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Design and development of advanced NAnomedicines to overcome Biological BArriers and to treat severe diseases

Periodic Reporting for period 2 - NABBA (Design and development of advanced NAnomedicines to overcome Biological BArriers and to treat severe diseases)

Reporting period: 2017-01-01 to 2019-02-28

Nanomedicine is an emerging and promising field to combat currently untreatable diseases. Many therapeutic targets are shielded behind biological barriers, limiting the possibility to reach them with conventional drugs, which therefore are administered in large quantities, so that hopefully at least a small amount of them will reach the pathological target. Biological barriers are even more problematic for most biological pharmaceutics, such as recombinant proteins, antibodies and gene therapeutics, dramatically reducing the effectiveness of new generation treatments and personalised medicine. The most promising solution to this challenge is the use of “nano-shuttles” able to safely navigate into the body, overcoming the biological barriers and finally releasing the drug where is required.
NABBA project intends to address this challenge developing innovative nanoparticles able to load the drugs and deliver them selective and efficiently at the pathological site. The NABBA project is part of the Marie Sklodowska Curie Actions, and therefore the other relevant objective is to train Early Stage Researchers (Fig. 1) providing them with an expertise able to promote their future career and to enrich the competitiveness of the European Research Area (ERA) in a field with relevant perspectives for health and market. For this purpose the NABBA consortium has been created, actually composed by seven European academic research groups (Fig.2 in green) and four companies (Fig.2 in red), with the intent to develop this multidisciplinary research, train the fellows, and strengthen the interactions among the European research groups and companies.
There is no doubt that nanomedicine will have an impressive growth in the next future, as a more efficient therapeutic and diagnostic approach. It is expected therefore that it will also provide new job opportunities for young researchers with qualified expertise in the field.
NABBA project generated nanoparticles (NPs) for drug delivery based on different self-assembling biocompatible materials: lipids, polysaccharides, polymers such as polymethacrilate or polyisoprene. The materials must self-assemble in physiological medium (water) incorporating the greatest possible amount of drug. The requirement for self-assembling must be finely tuned, nature exploits liposomes, mainly composed by phospholipids in which the presence of both hydrophobic and hydrophilic moieties favour aggregation in water generating a bi-layer (Fig.3) in which both hydrophobic and hydrophilic groups can be incorporated.
Following the example of nature other polymeric biocompatible materials containing both hydrophobic and hydrophilic moieties have been developed, optimising the loading capacity and the conditions to deliver the drug. Alternatively, the self-assembly has been performed using a mixture of cationic and anionic biopolymers. The capacity to load the drug and the stability of the NPs at different pH were accurately tuned in order to allow them to release the drug at the pathological site, in the required amount. The problems faced are the following: i) how to increase the amount of drug loaded in the NP; ii) how to induce the collapse of the NPs selectively at the pathological site, in order to release the drug; iii) how to direct the NPs selectively at the pathological site (suppose the cancer cell). NABBA project developed different strategies for each problem. For example, in order to increase the amount of drug carried by the NPs, instead of encapsulate it, the drug has been covalently linked to a polymeric component, with a linkage that can be cleaved at the pathological site. Exploiting this strategy, even two different drugs can be inserted (Fig.4)
The goal to direct selectively the NPs at the pathological site, allowing them to overcome the biological barriers that stand in the way, is very difficult. The NPs have been decorated at the surface with molecules (targeting agents) selectively interacting with receptors overexpressed at the pathological sites, specifically at the vascular epithelia and tumor cell membrane. This interaction will favour the transport trough the biological barriers. In NABBA project different targeting agents have been attached at the surface on the NPs, in particular carbohydrates such as mannose that specifically interact with the mannose receptors of the dendritic cells, the cells of our immune system. The need to induce the collapse of the NPs and the release of the drug at the pathological site has been faced with different strategies, from one side the NPs were built in such a way that they collapse at specific conditions present at the pathological site (lower pH, specific enzymes, oxidative conditions). From the other side, the possibility to induce the collapse by external forces such as an ultrasound (US) (Fig.5) been focused at the pathological site, was successfully explored in the NABBA project.
In order to study the efficacy of the NPs for drug delivery, first of all in vitro models of the biological barriers and 3D microenvironments were setup for different pathologies. Then analytical methods were standardized to follow the NP fate in vitro and in vivo. Finally, the efficacy of NPs to spread into the pathological microenvironment, their capacity to overcome the biological barriers and to release the drug, were tested. NABBA project setup models of the BBB, lung epithelia and pseudomonas biofilms, and developed 3D spheroids mimicking the microenvironment of pancreatic cancer and models of lung infections.
Despite some nano-drugs are already in the market, they are mainly limited in the nature (mainly liposomes) and without targeting properties, in other words not selective. NABBA project generated NPs by self-assembling different biocompatible amphophylic monomers and studied their loading capacity, bioresponsiveness and controlled release properties. The most challenging goal was promote a specific “active targeting”, in other words the capacity to release the drug selectively where is needed. Very interesting and promising results have been obtained, in particular targeting different tumor tissues and brain pathologies, considering that the blood brain barrier is the most difficult biological barrier to overcome. The translation of the scientific results in the area of therapeutics is not easy, 10-15 years are required for a new drug from the discovery to reach the market, with an investment of 2 billions of USD, and a very high percentage of failure. Nevertheless, a class of nanoparticles developed by one of the NABBA partners (University of Milano - Bicocca), registered as Amyposomes®, gave rise to the spin-off company Amypopharma®, in which recently a private “venture capital” company (Biovelocità) entered in the shareholding. The development of Amyposomes® as therapeutic against peripheral amyloidosis neuropathy is planned, with further perspectives to develop it as therapeutic against Alzheimer’s disease.
a) Two drugs, 2’-difluorocytosine and doxorubicine, connected via a polyisoprene chain, and b) Cryo-
The NABBA consortium. In green the academic groups, in red the companies
Ultrasound induced collapse of drug delivery systems
The 12 ESRs of NABBA Project