Main Findings

Work Package 1: Nanocarrier design, development and characterization

 

Objectives

  • Nanocarrier selection and development nanotechnologies (Phases A-F)
  • Nanocarrier characterization (Phases A-F)
  • Nanocarrier optimization (Phases B-D)
  • Final processing of nanocarriers and pharmaceutical presentation (Phases D-E)
  • Nanocarrier production for advanced clinical and GLP toxicology studies

Work plan and conclusions

The main objectives of WP1 comprise the development and characterization of empty and fluorescent dye-labelled nanocarrier candidates. The criteria for the selected nanotechnologies have included e.g. the use of safe biomaterials, simplicity and scalability of the technology used for the preparation of the nanocarriers, and stability in intestinal fluids.

 

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Trans-int nanocarriers including nanocapsules, nanoparticles and micelle-based drug delivery systems

 

The characterization of the prototype families within WP1 have included e.g. production yields, size and zeta-potential distributions, stability in simulated intestinal fluids and cell culture media, shelf stability, stability during freeze-drying, fluorescent labelling and dye labelling stability. In addition to the nanocarrier development and characterization process, the work in WP1 has also comprised development and validation of analytical assays which have been used for the characterization of the nanocarriers.

 

Studies of nanocarrier-mucus interactions

While an extensive physico-chemical characterization of the nanocarriers has been performed, not much data has been obtained so far regarding the mucus-interaction properties of the different prototype families. The main reason for this has been the lack of a suitable method to analyse the interactions between mucus and nanocarriers. Different strategies have been investigated, including e.g. Transwell systems, mucus filled micro-capillary diffusion systems and DLS.

 

Recently a microscopy based technique measuring fluorescence recovery after photo-bleaching (FRAP) has been applied for the analysis of mucus-interactions within the consortium. A FRAP experiment measures the rate at which fluorescent particles diffuse back into a volume that has been photo-bleached by a high intensity laser beam. The quicker the recovery of fluorescence in the bleached region, the faster is the diffusion of the carrier in the mucus. In addition to providing diffusion constants in mucus, the FRAP experiment gives a measure of nanocarrier mucus-adhesion properties, through the determination of a parameter called the trapping constant.

 

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Illustration of a fluorescence intensity plot for the bleach region (inside the circle) during a FRAP experiment, which is used to calculate the diffusion constant and the mucoadhesion.

 

As an alternative, a microfluidic chip-based technology has also been optimised for tracking the behaviour of the different nanocarriers upon their contact with mucus. This technology provides information through the direct monitoring of particle diffusion by fluorescence microscopy in order to compare the muco-penetration capacity of the different nanocompositions developed within the consortium.

 

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Schematic representation of the microfluidic device developed by UCD for mucopenetration studies

 

Work Package 2: Formulation of peptide drug candidates

 

Objectives

  • Peptide characterization and stability profiles
  • Development of peptide-loaded nanoparticle formulations
  • Evaluation of the peptide release and in vitro/ex vivo stability of the peptide-loaded nanocarriers
  • Optimization of the peptide-loaded nanocarriers
  • Stability of peptide-loaded nanocarriers during storage
  • Final processing of nanocarriers, pharmaceutical presentation, and stability

Work plan and conclusions

The activities of WP2 build on the knowledge gained on the physico-chemical and biological properties of the nanocarrier prototypes developed in WP1, combining this with the understanding of the characteristics of the therapeutic peptides to be delivered (e.g. physical chemistry, stability). This close collaboration between the two work packages has been critical for the creation of peptide-loaded nanoparticle formulations.

 

Since the start of the project, thirteen prototype families including more than 1000 nanocarrier formulations have been explored by the WP1 partners at the University College of Cork, University College of Dublin, University College of London, University of Angers, University College of Louvain and the University of Santiago de Compostela. A wide range of technologies and raw-materials (polymers, oils and surfactants) has been applied in the development of the prototypes, which include nanocapsules, nanoparticles, polyelectrolyte complexes and a micelle based drug delivery system.

 

New prototype families and subtypes to existing prototype families have been added continuously throughout the project. The addition of new prototype families and subtypes is valuable as it creates a wider basis for prototype selection, and extends the characteristics of the carriers investigated by the consortium. The development of new formulations also aims to integrate the knowledge that is emerging from the consortium’s activities.

 

In relation to this, recent activities of this work package included the characterization and analysis of the peptides, together with the optimisation of their loading into the different nanocarrier prototypes developed in WP1. The consortium is currently working with five different peptides with different physico-chemical properties (molecular weight, solubility, hydrophilia etc.). A sixth peptide is expected to be available by early 2016.

 

From the information accumulated within WP2, it can be concluded that from a formulation standpoint not all of the aforementioned peptides will be equally well suited for all prototypes. Notably, the solubility and isoelectric point have a high impact on their encapsulation efficiency. Nevertheless, the partners have been able to adapt and modify the formulation parameters so that a considerable number of formulations fulfilling the initial requirements have now been identified. These requirements include high loading, controlled release and efficient protection of the peptide in the harsh environment of the gastrointestinal tract.

 

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Transmission electron microscopy (right) and atomic force microscopy (left) images of a peptide-loaded polymeric nanocarrier prototype developed at the USC.

 

At present, the selected prototypes from the majority of the thirteen families (WP1-WP2) have been transferred to the downstream work-packages for detailed in vitro-in vivo evaluation, while a few are still being subjected to final characterization and/or optimisation.

 

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Macroscopic image of a peptide loaded polymeric nanocarrier prototype in a liquid form (right) and as a freeze-dried powder (left)

 

Work Package 3: In vitro mechanistic and toxicity studies

 

Objectives

  • Evaluation of cellular toxicity and functionality
  • Evaluation of the interaction of nanocarriers with the intestinal epithelium and transport of the associated peptides

Work plan and conclusions

When encapsulating a drug/peptide into a nanoparticulated drug delivery system, the nature of the nanoparticle composition, the geometry and the surface properties can altogether exert a toxic effect. The toxicity screening of nanocarriers coming from WP1 and WP2 are evaluated in WP3 in vitro in Caco-cells.

 

Nanotoxicology is evaluated at different time points (2 h and 24 h) using validated assays: lactate dehydrogenase (LDH) release, ATP content, neutral red assay, and MTS cell proliferation colorimetric assay. All the prototype families were tested. The cytotoxicity studies indicate that most of the prototypes tested induce a very low to low cytotoxicity with EC50s ranging from 0.1 to >8 mg/ml.

 

A toxicological screening of nanocarriers is also carried out based on cellular immunological responses. In dendritic cells (DCs), the toxicity of the prototypes is measured by using Trypan Blue staining and flow cytometry (morphologic gate + 7AAD staining). Moreover, the role of the nanoparticles on DCs maturation is also evaluated in vitro. The prototypes are incubated with bone-marrow cells from C57BL6 mice to study their effects on DCs generation, vitality and maturation. The prototypes tested did not affect DCs at non-cytotoxic concentrations.

 

Nanoparticle size and surface properties, among other physicochemical properties of nanoparticles, strongly influence the mechanisms involved in nanoparticle cell internalisation. Increasing our understanding of the mechanisms and processes involved in nanoparticle transport across the intestinal barrier and the factors limiting their transport across this barrier could help to improve the nanocarriers in order to enhance peptide transport. The transport of WP1 and WP2 nanocarriers across the intestinal barrier is evaluated in vitro across different in vitro cell models: Caco-2 monolayers (enterocyte-like model), Caco-2 and Raji cell co-culture monolayers (M cell-like model) and Caco-2 and HT29-MTX co-culture monolayers (mucus secreting model). Nanoparticle transport can be evaluated by assessing the fluorescence detected in the basolateral compartment. Peptide quantification in both culture media resulting from cell culture studies is carried out using a previously validated LCMS/MS technique. The apparent permeability coefficient (Papp, cm·s-1) across cell monolayers is calculated. Flow cytometry and fluorescence microscopy are used to quantify and visualise the uptake of the nanoparticles by the intestinal cells.

 

The results of the in vitro transport studies indicate that the uptake by Caco-2 cells and the transport of fluorescent nanoparticles across Caco-2 monolayers is strongly influenced by their composition and surface properties.  For several prototypes, a significant transport of insulin was detected.

 

Nanoparticle and peptide transport has also been evaluated across living tissue in Ussing chambers: (i) in rat colonic epithelium by measuring epithelial membrane properties and quantifying transport and barrier functions of the tissue and (ii) in human jejunal and colonic tissues from biopsies. None of the nanoparticles tested induced toxicity. The transport of the nanoparticles and the associated peptide was also influenced by the prototype family.

 

Work Package 4: Preliminary in vivo mechanistic and toxicity studies

 

Objectives

  • Evaluation of particle interaction with epithelial barriers and biodistribution
  • Preliminary immunological evaluation

Work plan and conclusions

WP4 is examining selected peptide-nanocarriers in vivo using mouse and rat models to assess the fate of the particles in the intestine and to see if they are absorbed across the gut wall or if they release their peptides in advance of it. This involves advanced imaging modalities from four of the labs in TRANS-INT.  There is also an opportunity to gain preliminary data on whether the peptide-loaded particles actually produce a biological effect in the animal and to associate this with blood levels of the peptide.  Finally, this WP is also examining the effects of the peptide-loaded nanocarriers on local immunology in the mouse gut; this has never been looked at before for oral systems, but it is an important aspect of preclinical toxicology.  To do that, partners have to ensure that the nanoparticle constructs are free from any contamination that could trigger a local immune response, so this means that ingredients that make up the construct need a high level of purity and the synthetic process must be essentially free of bacterial contamination.  All studies in this WP have undergone local ethical committee review and are licenced by National Authorities; are carried out by individuals with appropriate training and competency according to legal standards. The WP began in mid-2015 with the establishment of the model assessment systems and in Q4, particles have been arriving at the reference laboratories.

 

At UCD, researchers have been using a published rat intestinal instillation model (Brayden, DJ & Walsh, E. (2014). AAPS J. 16:1064-1076) to examine the fate of particle constructs in different regions of the intestine, especially the major site for nutrient uptake in the intestine, the jejunum. This model allows study of fluorescently-labelled particle uptake by the gut wall and also to assess if the peptide-loaded particles generate blood levels that relate to a change in a biological parameter (e.g. reduction in blood glucose if insulin was the payload).  The rationale for this model is that by locally-instilling the peptide construct into the target region, we do not have to factor in the impacts of dilution, stomach transit, and particle dispersion.  As a screening tool it therefore gives the particle the best chance to succeed; if successful at this stage, then we have to further formulate to take into account those other aspects. If it fails, then the particle needs a redesign or should be abandoned.  In one example (Figure 1), we established the bioassay for a simple peptide and assessed an established gut permeation enhancer to provide positive control data for instillations of a candidate peptide. 

 

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Figure 1.  Rat jejunal instillations over 120 min under anaesthesia.  Pharmacodynamic effects are on the y-axis. Enhancer I is a novel excipient; Enhancer II is a gold standard one, sodium caprate. The peptide was also assessed by the sub-cutaneous delivery at 50-fold lower concentrations.  An ELISA to measure the peptide in plasma has been established in parallel. This data is being used as an aid to benchmark TRANS-INT nanoparticle constructs.

 

MHH have been labelling one of TRANS-INT’s candidate peptides using a novel isothiocyanate-based dye. The label is robust and has been developed with a nanocapsule prototype. By employing multimodality imaging probes or different dyes in fluorescence tomography, an understanding of the biokinetics and tissue distribution of the nanocarrier and its payload following oral delivery to mice will be obtained using bioluminescence (Figure 2).

 

MHH is also labelling the peptide for analysis in rodents by SPECT/CT imaging, examples of which are seen in the literature for exendin-4:  (Chuang EY et al (2013) Biomaterials. 34:7994-8001).

 

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Figure 2.Bioluminescence study of the fate of fluorescently labelled nanoparticles in a mouse following oral delivery. Note the high levels in the stomach (white).

 

At the University of London, one of the fluorescently-labelled constructs has been assessed by fluorescent microscopy following oral delivery to rats with some initial evidence of particle uptake.

 

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Figure 3.  Fluorescent particles beneath the intestinal epithelium.

 

Work Package 5: In vivo pharmacokinetic/pharmacological evaluation of nanomedicines

 

Objectives

  • In vivo evaluation of the pharmacological profile
  • In vivo evaluation of the drug pharmacokinetic profile

 

Work plan and conclusions

This WP’s activities were due to start in April 2015, however, they were started early because it was felt to be quite relevant to provide biological data to other WPs in relation to the formulations to be developed.  We undertook several proof of concept experiments ahead of the expected start of this WP.

 

In practical terms, this involved testing the effects of 6 different formulations in direct intra-intestinal instillations and then assessing glucose levels for at least 8 hours. These experiments provided relevant information in relation to a) the degree of activity (hypoglycaemia) of the formulations tested; b) the length and dynamics of their biological effect; c) the quality control parameters needed to minimise inter-assay variations between experiments when a large number of formulations need to be tested. These data were shared with all of the partners involved in this WP in order to minimise variations in experimental protocols between the partners.

 

Work Package 6: Preclinical efficacy, toxicological, immunological evaluation

 

Commences in May 2016

 

Objectives

  • In vivo advanced preclinical pharmacokinetic evaluation
  • In vivo advanced immunological evaluation
  • In vivo exploratory and GLP-toxicity evaluation

 

Work Package 7: Training and education

 

Objectives

  • Development of a modular education program in oral nanomedicine
  • Organization of a young innovative debating school to discuss advances in oral nano-drug delivery
  • Development of a training program involving personnel exchange between the member institutions of the consortium

Work plan and conclusions

  • The 4th Annual TRANS-INT training for the modular programme in nanomedicine took place in Barcelona in May 2015. This included a workshop session on peptide synthesis and the mass spectrometry of peptides at IRB Barcelona, as well as talks on what the product development pathway might look like for an oral nanomedicine from Sanofi, and a review of tools for in vivo imaging in small animals from MHH.  The programme has been set for the 5th Annual TRANS-INT training programme at the annual meeting in Cork in April 2016 and it is open to non-TRANS-INT scientists and to the public.
  • In relation to the Crossing Biological Barriers meeting in Dresden, held in November 2015, there was a large attendance, of 30, from TRANS-INT. PhD students and postdoctoral fellows were assigned eight podium slots to discuss their research in front of the COMPACT and ALEXANDER consortia, while the remainder were assigned posters.  This was an excellent training experience for this group.
  • The fourth young debating school was held in Barcelona in 2015. It included a poster session where the students and postdocs debated the merits of their data with the PIs of the consortium and with the members of the Scientific Advisory Board (SAB). They also debated a paper on oral insulin in nanoparticles during the modular session.
  • The current student/postdoc committee, with representatives from WPs 1-4, has completed its period and representatives for this committee are being sought from later WPs.
  • In the training programme, four exchanges took place in 2015, taking the total to 14.  These cover student and staff visits to TRANS-INT laboratories for periods of days to months to learn new techniques and to carry out research that they cannot do in their home institutions.

 

Work Package 8: Exploitation and Dissemination

 

Objectives

  • To define and implement an integrated strategy for TRANS-INT dissemination and exploitation
  • To regularly inform all stakeholders about TRANS-INT and the project results
  • To promote the (use of) TRANS-INT results and advertise the benefits of the project

Work plan and conclusions

An Exploitation and Dissemination plan has been prepared for the TRANS-INT project. The plan explores the means and approaches to be used to maximise the visibility of the project findings to targeted audiences.

 

The project website is used as the main online tool for dissemination of the project’s outputs. Online social media networks have also been setup: Facebook, Twitter and LinkedIn.

 

A successful joint Conference with the ALEXANDER and COMPACT projects took place in Dresden on 9-11 November 2015. TRANS-INT partners participated actively in the conference with 7 oral presentations, 2 posters and with the chairing of 4 sessions. 

 

A considerable number of dissemination activities took place at a national and international level:

  • Four scientific publications
  • Twelve oral presentations
  • Two poster presentations

As the consortium proceeds with achieving its scientific objectives a constant focus on how to best disseminate and exploit results will be taken with the aim to assure maximum visibility of the project and full exploitation.

 

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