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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - TROJANANOHORSE (Hybrid immune-eluding nanocrystals as smart and active theranostic weapons against cancer)

Teaser

Summary

Cancer, together with circulatory diseases, is one of the primary causes of mortality worldwide and its incidence is expected to rise by about 70% (from 14 million to 22 million new cases/year) over the next 2 decades. There is a continuous growth of scientific studies on the genetic and molecular bases of this severe disease on one side, and on the development of new smart and nanosized materials as therapeutic platforms on the other. However there is still a huge disproportion between these nanotherapeutic treatments proposed to patients with respect to the conventional ones (i.e. surgery, chemotherapy, radiotherapy, immunotherapy and their combinations). Indeed several kinds of nanomaterials for theranostic (i.e. therapeutic and diagnostic) applications were developed so far, by many research groups worldwide, showing several smart characteristics, such as 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 - are still important challenges. Similarly, very little attention is paid at present 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. This ERC Starting Grant project TrojaNanoHorse (TNH) aims to cover the gap between the present nanomedicine tools and the clinical requirements, and strives to develop a non-immunogenic hybrid equipped nanosystem. It 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. Therefore, 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 idea is that of the Greek myth of the Trojan horse, which is not recognized as dangerous, hence is taken inside the city (i.e. the tumor). The TNH is then completed by targeting ligands in order to be safely and precisely directed to the cell objective. The action is 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.

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 pathology.
Furthermore the high flexibility of the proposed 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. Therefore the useful implication of this project is clear, when successful, even in the clinical fields to improve the quality of patientsâ€™ life.
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 standard

Work performed

Started in March 2016, with an overall budget of 1\'489\'219 euro by the European Research Council for 5 years, this project is now in the middle of its running course. The first 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 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 characterization of the biovesicles derived from different cell lines, in particular from both cancer and healthy cells cultured in vitro.
The TrojaNanoHorse (TNH) construction was also 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. 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 is still working to optimize the yield of the final TNH and the first cytocompatibility and cell internalization tests are on-going.
The hemocompatibility and colloidal stability of both the nanocrystals and the whole TNH were studied. In particular the Z-potential and colloidal stability of the nanocrystals were observed in many different media, both inorganic, organic, simulated and biological fluids, as well as the TNH in phosphate buffers, cell culture media and blood plasma. The performed hemocompatibility tests of the pristine nanocrystals show no coagulation activity. Those on the TNH are running to measure the absence of any thrombogenic effect.
The team also started to plan the targeting ligands and the way to anchor them on the biomimetic shell of the TNH. In particular, the anchoring of monoclonal antibodies to the lipid shell is in progress with specific and unconventional characterization methods also under development to assess the successful antibody binding. Thereafter the targeting mechanism and cell internalization mechanism will be proven.
Many efforts were also developed to set-up the instrumentation for the stimuli-responsive therapy, which is now under patenting thanks to the successful results achieved so far. In particular, a novel, unconventional and safe methodology of nanocrystals activation to develop highly toxic species directed against cancer cells was developed, first in acellular media and then with cancer cells. Two commercially available and clinically relevant instruments were purchased/hired and, in the meanwhile, a lab-made equipment was designed, set-up and tested for this therapy. We are on the way to fully understand the different key parameters for controlling the stimulation, the nanocrystals activation and their complex biological effects on cancer cells. In particular, many cell cytotoxicity tests were carri

Final results

There are several progresses beyond the state of the art and several groundbreaking results so far already achieved within the TrojaNanoHorse project activities:
1. A novel synthesis for nanocrystals was successfully developed and so far patented, providing highly reproducible spherical nanocrystals with uniform size distribution, outstanding colloidal stability for long time period in various media, morphology, crystallinity and surface chemistry. A great control over their biocompatible behavior, low cellular toxicity, haemocompatibilty, and imaging capability was also obtained;
2. The successful construction of the TNH itself, using a biomimetic and non-immunogenic shell, never reported before, is successfully accomplished and also patented. This approach will render the TNH an injectable and immune-eluding biosystem, showing high colloidal dispersion and stability in biological fluids and absence of haemolytic and thrombogenic effects;
3. The anchoring of targeting ligands, in particular monoclonal antibodies, to the TNH is under achievement with a novel and unprecedent way. Furthermore, specific and unconventional characterization methods are also under development to assess the successful antibody binding, opening novel protocols and methodologies for this specific task.
4. A novel, unconventional and safe methodology of nanocrystals activation to develop highly toxic species directed against cancer cells was developed and is under patent application. Both commercially-available and lab-made equipment has been set-up for this therapy, and we are on the way to fully understand the key parameter to control the stimulation, the nanocrystals activation, and their biological effects on cancer cells. Therefore, the TNH would exploit its cancer killing action only upon an appropriate stimulus after cell internalization. This implies the absence of carried drugs, thus avoiding any unwanted and harmful release and side effects.

5. The TNH per se, i.e. without the activation stimulus, is prepared to be safe-by-design, that is highly safe, bio- and haemo-compatible, biodegradable in safe by-products at the end of its life when in contact with biological fluids and cells.

6. Furthermore, protocols for the safety assessment of nanosized materials in biological media are under development, contributing to a UE standardization in the nanomaterials safety assessment.

7. The TrojaNanoHorse construct would also work as a bioimaging multimodal system for the direct visualization of treated cancer cells. In particular, one imaging modality derives from a very novel and unconventional stimulation, here exploited for the first time to visualize cancer cells.

8. All in all, the TNH project would promote the construction and application of a novel theranostic biomimetic nanodevice, never reported previously.