine tools and the clinical requirements, and strived to develop a non-immunogenic hybrid nanosystem. The ERC project was divided in three objectives aiming to: (1) construct the TNH and proof the therapeutic capability and targeting action; (2) test the biodegradation and safety of the nanoconstruct; (3) proof the bio-imaging and diagnostic capabilities in a multimodal way, thus rendering the whole TNH a complete theranostic tool. As a result, the developed TNH nanosystem is equipped by a therapeutically active nanocrystalline core with multifunctional features, such as bio-imaging capabilities and stimuli responsive action for therapy, as well as a biodegradation and safe-by-design behaviour to be not harmful per se for the biological system. The TNH is programmed to kill the tumor cells without the use of potentially toxic drugs, i.e. chemotherapeutics. Furthermore, the TNH have a biomimetic lipid bilayer shell autologously derived from the patients, thus rendering the whole nanoconstruct fully biomimetic, haemocompatible (thus injectable) and naturally non-immunogenic. The TNH is then completed by targeting ligands in order to be safely and precisely directed to the cell objective.
The main focus of the project is anticancer therapy, in particular against leukaemia, having of course a fundamental impact in the fight against this tremendous disease. The high flexibility of the TNH concept with its hybrid core-shell nanostructure can be successfully applied to other nanomaterials of suitable nanodimensions and to solid tumors (upon development of efficient targeting), but also to cardiovascular diseases, kidney infections, gastrointestinal illnesses, wound therapy or even extended to animal care and other domains, as for example antibacterial treatments.
In a Nanomaterial or Nanotechnology era, as we are now, it is still difficult to recognize and standardize the risks associated to the exposure of living systems to nano-objects. Therefore, the safety and biodegradation assessments performed during this project will contribute in defining standardized nanotoxicology essays, new protocols and guidelines which at present are lacking, leading to a strong input on the regulatory aspects and standards of next-generation nanomedicine therapeutics.
The new methods and design of the instrument set up developed under this project were patented so they can be transferable to industry or generate spin-offs for the systematic use of therapeutically stimuli-responsive nanoparticles and diagnostics for clinical cancer therapy. In particular, the project have proposed also the use of already commercially-available instruments in clinical practice for the stimuli application to this novel TNH nanoconstruct, thus establishing new applications of these machines against cancer and speeding up the research in this field.
Concerning the impact of the TrojaNanoHorse project in science, we expect to open new horizons in the nanomedicine field with strong inter- and multi-disciplinary developments.
Started in March 2016, with an overall budget of 1'489'219 euro by the European Research Council for 5 years, this project is finished reaching all the proposed aims. The obtained results have impressively confirmed the high-risk hypothesis at the base of the project but, thanks to the top-level and multidisciplinary competences of the research team, also its feasibility.
At the beginning the project started with the laboratory set-up, instrument purchase and team member recruitment. In the meantime, the first research activities were carried out in the direction of developing and optimizing a synthesis of round-shaped, smooth edges nanocrystals with 20-50 nm in diameter. We particularly strived to control and obtain repeatability on the morphological and structural properties of the nanocrystals as well as having a good yield and easy synthetic approach. For this reasons, two kinds of wet-chemical synthesis of the nanocrystals were successfully developed: the first method following a conventional approach and the second method showing in contrast a high level of innovation, both already patented and published so far. Actually, the nanocrystals obtained with this new method demonstrate highly reproducible data in terms of size distribution, morphology and surface chemistry, but also of biocompatibility with respect to the former nanocrystals, and low cellular toxicity with outstanding colloidal stability in ethanol and water media.
The team proceeded also to the extraction and full characterization of the biovesicles derived from different cell lines, in particular from both cancer and healthy cells cultured in vitro. The biological role of extracellular vesicles (EVs) was also studied in terms of intrinsic homing capability towards healthy (B lymphocystes) and cancer cells, both Daudi and HL60 cell lines. We also proposed the bioconjugation with the monoclonal antibody anti-CD20 Rituximab in order to have a specific cancer cell targeting. We highlighted indeed that EVs extracted from healthy B-cell lymphocites have natural homing capability towards their parental cells and Daudi cancer one, while reduced homing towards HL60. Furthermore, upon specific bioconjugation with antiCD20, a specific targeting is verified mostly only the CD20+ Daudi cancer cell line, and not towards B-lymphocites.
The TrojaNanoHorse (TNH) concept design and construction was fully developed: since this task is of particular high-risk, many different procedural, physical and chemical parameters were varied to obtain an optimized preparation protocol for the TNH. We first studied the use of artificial lipids to coat the synthesized nanocrystals and evaluated their long-term biostability in biological fluid, cell internalization and the stimuli responsive capability upon UV light exposure, as a preliminary study. Then we moved to the use of cell derived EVs to coat the ZnO NCs. Several characterization techniques were also used and developed specifically to properly characterize the nanoconstruct. We noticed that the process is a consequence of various mechanisms, including thermodynamic, kinetic and electrostatic ones. The team first worked on a preliminary TNH were EVs are derived from KB adenocarcinoma cell line to optimize the TNH preparation yield and the first cytocompatibility and cell internalization tests. A second version of the complete TNH concept, prepared with an advanced active encapsulation method of the extracellular vesicles extracted from healthy B lymphocytes, was then adopted. High yield and high level of ZnO nanocrystals internalization in the EVs was performed. The hemocompatibility and colloidal stability over long time periods of the whole TNH was successfully assessed. Furthermore, the monoclonal antibody antiCD20 was also bioconjugated to the TNH, creating a TNHCD20: the targeting selectivity towards Daudi cancer cells, sparing healthy B lymphocytes (also CD20 positive) and cancer HL60 cells (which are CD20 negative cells) was successfully demonstrated. Finally, the stimuli responsive ability towards an ultrasound stimulation was also achieved, demonstrating the synergism between the TNHCD20 and the applied shock-waves to kill Daudi cancer cell and spare the healthy ones.
Concerning the stimuli-responsive therapy, many efforts were devoted to set-up the instrumentation for ultrasound irradiation (now patented) and to deeply understand the mechanism of interaction between ultrasonic waves and zinc oxide nanocrystals. Actually, two commercially available and clinically-relevant instruments were purchased or exploited and, in the meanwhile, a lab-made equipment was designed, set-up and tested for this therapy. A novel, unconventional and safe methodology of nanocrystals activation to develop highly toxic species directed against cancer cells was developed, first explored in simple water-based media using commercial dyes, then in more complex biological relevant media and using soft tissue to simulate the US penetration in the body, and finally against cancer cells. The different key parameters for controlling the stimulation, the nanocrystals activation and their complex biological effects on cancer cells were explored and understood, showing an effective synergy between the US and ZnO NCs. A full understanding of the inertial cavitation and interaction with the surface of the ZnO nanocrystal was achieved, opening also a novel possibility to use the nanomaterials not only for the therapy but also for imaging, in particular as ecographic nanoconstrast agent. As mentioned above, also the final targeted TNHCD20 successfully underwent to a synergistic activation by ultrasound, in particular shock-way using a clinically-approved machine.
The cytotoxicity, the dissolution and biodegradation tests of the TNH and also of ZnO NCs were carefully designed and actuated in different fluids and cell conditions, establishing protocols and two guideline reviews for operation on cytotoxic behaviour of ZnO and related shielding.
Finally, the study of the bioimaging and nanodiagnostic capabilities of the TNH, was explored, starting with the intrinsic properties of ZnO as semiconductor. The optical properties of the synthesized nanocrystals were studied, also depending on the synthetic procedure, the chemical functionalities and dimensions in a paper.
Based on the strong expertise gained to set up the stimuli-responsive therapy and related equipment, important advancements were achieved to exploit a novel, unconventional imaging procedure, based on sonoluminescence. A new methodology and instrument set-up are described in a patent submitted to the Italian patent office and now under evaluation. A scientific paper related on the use of ZnO nanocrystal as contrast agent to enable sonoluminescence as a imaging technology for in vitro cell imaging is at present under submission.
Scientific dissemination in international congresses, broad-audience dissemination to non-specialized public, papers in peer-reviewed international journals with open-access modality, pitch to multinational companies and investment funds, two granted patents and one additional patent applications of the most relevant results were also accomplished.
At the beginning the project started with the laboratory set-up, instrument purchase and team member recruitment. In the meantime, the first research activities were carried out in the direction of developing and optimizing a synthesis of round-shaped, smooth edges nanocrystals with 20-50 nm in diameter. We particularly strived to control and obtain repeatability on the morphological and structural properties of the nanocrystals as well as having a good yield and easy synthetic approach. For this reasons, two kinds of wet-chemical synthesis of the nanocrystals were successfully developed: the first method following a conventional approach and the second method showing in contrast a high level of innovation, both already patented and published so far. Actually, the nanocrystals obtained with this new method demonstrate highly reproducible data in terms of size distribution, morphology and surface chemistry, but also of biocompatibility with respect to the former nanocrystals, and low cellular toxicity with outstanding colloidal stability in ethanol and water media.
The team proceeded also to the extraction and full characterization of the biovesicles derived from different cell lines, in particular from both cancer and healthy cells cultured in vitro. The biological role of extracellular vesicles (EVs) was also studied in terms of intrinsic homing capability towards healthy (B lymphocystes) and cancer cells, both Daudi and HL60 cell lines. We also proposed the bioconjugation with the monoclonal antibody anti-CD20 Rituximab in order to have a specific cancer cell targeting. We highlighted indeed that EVs extracted from healthy B-cell lymphocites have natural homing capability towards their parental cells and Daudi cancer one, while reduced homing towards HL60. Furthermore, upon specific bioconjugation with antiCD20, a specific targeting is verified mostly only the CD20+ Daudi cancer cell line, and not towards B-lymphocites.
The TrojaNanoHorse (TNH) concept design and construction was fully developed: since this task is of particular high-risk, many different procedural, physical and chemical parameters were varied to obtain an optimized preparation protocol for the TNH. We first studied the use of artificial lipids to coat the synthesized nanocrystals and evaluated their long-term biostability in biological fluid, cell internalization and the stimuli responsive capability upon UV light exposure, as a preliminary study. Then we moved to the use of cell derived EVs to coat the ZnO NCs. Several characterization techniques were also used and developed specifically to properly characterize the nanoconstruct. We noticed that the process is a consequence of various mechanisms, including thermodynamic, kinetic and electrostatic ones. The team first worked on a preliminary TNH were EVs are derived from KB adenocarcinoma cell line to optimize the TNH preparation yield and the first cytocompatibility and cell internalization tests. A second version of the complete TNH concept, prepared with an advanced active encapsulation method of the extracellular vesicles extracted from healthy B lymphocytes, was then adopted. High yield and high level of ZnO nanocrystals internalization in the EVs was performed. The hemocompatibility and colloidal stability over long time periods of the whole TNH was successfully assessed. Furthermore, the monoclonal antibody antiCD20 was also bioconjugated to the TNH, creating a TNHCD20: the targeting selectivity towards Daudi cancer cells, sparing healthy B lymphocytes (also CD20 positive) and cancer HL60 cells (which are CD20 negative cells) was successfully demonstrated. Finally, the stimuli responsive ability towards an ultrasound stimulation was also achieved, demonstrating the synergism between the TNHCD20 and the applied shock-waves to kill Daudi cancer cell and spare the healthy ones.
Concerning the stimuli-responsive therapy, many efforts were devoted to set-up the instrumentation for ultrasound irradiation (now patented) and to deeply understand the mechanism of interaction between ultrasonic waves and zinc oxide nanocrystals. Actually, two commercially available and clinically-relevant instruments were purchased or exploited and, in the meanwhile, a lab-made equipment was designed, set-up and tested for this therapy. A novel, unconventional and safe methodology of nanocrystals activation to develop highly toxic species directed against cancer cells was developed, first explored in simple water-based media using commercial dyes, then in more complex biological relevant media and using soft tissue to simulate the US penetration in the body, and finally against cancer cells. The different key parameters for controlling the stimulation, the nanocrystals activation and their complex biological effects on cancer cells were explored and understood, showing an effective synergy between the US and ZnO NCs. A full understanding of the inertial cavitation and interaction with the surface of the ZnO nanocrystal was achieved, opening also a novel possibility to use the nanomaterials not only for the therapy but also for imaging, in particular as ecographic nanoconstrast agent. As mentioned above, also the final targeted TNHCD20 successfully underwent to a synergistic activation by ultrasound, in particular shock-way using a clinically-approved machine.
The cytotoxicity, the dissolution and biodegradation tests of the TNH and also of ZnO NCs were carefully designed and actuated in different fluids and cell conditions, establishing protocols and two guideline reviews for operation on cytotoxic behaviour of ZnO and related shielding.
Finally, the study of the bioimaging and nanodiagnostic capabilities of the TNH, was explored, starting with the intrinsic properties of ZnO as semiconductor. The optical properties of the synthesized nanocrystals were studied, also depending on the synthetic procedure, the chemical functionalities and dimensions in a paper.
Based on the strong expertise gained to set up the stimuli-responsive therapy and related equipment, important advancements were achieved to exploit a novel, unconventional imaging procedure, based on sonoluminescence. A new methodology and instrument set-up are described in a patent submitted to the Italian patent office and now under evaluation. A scientific paper related on the use of ZnO nanocrystal as contrast agent to enable sonoluminescence as a imaging technology for in vitro cell imaging is at present under submission.
Scientific dissemination in international congresses, broad-audience dissemination to non-specialized public, papers in peer-reviewed international journals with open-access modality, pitch to multinational companies and investment funds, two granted patents and one additional patent applications of the most relevant results were also accomplished.
Several groundbreaking results were achieved so far:
1. A novel synthesis for nanocrystals
2. The successful construction of core-shell hybrid TNH
3. The anchoring of targeting ligands to the TNH in a novel and unprecedent way.
4. A novel, unconventional and safe methodology of nanocrystals therapeutic activation using commercially-available and lab-made equipment.
5. The safe-by-design properties of the TNH per se, i.e. without the activation stimulus,
6. The establishment of protocols for the safety assessment of nanosized materials in biological media
7. The bioimaging multimodal capabilities of the TNH for the direct visualization of treated cancer cells.
8. All in all, the construction and application of a novel theranostic biomimetic nanodevice, never reported previously.
1. A novel synthesis for nanocrystals
2. The successful construction of core-shell hybrid TNH
3. The anchoring of targeting ligands to the TNH in a novel and unprecedent way.
4. A novel, unconventional and safe methodology of nanocrystals therapeutic activation using commercially-available and lab-made equipment.
5. The safe-by-design properties of the TNH per se, i.e. without the activation stimulus,
6. The establishment of protocols for the safety assessment of nanosized materials in biological media
7. The bioimaging multimodal capabilities of the TNH for the direct visualization of treated cancer cells.
8. All in all, the construction and application of a novel theranostic biomimetic nanodevice, never reported previously.