nanohealth

Workpackages

Targeted NPs (Liposome, HSA-PLA, Tiomers, Proticles)


Objectives

1. Optimisation of NPs for signal enhanced imaging
2. Coupling of targeting ligands to NPs for specific targeting
3. Development and characterisation of novel thiomer-based NPs for drug delivery

Background/Summary

The first objective of this WP will be the elaboration of novel strategies for NP derivatization and modification using iterative improvements as necessary. The modified NPs will be run through various model systems e.g. human placental cotyledon which will serve as test system to optimize MRI using lectin-coated NPs to target GlcNAc structures on endothelial cells and so to define the number of gadolinium chelates and targeting molecules necessary for visualisation by MRI (see WP3). Fluorochrome-labelled NPs will be used to assess ligand-dependent cell targeting using confocal fluorescence microscopy Imaging properties of NPs will be improved to lead to an enrichment of nanostructured Gd complexes in stem cells. Thus, acting as intracellular MR contrast enhancer.
In the second objective we will design and assemble targeted surface-engineered NPs to detect unstable atherosclerotic plaques in vivo. The targeting sequences of interest are peptide- and / or antibody-based and will include sequences defined in WP2.  Up to now, we could show that one target molecule is highly efficient to stain plaque regions in aortas of apoE deficient mice fed a Western diet.  Based on these promising results (proof of concept) the approaches on coupling the target-molecule to NPs will be further intensified. Another strategy will be to use monoclonal antibodies against a highly overexpressed structure in advanced plaques. In each case the targeted NPs will be iteratively optimised. 
With regard to the third objective of this WP, results obtained within the running project showed that the permeation enhancing properties of thiolated polymers (“thiomers”), based on the transient opening of tight junctions, can be even further improved. The higher the molecular weight of the thiomer the higher are its permeation enhancing properties. It is therefore a major aim of this third objective to perform more detailed investigations focusing on this phenomenon. In analogy to permeation enhancement thiomers might show a much higher efflux pump inhibition, when being formulated as NPs. It is therefore also an aim of this objective to discover the underlying mechanism of inhibition and to evaluate the potential of thiomer NPs on efflux pump inhibition (cf WP4). Another major aim is the development, synthesis and characterisation of novel thiomer-coated liposomes with enhanced mucoadhesive properties.

 


Development of targeting molecules for cancer and artherosclerosis

Objectives

1. Establishment of screening assays
2. Generation of target sequences suitable for incorporation into NPs
3. Generation of target sequences ligands containing improved efficencies suitable for incorporation into NPs
4. Generation of efficient antagonistic binding peptides
5. Generation of efficient antagonistic binding peptides

Background/Summary

This WP will develop novel targeting ligands for NP-based early detection and therapy of vascular disease and cancer, more specifically through the detection of neovascularisation (vulnerable atherosclerotic plaque and cancer) and cells over expressing specific G-coupled receptors.
To maximise the chances of generating useful results within this short project period we have selected two well characterised G-coupled receptors for tumour detection.
We will start with known molecules and modify them to enable conjugation to NPs. NOVEL targeting molecules will be developed by an approach based on a combination of molecular modelling (similarity design) and randomised production and screening techniques. Using known sequences selected amino acid residues of adequate z-score values will be substituted to generate new analogues. Potential targeting molecules will then be produced synthetically and validated for selectivity and specificity. An analogous approach will be used for v3 integrin ligands.

 


Evaluation of targeted NPs for imaging using MRI and PET/SPECT

Objectives

1. Evaluation of non-toxic NPs for modular targeting and theranostics
2. Application of NPs for Molecular Diagnostics Imaging in atherosclerosis
3. Optimising imaging protocols for stem cell tracking

Background/Summary

This work package undertakes translational R&D in magnetic resonance imaging (MRI). Participating in iterative development cycles with all other five work packages in years 5-7, the work package will test NPs optimised to bear targeting groups for use in molecular imaging by MRI. In a further step, NPs loaded with drugs and modified to carry the active agents into target cells („theranostics“) will be tested. Linkers will be used which permit simple attachment of the targeting groups, allowing the same NP carrier to be used for molecular imaging of a range of targets („modular targeting“). In addition to improvements in the NP carriers, carried out by the academic partners, the work package takes two clinically focused approaches to nanomedicine, reflecting the interests of the industrial partners (diagnosis of vulnerable atherosclerotic plaque and tracking of stem cell biodistribution over time after intravenous application of the cells in an animal model („4-dimensional tracking“). The WP thus has three objectives, namely (1) the evaluation of non-toxic NPs for modular targeting and “theranostics”, (2) the application of NPs for Molecular Diagnostics Imaging in atherosclerosis and (3) the optimising of imaging protocols for stem cell tracking.

 


Oral delivery, topical depot delivery

Objectives

1. Design and synthesis of API (peptide analogues)
2. Characterisation of API-analogue stability and biological activity in-vitro
3. Design of nanoparticulate delivery systems for API-analogues providing a sustained drug release
4. Therapy of hind-limb ischemia by the API-analogue NP vector in-vivo
5. Testing of thiomer NPs with optimised permeation enhancing and efflux pump activities

Background/Summary

This WP will develop a novel localised therapy for ischaemic tissue associated with diabetes and other disorders involving the local injection of thiomeric NPs containing an angiogenic peptide (API) able to provide localised long term release of the peptide through a depot effect.  Formulation of the peptide in this manner will enable facile administration by standard needle injection and also improve the local biodistribution by promoting the distribution in the target muscle tissue through enhanced diffusion. As well as exploring NP formulation, modification of the peptide will be explored as a way of enhancing this effect (for instance through increased peptide stability).
Since the oral route of administration is certainly the most favoured route of drug administration for both patients and clinicians alike, the second goal of the WP will be to build on the potential of thiomers for the oral delivery of drugs, based on work performed to date within the consortium with the goal of gaining a better understanding and improving the observed permeation enhancing and efflux pump inhibitory activities. Within the WP novel more potent nanoparticulate delivery systems for drugs such as therapeutic peptides and efflux pump substrate drugs will be generated providing high permeation enhancing properties, high efflux pump inhibitory properties, high mucoadhesive properties and protection against peptidases.

 


New in-vitro models for chronic toxicity and testing of all NPs within Nano-Health

Objectives

1. Assessment of toxicity in-vitro
2. Toxicity in-vivo
3. Predictive value of in-vitro tests 

Background/Summary

The term “Nanotoxicology” was introduced in 2004 by Donaldson and colleagues who realized that nano-scale particles behave very differently from their larger counterparts. The effects of the exposure and the mechanism of toxicity are much better documented for environmental combustion-derived NPs than for NPs designed for medical use. Inhalative exposure to these NPs increases all-cause mortality, asthma attacks, chronic obstructive pulmonary disease exacerbations as well as deaths and hospitalisations for cardiovascular diseases and cardiac deaths. The presumed mechanism of these pulmonary and extra-pulmonary effects is the generation of oxidative stress with subsequent activation of NF-kB/AP-1 and other redox signalling pathways and the release of pro-inflammatory mediators. Size, composition, shape and functionalisation (hydrophobicity and charge) are decisive factors for the toxicity of a particle.
In this WP, safety and toxicology will be addressed using a panel of in-vitro screening assays to prevent further development of toxic nanoparticles. Diverse aspects of toxicity including cytotoxicity, hemocompatibility and genotoxicity will be addressed. NPs with no obvious toxicity in the dose range of interest will be screened for in-vivo toxicity in rats and mice. For these applications standardised protocols will be used and adapted to the application of NPs. Tests investigating organ specific toxicity will be added if indicated by blood analysis or histopathological evaluation of in-vivo experiments. The effect on the immune system is studied in more detail to characterise the allergenic potential of NPs. As particles after a single exposure may be retained in the body for a prolonged time a new testing system will be developed addressing chronic cytotoxicity. The relationship between in vitro and in vivo toxicity will be explored for selected particles.

 


NP scale up and modelling

Objectives

1. Development of a suitable production method for thiolated polyacrylic acid micro- /NPs
2. Characterisation of thiolated polyacrylic acid micro- / NPs
3. Development of a suitable production method for PLA-HSA NPs
4. Characterisation of PLA-HSA NPs

Background/Summary

NPs and small micro-particles with uniform properties and defined structure are required for many pharmaceutical applications, such as individualized therapies and for the delivery of insoluble APIs. They also allow efficient surface modification, for the production of long-circulating drug carriers or targeted delivery vehicles. The main problem and the barrier for many large-scale implementations is the development of scaleable methods that result in reproducible batches that have a relatively narrow size distribution with uniform shape and/or drug loading and distribution. Currently, there exists a variety of available laboratory methods to make nano-suspensions, including top-down methods (e.g., nano-milling, jet-milling, dispersion of a liquid-phase in another liquid followed by solvent extraction), bottom-up methods (e.g., chemical polymer co-linking, in-situ gelation, crystallization and precipitation caused by a sudden shift of solubility due to a change in the physical or chemical properties, such as a acid-base reaction) or a combination of both.
However, all these methods impose major limitations for use in pharmaceutical applications due to (i) stability issues, (ii) contamination during milling, (ii) toxicity issues due to considerable surfactants, polymer or solvent use and (iv) scale-up difficulties arising from low mixing rates in large-scale vessels, which do not allow sudden shifts in a parameter to induce controlled and simultaneous phase inversion or nucleation. Thus, for the project, significant research is required to safely scale-up the NP production steps with the goal to obtain NPs with uniform controllable properties that can be manufactured in a reproducible manner. In summary, the main desired characteristics of the process are uniformity of the particles, the ability to control particle characteristics and reproducibility. 
Process understanding is the key factor for scaling up production processes and for controlling the product characteristics. Thus we will seek to understand the main factors and effects controlling   size distribution, particle shape, drug uniformity, etc. Process modeling will occur on the basis of the physical and chemical effects described in WP1. Using the mathematical tools developed above, recommendations will be made regarding the scale-up of the production processes. Alternative methods may be suggested as well (e.g., in the case of the emulsification process).