Community Research and Development Information Service - CORDIS



Project reference: 286125
Funded under: FP7-PEOPLE

Final Report Summary - NANORESISTANCE (Management of Resistance to Tyrosine Kinase Inhibitors with Advanced Nanosystems)



The work carried out by the NANORESISTANCE researches defines an unprecedented integrated approach for the comprehensive management of failure to anti-Tyrosine Kinase Receptor (anti-TKR) therapy and treatment monitoring. Tyrosine Kinase Receptors are epitomised by the Epidermal Growth Factor Receptor (EGFR) superfamily, the mostly studied and validated cancer biomarker. Defining and overcoming resistance to anti-EGFR has been a major compelling need in clinical oncology with significant impact on overall survival and associated public health burden. The first uniform clinical definition of acquired resistance to EGFR-inhibitors was reported in January 2010. Ever since, intrinsic and acquired resistance to EGFR inhibitors is increasingly well-recognized. This partnership has played a structuring role by allowing researchers to acquire key skills equally relevant to the public and private sectors including (i) cutting edge nanobiotechnology techniques for fabrication of nanotheranostic conjugates for targeted nuclear drug delivery and imaging, (ii) pioneering approaches for intracellular targeting with carbon nanotubes (CNT), (iii) innovative mathematical models and assessment of biodistribution and drug release kinetics, (iv) advanced image processing algorithms for prediction model design, (v) state-of-the-art Surface Plasmon Resonance for assessing drug-target interactions, (vi) emerging technologies for in vivo protein-protein and theranostic compound-protein interaction and (vii) novel in vivo models of major malignancies such as colon cancer and gliomas. These parallel approaches have led to a series of promising innovative solutions in the multifaceted challenge of the overall resistance to anti-EGFR therapies, as extensively described below. Most importantly, the partnership has offered and a well-structured scheme of complementary skills driven by the entrepreneurial spirit of academicians and research commitment of the industrial partners securing significant impact on their employability in their sector.


Having joined forces in a vibrant partnership and having achieved synergies across their main disciplines within an intensive research and training program, NANORESISTANCE partners have managed to meet successfully the project’s milestones and objectives thus making available to the European scientific community and the general public a complete substantial set of deliverables, most of which highly innovative and with notable European added value.

Major milestones and critical decision-making time points for the project have included (i) the availability of a set of prototype fluorescent aniliniquinazolines, (ii) the availability of nanometric delivery platforms, (iii) the validation of nanometric delivery platforms and ultimately (iv) the selection of most efficacious and specific prototype for further development and have been successfully achieved at the indicated time check points.

NANORESISTANCE’s Major Research objectives have included (i) determination of EGFR and EGFRvIII (mutant variant overexpressed in malignant brain tumors) binding efficacy of proprietary fluorescently conjugated anilinoquinazolines with diagnostic and therapeutic attributes; (ii) development and study of carbon nanotubes for cytosolic delivery of fluorescent aniliniquinazolines with high affinity, avidity and specificity for EGFR and/or EGFRvIII-kinase, independent of receptor-mediated uptake; (iii) new magnetic silica core-shell nanoparticles functionalized with fluorescent anilinoquinazoline TKI for nuclear anti-EGFR delivery and monitoring; (iv) fabrication of nanofibrous membranes composed of TKI-loaded biodegradable polymer with coaxial electrospinning for in situ drug delivery; (v) silencing of EGFR-pathway (e.g EGFR, K-Ras) mutations with siRNA grafted or complexed with CNTs; (vi) modeling of the interactions of the nanometric delivery platforms with the living systems, including transportation kinetics across barriers and finally (vii) evaluation of the theranostic efficacy of nanometric systems in vitro and in vivo. Through a prudently designed and comprehensively implemented workplan, all major objectives have been achieved effectively also introducing experimental and technological advancements much beyond those initially proposed.


As a prerequisite to the successful development of the distinct groups of nanosized carriers has been the de novo synthesis of a library of functionalized, target-specific tyrosine kinase inhibitors for mutant EGFR, including the EGFRvIII variant. Docking of candidate structures with both wild type EGFR and G719S mutant resulted in a number of useful remarks regarding the anilinoquinazoline core and the theoretically predicted interaction that core might have with the active site of the kinase. Of significance for the work for nanoparticle functionalization has been the finding that the existence of a Tetra-Ethylene-Glycol (TEG) or Hexa-Ethylene-Glycol (HEG) tail is not prohibitive for binding of the inhibitors in the enzyme binding pocket. For TKI synthesis the three points of diversity have included the aniline moiety, the Michael Acceptor and the oligoethylene glycol chain. Depending on the Michael acceptor stability, the selected synthetic approach has led to a plethora of different inhibitors for grafting onto the nanoparticles. Synthetic access to NPs compatible grafting methodologies were investigated successfully and grafting alternatives for all three delivery systems, i.e. (i) carbon-nanotubes, (ii) silica-coated magnetofluorescent nanoparticles and (iii) nanofibrous membranes have been identified. Besides their use as active targeting moieties in nanostructured carriers, these pegylated TKIs have shown potent and irreversible anti-EGFR efficacies in TKI-resistant brain, colon and breast malignancies. Besides applicability in (nano)pharmaceutical development, the new synthetic approaches provide a strong basis for researchers in the field for further advancements in medicinal chemistry.


Following the rationale design of new 4-anilino-quinazoline derivatives active on the TKRs, partners improved their efficacy on in vitro human glioblastoma, the most aggressive type of brain tumors, with covalent conjugation onto carbon nanotubes. Introducing cleavable chemistry for the synthesis of CNTs-TKIs conjugates, partners have obtained a suitable scaffold for the selective transport and release of TKIs to the inside cells. Following results obtained on in vitro models of rationally developed TKI-resistant cells and in advanced orthotopically implanted in vivo models, CNT-TKIs conjugates represent a valuable option for next clinical trials in glioblastoma patients showing resistance to conventional therapies. Furthermore the new 4-anilino quinazoline molecules displayed a discrete efficacy on TKI-tolerant glioblastoma cells making them potentially suitable for patients with acquired resistance and mutation on the EGFRs target as supported by the modulation of the EGFRvIII variant. Most importantly, these nanosystems induced apoptosis both in vitro and vivo followed by prolonged animal survival and improvement of neurological signs. In an additional contribution of NANORESISTANCE partnership, CNTs were complexed to siRNA anti-hEGFR capable of silencing wild type EGFR and seven different mutant forms, including EGRvIII. Within this work, CNTs complexed to anti-hEGFR siRNA have shown to be capable of silencing wild type EGFR and EGRvIII and inducing cancer cell death in anti-EGFR drug resistant GBM. Furthermore, siRNA anti-hEGFR complexed to CNTs induced significant cell growth inhibition in glioblastoma cells highly expressing the EGFRvIII variant and demonstrating resistance to the FDA-approved and clinically used TKI Gefitinib.
The promise of carbon nanotubes, is to achieve targeted therapies by using lower amounts of the indicated therapeutic agents. This will decrease the possible side effects in glioblastoma patients and the evolution of cells with acquired tolerance. Considering the high morbidity of GBM and associated disabilities during the patients’ disease course, it becomes evident that NANORESISTANCE results are expected to have significant impact on patients’ quality of life and the health care system. Swift translation of these data to the clinic would be of major interest for the International Brain Tumor Alliance support group and their role in raising awareness for the hope of nanomedicines is important.


Nuclear, as well as mitochondrial delivery, is of major significance for the management of resistance to anti-EGFR therapies since nuclear translocation and constitutive expression of phosphorylated EGFR is associated with cancer aggressiveness and treatment unresponsiveness.
Magnetic nanoparticles show great promise for a huge range of applications. The major advantages correspond to their magnetic nature and ease of biofunctionalization. By modulating major physicochemical characteristics such as biomaterial content, zeta potential, size and shape, magnetic nanoparticles rationally designed and formulated by NANORESISTANCE partners have shown favorable cell trafficking properties that have enabled the targeted delivery to the cell nucleus in a panel of major cancer cells, including colon, glioblastoma and, most notably, triple-negative breast cancer cells. Magnetic materials were silica coated by two different approaches to provide them higher stabilities, as well as to facilitate their functionalization. Partners were able to synthesize silica coated, magnetite (Fe3O4), cobalt ferrite (CoFe2O4) and maghemite (gamma-Fe3O4) nanoparticles that were further functionalized with DNA for increased biocompatibility. By an additional approach, Infrared Fluorescent or Rhodamine dye-doping added fluorescent attributes for bimodal imaging without compromising magnetic functionalities. Magnetic properties were improved by a size sorting process made on the first steps of the synthesis. In order to achieve grafting of the TKI on the surface of the nanocomposites, the partnership developed a method to calculate the amino groups anchored onto the surface of the silica shell coating magnetic cores. The concept of “smart probes” has been introduced using a quiescent fluorophore (fluorescamine) which becomes fluorescent once it reacts with primary amino groups. This is a significant achievement of the partnership as it has enabled very accurate assessment of TKI grafting. Reverse microemulsion methods have enabled the manufacturing of silica coated magnetic nanoparticles with different shell thickness coating magnetic cores, a development that influences the final magnetic behavior of the systems. With regards to the second main application, that of hyperthermia, it has been found that size increase of the magnetic core increases the specific lost power (SLP), an indicator of successful hyperthermia which does not seem to be affected by fluorophore encapsulation.

The grafting of Pegylated TKI onto the end-functionalised silica-coated NPs has been the outcome of unprecedented synergy between partners UPMC and ProActina, with considerable impact on both teams. The collaboration has yielded a wide panel of esterified NPs with the general formula gamma-Fe2O3 @-SiO2 @ NH2 @ TKIs or gamma-Fe2O3 @-SiO2 @ COOH @ TKIs. Grafting was confirmed and quantified with Thermogravimetric (TGA) analysis that has yielded 30-40% grafting efficacy with more favorable grafting yield in COOH-end-functionalised NPs. Nanotoxicologic assessment with a clonogenicity assay confirmed nanosafety for long term use. Kinase –inhibiting properties have been effectively preserved with grafting and compare favorably to clinically applicable TKIs in highly malignant glioblastoma cells.


Irrespective of disease or nanoplatform used, biological targeting and specificity of action, combined with low toxicity, is commonly desired in nanomedicine. To achieve this goal, the big number of functionalized nanoparticles (NPs) produced for each biological target, create a need for efficient and cost effective in vitro methods to screen their functionality, before they are applied to animal models. NANORESISTANCE partners have expanded in vitro methods of assessing the biological specificity of NPs, emphasizing on solution methods, on chip biosensor analysis using Surface Plasmon Resonance (SPR) and cellular imaging. Partners have demonstrated the development of a Surface Plasmon Resonance (SPR) platform for real time in vitro studies of NP-protein interactions using ProteOn XPR36, a unique multiplex biosensor system. Initially, the NPs described above were assessed for NP dispersion and surface charge properties were assessed with Dynamic Light Scattering (DLS), highly sensitive fluorescent scans and Z-potential measurements prior to building the nano-SPR platform. Selective surface chemistry and immobilization protocols have been developed and have been used to obtain high density and stable tethering of both -COOH and –NH2 type NPs on biosensors chip surfaces. To further validate this platform, partners aimed to investigate the interaction of tethered NPs with recombinant EGFR kinase (mutant L858R). Selected -COOH and –NH2 type NPs which were functionalized with anilinoquinazoline derivatives, which were among the most potent EGFR kinase inhibitors have been specifically assessed. Non-functionalized NPs were used as control. In parallel, partners have successfully developed synthesis for the modification of NPs of –NH2 type with biotin groups. These NPs have been shown to interact with a previously modified neutravidin surface chip demonstrating the specificity of this grafting process. Such findings demonstrate the feasibility of the SPR approach and provide experimental avenues for future fine-tuning. More specifically, the partners have (i) established physicochemical stability and aggregation assays for all types of NPs (amino and carboxyl NPs and functionalized NPs); (ii) devised essentials methods enabling NP work on SPR platforms without the risk of damaging the biosensor; (iii) optimized the amino-linked immobilization of NPs on different SPR surface chips; (iv) established proof of concept protocols and methods; (v) co-optimized the purification of the EGFR kinase protein; (vi) optimized EGFR immobilization on SPR chip surfaces in sufficient amounts to perform studies with small molecules i.e. our TKIs ; (viii) set up efficient chemical protocols to modify NPs bearing –NH2 terminal groups to biotin terminal groups, which are able to react with neutravidin surfaces in SPR experiments; (ix) developed methods of NP biotinylation that use very small amounts of material ; (x) discovered chemical synthesis methods to modify COOH- bearing NPs to amino-type NPs and (xi) set up exact protocols to monitor the interaction with various NPs with EGFR and TKIs compounds. The NANORESISTANCE biosensor platform offers a rapid and efficient technology for a wide panel of in vitro interaction studies between functionalized nanoparticles and biological targets, thus progressing far beyond the initially set objectives.


A: DIFFERENTIAL INTRACELLULAR DISTRIBUTION AND SUB-CELLULAR UPTAKE OF gamma -Fe2O3 @-SiO2 NPs IS DETERMINED BY END-FUNCTIONALISATION AND ZETA POTENTIAL: As previously stated, nuclear translocation and constitutive activation of phosphorylated EGFR (npEGFR) defines an emerging resistance biomarker in highly morbid malignancies such as colorectal cancer (CRC), triple-negative breast cancer (TNBC), malignant gliomas (GBM) and certain subtypes of colon cancer (CRC). The novel system of theranostic nanoparticles with anti-EGFR sensing attributes developed, characterized and validated by NANORESISTANCE partners define a promising approach to image and inactivate npEGFR. Temporo-spatial trafficking and intracellular distribution in live (i) colorectal cancer cells, (ii) triple negative breast cancer cells and (iii) glioblastoma cells characterized by constitutive expression of pnEGFR incubated with gamma -Fe2O3 @-SiO2 @ NH2 @ TKIs or gamma -Fe2O3 @-SiO2 @ COOH @ TKIs was assessed under confocal and fluorescence microscopy. There has been strong evidence of zeta-potential dependent, energy-driven, endosome/vesicle-mediated nuclear delivery with the NH2-end-functionalized NPs demonstrating higher perinuclear uptake and COOH-end-functionalized NPs showing higher intra-nuclear predilection associated with homogenous biodistribution in the cytoplasm. In vivo biodistribution and pharmacokinetics studies enabled by quantitation of fluorescence, as explicitly discussed later have confirmed the impact of zeta potential on tissue uptake and intratumoral distribution. More importantly, TKI-grafted NPs carrying Michael Acceptor functionalities and having demonstrated irreversible binding in in vitro enzymatic assays, disclosed compromised fluorescent signals in anti-p-EGFR immunoassays associated with features of apoptotic nuclei, strongly demonstrating sustained EGFR-inhibiting efficacies and high anti-cancer potential. It is, therefore, evident that charged bimodal silica core-shell NPs with anti-EGFR attributes offer a promising solution for the management of resistance attributed to npEGFR. Their tunable fluorescent and magnetic properties enable concurrent tracking and validation of biomarker expression in real time, in vivo. Rationally designed orthotopic animal models for CRC, TNBC and GBM have been developed as enabling systems for such validation.

B: SPATIOTEMPORAL DISTRIBUTION OF CHARGED NANOPARTICLES IN ORTHOTOPIC GLIOBLASTOMA MODELS: TKI therapies for glioblastoma fail mostly due to inability of these small molecule drugs to cross the blood brain barrier which is intact in tumors areas of high proliferative rate, such as the tumor margins. Assessing the biodistribution and brain tumor upake characteristics of nanosystems carrying TKI functionalities is of high clinical relevance. The temporal and spatial distribution and availability of both positively and negatively charged gamma -Fe2O3 @-SiO2nanoparticles were quantified in normal and glioblastoma-bearing mouse brains to determine the distribution characteristics of these nanosystems and their relevance to tumor pathology. Nanoparticles were injected intravenously, the animals were sacrificed at different times post injection, and frozen sections of the brain were collected. The sections were subsequently imaged using an epi-fluorescence. The nanoparticle accumulations and tissue area were segmented using automatic, unsupervised, thresholding. The nanoparticle fluorescence intensity per tissue area, which is analogous to nanoparticle concentration, was subsequently estimated. The results for both negatively and positively charged nanoparticles, for time points ranging from 1 to 48 hours were extracted. Given these results, it was evident that the nanoparticles, independent of charge, reached their maximum concentration by 10 hrs and are maintained at that concentration for at least 14 hrs. In order to further evaluate the spatial distribution of the nanoparticles in glioblastomas, the same biodistribution study was conducted in mice bearing highly vascularized, orthotopically implanted glioblastoma tumors. At 10 hrs post injection, the animals were sacrificed and frozen sections of the brains where imaged with an epi-fluorescence microscope. The area infiltrated with tumor cells was automatically segmented and at the same time the tumor boarders were identified and the tumor area was divided into regions separated by 25 μm progressively deeper into the tumor. The areas of nanoparticle accumulation were also automatically identified using a similar approach. With these two types of information, the nanoparticle fluorescence intensity per tissue area was calculated both outside the tumor and inside as a function of distance from the tumor surface. This work indicated that the negatively charged nanoparticles preferentially accumulate at the boarders of the glioblastoma tumor more so than positively charged nanoparticles.
In order to explain the spatial patterns estimated above, frozen sections of the glioblastoma tumor were stained with CD31 (to identify the blood vessels) and Hoechst (to identify the tumor cell nuclei). Several blood vessel characteristics (diameter, area, density (vessel area per volume) and wall thickness) were estimated per tissue area as a function distance from the surface of the tumor. The results imply that the vessels are bigger with thicker vessel walls closer to the surface as opposed to the deeper layers of the tumor. This finding has been of high correlation with the clinical condition where hyperplastic vessels with thick wall, the so-called “garland vessels” are pathognomonic of glioblastoma multiforme (GBM). Comparing the nanoparticle concentration with the vascular density (i.e. the vessel cross-sectional area per tissue volume) as a function of the distance from the tumor surface, a clear correlation is evident. This outcome is consistent with the numerical modelling results. The therapeutic implications suggested by the quantitative spatial analysis of the nanoparticle distribution, i.e. higher concentration in the periphery of the tumor, are quite significant for the effectiveness of glioblastoma management and delivery of TKI to this tumor. The property of the proposed nanoparticles to concentrate in the periphery of this tumor offer the possibility of eradicating the highly proliferative cancer stem cell sub-population which is resistant to conventional therapies and further minimize the residual malignancy after surgery. This work provides significant insights into a major clinical challenge, that of drug delivery across the blood-brain-barrier in tumors and further contributes to the evolving field of tumor nanopharmacology.

C. MATHEMATICAL MODELING: A set of “design maps” have been constructed and a series of ectopically grown epithelial tumors have been used for constitutive modeling and the mechanical properties of tumors and how these parameters affect tumor growth. A biomechanical model of tumor growth has been derived and has been validated with the available functionalized NPs. Part of this work has already been published. This model has been expanded and has been applied in the analysis and interpretation of the biodistribution of charged NPs in glioblastoma tumors. A spherical and symmetric tumor was assumed and thus, an one-dimensional problem along the radial direction of the tumor was considered. The delivery of nanoparticles to tumors involved both the transport of fluid from the blood vessels into the tumor interstitial space, as well as the transport of the nanoparticles. The fluid transport problem was solved first for steady state to calculate the interstitial fluid pressure distribution and subsequently the transient nanoparticle transport equations were solved for the intratumoral concentration of the nanoparticles. An in-house computer program implementing a finite differences method was developed for the solution of the model equations. This work is currently under publication in “Nanomedicine” and addresses a wide scientific audience while it provides design guidelines for the nano-pharmaceutical society.


Electrospinning has gained huge attention due to its capability to prepare nanofibers with tailored characteristics, rendering drug-loaded membranes suitable for biomedical applications, addressing timely clinical challenges. Such membranes enable us to treat locally minimally residual disease along surgical margins in very invasive and malignant tumors, where biological barriers keep them inaccessible to conventionally delivered targeted therapies. NANORESISTANCE tasks have entailed the application of co-axial electrospinning and the production of coaxial nanofibers where the core has been TKI-loaded biodegradable polymer. Partners have managed to (i) achieve the fabrication of functional Tyrosine-Kinase Inhibitor (TKI)-loaded electrospun nanofibrous membranes, and (ii) provide evidence that they may be further developed for biomedical applications.
Prior to the assessment of TKI delivery and anti-cancer efficacy of these membranes, drug release studies were conducted by UV-visible spectroscopy, in an environment mimicking tissue culture conditions. Mechanisms of drug release, shot-term and long-term release kinetics and released drug quantification have been explicitly assessed. Long-term clonogenecity assays have confirmed nanosafey for extended applications. Colon cancer and glioblastoma cell lines with differential expression of EGFR have been exposed to nanomembrane-released quantities of EGFR inhibitor equivalent to those routinely used in in vitro cell viability assays. Nanosafety of the drug-free nanofiber scaffolds was further documented with in vitro cytotoxicity colorimetric assays for short term and clonogenic assay for long-term (up to 3 weeks) application. Polymeric (PEO/PLLA) TKI-loaded scaffolds demonstrated comparable antiproliferative and cancer cell death efficacies when tested against equivalent concentrations of free TKI, shown to be attributed to the released TKI but not from scaffold degradation products.

It can be, therefore, concluded that drug-loaded PEO/PLLA nanofibrous membranes comprise a promising drug delivery system in surgically non-amenable malignancies as well as in those malignancies where biological barriers prevent systemic drug delivery, such as brain tumors and intraperitoneal metastasis of gastrointestinal or ovarian cancer. Additionally, having incorporated the silica-coated magnetic nanoparticles within the nanofibrous meshes, a novel system for amplified localized hyperthermia has been made available. It becomes evident that the surgical community would gain significantly from the availability of our nanomembrane scaffolds loaded with the indicated targeted therapeutic and/or combinations with sensitizing agents (e.g. natural products) while the direct impact on patients’ survival and the health care burden from the relief from long hospitalisations would be tremendous.

Overall, NANORESISTANCE IAPP has offered an integrated nanomedicines platform for diverse malignancies which fail currently available targeted therapies, with documented added value and notable impact for the European patients, EU-based enterprises and the scientific community across sectors and disciplines.


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Record Number: 184990 / Last updated on: 2016-06-23