Final Report Summary - NANOTHER (Integration of novel nanoparticle based technology for therapeutics and diagnosis of different types of cancer)
Executive Summary:
The NANOTHER project was aimed to provide further development in nanotechnology applied to nanomedicine. In particular, the focus was to enhance and improve cancer therapies and diagnostics using these new tools that have arisen in the early twenty first century. The objectives of the project were i) to synthesize, based on previously existing methods, new nanoparticles (NPs) capable of interacting with living tissues, non-toxic and able to carry molecular drugs and other therapeutic molecules (siRNA), and ii) to make a proof of concept that efficacy of NPs with such properties would be better than the current therapies and could be potentially applicable to several types of cancer treatment.
Project Context and Objectives:
Over the last few decades, healthcare costs have been increasing due to a number of factors such as improved diagnostics, generalised screening, improved treatments and the general aging of the European population, which puts ever increasing pressures on healthcare budgets. There is significant pressure on the diagnostic and pharmaceutical industries to maintain innovation and product enhancement in the face of relatively restricted market growth. Nanomedicine and theranostics are emerging markets in which developing technologies and capabilities in nanotechnology, and diagnostic and therapeutic sectors are increasingly converging to improve efficiency and competitiveness of the discovery, development and marketing of diagnostics and therapeutics for various diseases such as cancer.
Concept
Two of the most important advances in nanotechnology rely on the development of novel technologies for new and effective medical treatments and diagnostic methods. Nanodiagnostics aims to identify disease at the earliest stages - at the cellular level, and perform drug delivery using nanoparticles (NPs) which will enable reduced toxicity of therapeutic drugs by delivering them to the disease site, and the consequent activation of the particles when they reach their target.
Overall NANOTHER objectives
NANOTHER intersects biomedical, health and nano industries, and R &D sits at the interface of chemical, biological and physical sciences and engineering. The main NANOTHER objective is therefore based on the integration of 5 key elements of current technology:
a) NP functionalisation technology,
b) fluorescence / contrast agent and specific antibody diagnostic techniques and imaging equipment,
c) novel drug-delivery and activation systems
d) new uses for electromagnetic based technology and medical equipment.
e) Another important innovation is RNAi technology, and the objective is to investigate the successful formulation and application of nanocarriers including siRNA as the therapeutic agent.
Characteristics and main research routes of the proposed project:
Functional nanocarrier = NANOTHER will focus on two of the most promising applications for cancer treatment:
i) polymer micelle core-shell NPs and
ii) magnetic NPs T
They were to be functionalised by linking several antibodies or ligands that bind distinctive targeted cancer antigens, and by linking therapeutic agents such as anticancer molecules and siRNA.
DIAGNOSIS = An important part of the NANOTHER project was the synthesis of NPs loaded with contrast agents or fluorescent labels to localise and detect the cancerous cells to be visualised by imaging systems such as MRI, SPECT, PET or fluorescence based imaging.
THERAPY = Hyperthermia was used to trigger drug release from hybrid magnetic NPs. For polymer micelle core-shell NPs, release mode is via biodegradable, bioerodible or pH sensitive systems. Pharmaceutical partners provided drugs already used, such as Doxorubicin, Taxol, Camptothecin (and derivatives). One new drug compound (from Pharmamar) in clinical trials Aplidin® NPs was also to be functionalised with siRNA to investigate nano delivery for the silencing approach. These investigations of different therapeutic agents on NPs for these specific applications contributed to the originality of this project.
THERANOSTICS = NANOTHER equally investigated a combined diagnostic and therapeutic approach. Initially, magnetic nanoparticles, properly functionalised and drug loaded, or furthermore, used only for their hyperthermic behaviour, were introduced in the body with an antibody specific for a chosen type of cancer. If a tumour is present, it will be automatically detected and the particles accumulate in the tumour area, at first by the passive enhanced permeability retention (EPR) process, followed by more specific binding through the appropriate antibodies to tumour cells. By applying an electromagnetic field, a hyperthermic effect is induced which will a) directly kill the tumour cells and b) trigger drug release of drug loaded NP simultaniously assisting with temperature increase, and thus with destroying tumour cells. This is the NANOTHER theranostic approach as there is a positive diagnostic (identification of tumour presence) and therapy by drug molecules, or hyperthermia, or both. An essential part of NANOTHER is the selection of drugs, biological determination of nanocarrier efficiency and determination of toxicology at different stages of the project
Project Results:
SP1: NP Functional specifications and selection (LCPO)
Sub-Project 1 was dedicated to the development of the new polymeric and hybrid (organic/inorganic) nanovectors. The first step in this work was the elaboration of polymeric and magnetic inorganic materials that constitute the scaffold of the proposed nanoparticule drug delivery systems. The idea was to synthesize advanced polymers with properties that will provide multifunctionality to the new drug carriers, such as thermo and/or pH responsiveness (for controlled release of chemotherapeutics), or ability to complex nucleic acid (for optimal siRNAs loading). Then, starting from these materials, a large panel of processes was investigated for the preparation of nano-objects with a focus on reproducibility and scalability. A third aspect that was studied in this SP is the drug loading, aiming at the higher loading contents and loading efficiencies to provide systems that can be realistically transferred to the industrial field. Finally, a last work package was devoted to preliminary studies of particles functionalization (for active cancer targeting) and an initial selection of the best systems based on objective criteria such as stability and toxicity.
WP1.1 Synthesis and characterisation of pH and/or temperature responsive polymers and block copolymers
A wide range of polymers was synthesized as potential material for nanoparticles preparation, mainly by INSTM and LCPO. INSTM explored many polymer families such as poly(amido amine)s, poly(ester)s, poly(ether-ester urethane)s, hemiesters of poly(maleic anhydride-co-n,butylvinyl ether)s, phosphonate polysaccharides and poly(hydroxypropylmethacrylamide-co-methacryloylglycylglycine-OH). Focusing on poly(carbonate)s, poly(amino acid)s and poly(saccharide)s blocks, LCPO performed the controlled synthesis of hydrophobic homopolymers and amphiphilic diblock copolymers. From this vast pool of biocompatible polymers, the selected materials were mainly amphiphilic copolymers with a diblock, multiblock or grafted structure.All the synthesized polymers have been extensively characterized, aiming to establish a relation between molecular properties and self-assembly behaviour.
These polymers were considered to be the most interesting for the preparation of novel nanotherapeutics.
Polymer name and chemical structure Developer Synthetic path Main properties
WP1.2 NPs formulation and characterisation
Methods explored for the preparation of polymer and hybrid nano-objects included supercritical CO2 processes (RESS/RESSAS, anti-solvent spraying), co-precipitation and nanoprecipitation (with or without the addition of stabiliser). Supercritical CO2 techniques developed by FEYECON produced particles in the range 200-400nm for some of the tested polymers but the cold powders obtained after spraying attracted moisture, hampering the harvesting step and causing particles agglomeration. The preferred method was nanoprecipitation, which allows the preparation of drug-loaded nanoparticles in one step with a good control over size and size dispersity with reasonable reproducibility and scalability (as demonstrated by ARGUS in SP4). Hybrid particles were also elaborated by this process, starting from the same polymer materials and magnetic NPs (mainly magnetite NPs provided by COLORITA).
WP1.3 Polymer and hybrid nanoparticles drug loading and characterisation
(drug, peptide, siRNA)
Anti-cancer drugs of various natures (small chemotherapeutic, peptide, siRNA) were successfully loaded in both polymer and hybrid nanoparticles. For each drug, the particles that showed the higher loading capability (usually one or two systems) were selected for further optimization (performed in SP4). Interestingly, some nanoparticles, like PTMC-PGA vesicles were able to load drugs of different natures.
Characterization of the new nanomedicines included classical physical-chemistry methods such as DLS (for hydrodynamic size determination), TEM, SEM, AFM (morphology and structure study) as well as chemical assays, spectroscopic and chromatographic methods for the quantification of loaded species. Specific extraction and quantification protocols have been set up for each drug and particle system.
In addition to magnetic NPs (that are used as negative MRI contrast agents), fluorescent dyes have been included in the proposed nanosystems, to serve as labels for either in vitro experiments (FITC, Rhodamine) or for in vivo biodistribution studies (near infrared dye).
WP1.4 Functionalization and selection of the 'best ' system
Functionalization strategies for ligand (folate, antibodies) grafting were developed in relation with polymer chemistry. Folate targeted PCL-PEG was prepared in bulk by INSTM and targeted particles were obtained directly from the functional polymer. For antibody grafting, amphiphilic polymers (PCL-PEG, PLGA-PEG, PTMC-PGA) were first modified using PEG heterobifunctional linkers. Antibodies were linked to the surface in aqueous medium after particles synthesis. Although requiring several steps, this strategy presents the advantage of being highly versatile since potentially any antibody, protein or peptide could be linked. Optimization of the most promising targeted systems has been performed in SP4.
For the selection of these 'best ' systems, objective criteria, listed hereafter, have been agreed upon by all partners and evaluated thanks to the work performed in different SPs: toxicity (SP2), stability in biological media (SP6), drug loading (SP1), scale up (SP4), magnetic properties (SP3, SP5), novelty.
Four nanoparticles selected for in vitro and in vivo testings: typical size, potential payloads and surface properties. All these particles are prepared by the nanoprecipitation method using either acetone or DMSO as organic solvent.
siRNA
Targeting Yes (folate, antibodies) Yes (antibodies) no Yes (antibodies)
Other functions Fluorescent labelling (FITC) Fluorescent labelling Fluorescent labelling (FITC, near infrared dye) Fluorescent labelling (FITC, Rhodamine, near infrared dye)
SP1 has provided a large supply of new nanosystems with promising features for cancer therapy and imaging (high loading capability, good stability, incorporation of imaging agents, targeting properties, stimuli responsiveness), that have been after a first pre-selection transferred to the other SPs for optimization and biological evaluation.
SP2 NP Toxicology and Biocompatibility (JRC)
PEG-PBLG unloaded NPs: CFE has shown that NPs have induced low cytotoxicity in all the cell lines examined, with the exception of MDCK where 100% toxicity (no colony formation) has been observed. No toxicity or very low toxicity has been measured by Alamar Blue in A549, HepG2, HaCaT, TK6 and Caco-2 exposed to PEG-PBLG unloaded NPs. No apoptosis/necrosis and no oxidative stress have been detected up to 72h exposure and in A549 cells PEG-PBLG unloaded NPs have induced significant secretion of the pro-inflammatory cytokines IL-6 and IL-8. In addition, MTT and Alamar Blue assays have not detected cytotoxicity induced by PEG-PBLG unloaded NPs in HSOC, as well as no apoptosis/necrosis has been measured.
PTMC-PGA unloaded NPs: CFE has not shown any cytotoxic effect exerted by PTMC-PGA unloaded NPs in any of the cell lines examined. PTMC-PGA unloaded NPs have not induced apoptosis/necrosis but, in contrast, they have significantly induced oxidative stress in TK6 and HepG2 cells and moderately in Caco-2 and A549 cells. Further studies have shown that the oxidative stress induced in TK6 and HepG2 was dose-dependent. In HSOC PTMC-PGA unloaded NPs has moderately induced cytotoxicity and cell death. In HaCaT, Caco-2, A549 and HepG2 PTMC-PGA unloaded NPs have induced inflammation as observed by detecting secretion of IL-6, IL-8 and TNF-alpha cytokines.
Magnetic PEG-PBLG NPs: CFE has shown high toxicity in all the cell lines investigated after 72h exposure to magnetic PEG-PBLG NPs. In contrast, Alamar Blue has not shown toxicity induced by these NPs in Balb/3T3 and MDCK cells.
Magnetic PTMC-PGA NPs: Alamar Blue has not shown toxicity induced by these NPs in Balb/3T3 and MDCK cells. In contrast, low cytotoxicity following 72h exposure to magnetic PTMC-PGA NPs has been observed by CFE assay, with the exception of HaCaT, where particles were highly toxic (100% toxicity).
NBRh13 NPs: CFE results have shown that NBRh13 NPs are highly cytotoxic for all the cell lines investigated; in particular they have induced 100% toxicity in Balb/3T3 and Caco-2 cells. By MTT and Alamar Blue has been shown that NBRh13 NPs have not induced toxicity, with the exception of Caco-2 where NPs resulted highly toxic already after 24h exposure. No cell death, detected as apoptosis and necrosis, has been observed, but NBRh13 NPs have induced significant oxidative stress in TK6, HaCaT, HepG2, Balb/3T3, Caco-2, A549 and MDCK cells. Since NBRh13 shows magnetic properties, it has been irradiated and the long-lived toxic intermediated have been investigated in vitro in Caco-2 and TK6 cells. Irradiated NPs have induced a reduction in the Caco-2 and TK6 viability. Reduction in Caco-2 survival has been measured also when short-lived toxic intermediates have been investigated NpsBlock_IS19b NPs: with the only exception of MDCK, where NpsBlock_IS19b NPs have induced high toxicity, low cytotoxicity has been observed in all the cell lines investigated by CFE following exposure to NpsBlock_IS19b NPs. Alamar Blue has shown that NpsBlock_IS19b NPs is toxic in Balb/3T3 cells, but not in Caco-2 and moderately toxic in TK6 cells. In addition, after 24 exposure NpsBlock_IS19b NPs were not toxic, did not induced cell death even after UVB irradiation and did not stimulated the secretion of pro-inflammatory cytokines in HSOC.
Block-M IS19b NPs: no cytotoxicity, investigated by MTT and Alamar Blue assays, has been observed in HaCaT cell line. Alamar blue performed on Caco-2 exposed to Block-M IS19b has shown that NPs are highly toxic and in TK6 cells.
MS60 NPs: CFE assay has shown that MS60 NPs are cytotoxic in all the cell lines investigated but not in A549 cells. Alamar Blue has shown that MS60 is not toxic in Caco-2 and low toxicity and oxidative stress has been detected in TK6 cells. No apoptosis or necrosis has been measured in all cell lines exposed to MS60 NPs, with the exception of HepG2 cells. In HSOC MS60 NPs resulted non toxic (MTT and Alamar Blue assays) and did not induce apoptosis. Following exposure to MS60 low risk of inflammation has been observed in HSOC. No oxidative stress has been observed in all the cell lines investigated following exposure to MS60, with the exception of TK6 exposed to MS60-batch6.
NBRh14 NPs: MTT and Alamar blue assay have shown that NBRh14 NPs do not induce cytotoxicity in Caco-2 cells but, in contrast, NBRh14 NPs impair the viability of TK6 cells.
VAM41-PEG NPs: CFE has shown that VAM41-PEG NPs are not cytotoxic in all the cell lines investigated. By Alamar Blue assay VAM41-PEG NPs resulted cytotoxic in Caco-2 and TK6 cells, but not in A549 and HepG2. In addition, no apoptosis and/or necrosis have been induced following exposure to VAM41-PEG NPs.
PBLG-b-Hyalunorate NPs: Alamar Blue assay has shown that PBLG-b-Hyalunorate NPs are not cytotoxic in A549, TK6, MDCK, Balb/3T3 and HepG2 cells. PBLG-b-Hyalunorate NPs, in addition, do not induce apoptosis and necrosis in A549, HaCaT, TK6 and Caco-2 cells.
PTMC-b-PGA-hybrid NPs: NPs have inhibited the viability of HT-29 and BT-474 cells already after 24h exposure and more severely after 72h, revealing thus a time- and a dose-dependent cytotoxic effect. Irradiating PTMC-b-PGA-hybrid NPs and analyzing the short-lived toxic intermediates in HT-29 cells has not shown any sigh of cytotoxicity, suggesting that these cells did not underwent to local heating during irradiation.
In vivo, the toxicological profiles of selected NPs have been investigated in CD1 mice via the Maximum Tolerated Dose (MTD) after single bolus administration and after multiple intravenous administrations (MTMD). Formulations have been selected taking into consideration the in vitro toxicity profiles and the future applications of the particles (investigated in other SP).
SP3 Targeting and Diagnostics (VICOMTEC)
Subproject 3 aimed at investigating and evaluating the selected nanosystems in terms of targeting and diagnosis. The first step in this work package was the production and attachment of antibodies and other targeting ligands to nanoparticles, and then the selection and the optimization of the best functionalization procedures. A task was thus dedicated to the nanoparticles characterization in terms of size, stability, etc. The second step in this work package was the in vivo preclinical evaluation of the functionalized NPs using MRI and SPECT/PET imaging. The functionalized NPs were evaluated from the point of view of their physical properties in the conditions of MR and SPECT/PET imaging. Then their targeting efficacy was tested first in vitro, and in vivo in mice, in both planar and tomographic modes.
WP3.1 Functionalization of NPs for targeting
This task has been confronted in its early stage to the large number of nanoparticles investigated and to the absence of a source of antibodies within the consortium, accentuated by the withdrawal of DKP.
In an initial phase, prior to the selection of the cellular targets and corresponding antibodies, various approaches have been developed for nanoparticle functionalization; they were optimized using model human antibodies and fusion proteins, with a minimal set of nanoparticles from LCPO, INSTM and Colorobbia, selected on the basis of toxicity evaluation.
WP3.2 Preclinical evaluation of the NPs in vivo using MR
The first thing to do in this work package was to establish the magnetic properties of the designed nanoparticles to verify which were suitable or not for MR imaging. To do so, measurement of longitudinal and transverse relaxivities (R1 and R2) of encapsulated nanoparticles provided by LCPO and of bare nanoparticles immersed in liquid DEG provided by COLORITA has been carried out. These systems were first loaded with magnetic core by their respective providers (Fe2O3 for LCPO NPs and CoFe2O4 or Fe3O4 for the COLORITA NPs). The experiment at high field were carried out by RMSB at 4.7T (200 MHz operating frequency) on a Bruker Biospec system, where transverse relaxivity measurement was made using a multiple spin-echo 2D imaging sequence and the longitudinal relaxivity measurement using an inversion-recovery 2D imaging sequence. The experiment at low filed were carried out by INSTM by means of two spectrometers, the using a saturation recovery sequence 90º -90º to measure the longitudinal relaxation time and the Carr-Purcell-Meiboom-Gill sequence for the measure of transverse relaxation time.
WP3.3 Preclinical evaluation of the NPs in vivo using SPECT/PET
In order to evaluate the selected NPs using SPECT/PET imaging systems, a first step was the design of a radiolabelling protocol that will be used through the project. TEIA initially received NPs produced by COLOROBBIA, and managed to successfully design a labelling protocol that preserves the nanoparticles stability. This protocol is a direct 99mTc labelling In which, typically 100 µl of the pertechnetate generator eluate is reduced with 40 μl of SnCl2 and the pH adjusted to 7-8 using NaHCO3 prior to the addition of 0.5ml nanoparticles solution and incubation at room temperature for 30min.
SP4 Pro-therapy (INSTM)
SP4 subproject aimed at the optimization of the drug loaded organic and hybrid nanoparticle formulations, being targeted or not, as well as at the NPs production scale-up. Objectives of SP4 included the preliminary in vitro and in vivo screening of the organic, hybrid and targeted, drug loaded nanoparticles; the satisfactory achievement of the objectives has guided the selection of the best formulation candidates employed in the biodistribution and efficacy tests performed in SP5 and SP6.
The main achievements of SP4 are summarised hereafter:
- 5 types of therapeutic agents, including three chemotherapeutics (Doxorubicin, Paclitaxel, Aplidin®), one biotherapeutic agent (siRNAs directed against the hypoxia inducible factor 1a) and two inorganic cores (magnetite NPs and maghemite Nanoparticles aimed at cancer treatment by hyperthermia and as MRI diagnosis tools, were loaded into the nanocarriers
- 3 different targeting ligands (1. anti-EGFR mAb for colon cancer model; 2. anti-Her2 mAb for breast cancer models; 3. Folate for breast cancer and general cancer targeting) were investigated and grafted to nanoparticles carrying the appropriate therapeutic agent for each cancer model.
- The production of polymeric materials, inorganic cores and their assembly into nanoparticles was performed under scale-up conditions.
- An in vitro and in vivo preliminary screening was performed in order to allow the selection of nanocarriers submitted to SP5 and SP6 studies. The characterizations performed pointed out the high potential of the developed nanovectors according to their chemical-physical, magnetic and relaxivity properties. The targeting ability using mAb was also demonstrated in vitro.
The activities performed within SP4 were divided into four Workpackages and are described, in the following paragraps, by each Partner role.
WP4.1 Optimisation of drug loaded polymeric nanoparticles and preliminary in vitro and in vivo screening.
Preliminary experiments were performed by loading retinoic acid (RA) as a hydrophobic model drug, later substituted by the loading of paclitaxel (PTX). Several polymeric matrices were screened and among those, the most successfully employed were the 2-methoxyethanol hemiester of poly(maleic anhydride-alt-butyl vinyl ether) 5% pegylated (VAM41-PEG), the poly(caprolactone)-block-poly(ethylene glycol) (PEG-b-P BLG) (IS19b) block copolymer, and novel polyammidoamines.
The applied processes were highly reproducible and the obtained nanoparticles had a monomodal diameter distribution with a mean diameter of 60-140 nm, depending on the material and the preparation method. The diameter distributions were assessed by dynamic light scattering technique (DLS) and confirmed by scanning electron microscopy (SEM), atomic force microscopy (AFM), and/or (TEM) Transmission electron microscopy. VAM41-PEG PTX-loaded nanoparticules formulation presented a 18% of loading and 77% of encapsulation efficiency. IS19b PTX-loaded nanoparticles formulation (Block-PTX) presented a 2.5 % of loading and an encapsulation efficiency of 86%. IS19b doxorubicin loaded NPs (Block-D) with average diameter of 140-160nm were obtained and the loading of the doxorubicin resulted in a 3.5% wt. Additionally, novel cyclodextrin /PAA cross-linked resins containing different amounts of ß- and cyclodextrin were prepared by copolymerization in aqueous media, at pH12, and converted into aqueous nano-suspensions by the High Pressure Homogenization technique (average sizes of about 320-370 nm).
WP4.2 Preliminary therapeutic evaluation of nanoparticles for hyperthermia and thermal assisted drug release
Efforts were devoted to refine the synthesis of two hybrid products coming from COLORITA's preexisting know-how (NBRh1 and NBRh13), which were produced with 10 and 5 liter-scale processes respectively. Products NCh14 and NCh15 have instead been produced with 400 mL-scale processes. In each case no significant stability changes occur by varying the synthesis scale.
According to the results came out from SP1, preliminary trials of drug loading on hybrid systems were carried out. Commercial paclitaxel (from Discovery Chemicals) was tested for encapsulation. Aiming to define the efficiency of loading, preliminary trials on the polymeric shell alone were investigated (NBRh13-blank; block-copolymer PLGA-b-PEG). The trials revealed that the polymer/paclitaxel weight ratio can be pushed up to 5/1 when the total amount of paclitaxel is maintained lower than 2.5 mg/mL. The maximum PTX loading capability found for hybrid NBRh13 was LCh = 1.78%, corresponding to polymer/PTX ratio = 4/1 and polymer/MAG ratio = 1/1. Doxorubicin loading was investigated as well, and according to the latest results the highest Doxo loading contents were assessed around 10% in hybrid systems containing 50% of magnetite as well Hybrid organic/magnetic nanoparticles were prepared by using the VAM41-PEG polymer and magnetite NPs provided by COLORITA. Human Serum Albumin and 0-glycidyl-2,3-0-isopropylidenglycerol-cyclodextrin were used as cofactors and coprecipitation method was applied. The prepared nanoparticles displayed a monomodal diameter distribution in suspension, with a mean diameter of 160nm. Paclitaxel loaded hybrid NPs (Block-MP) were also prepared under optimised protocols, without affecting the loading capability of the system and displaying preserved magnetic, relaxivity and hyperthermia properties.
Characterization of hybrid NPs.
The magnetic and relaxometric characterization of the provided nanoparticles was performed considering the main parameters commonly used to describe a magnetic system (i.e. blocking temperature, coercive field, remnant and saturation). The magnetic properties of the magnetite and/or maghemite core were well preserved after polymeric coating and functionalization (targeting moieties grafting and drug loading). In some cases, small differences in the coercivity and ZFC shape were observed, associated to a modification of the surface spin adjustment during the functionalization procedures. However, they were not so important and to produce a significant variation of the hyperthermic efficacy. The difference in the saturation magnetization has to be ascribed to the accuracy of evaluation of the magnetic material concentration, which was often very low in the case of the hybrid samples.
The optimisation of the experimental set-up using a coil suitable for future in vivo tests (f 10 cm) was accomplished. The new experimental conditions were adapted for applied field intensity and frequency of 22 kA/m and 186 kHz, respectively.
The efficacy of different synthesised hybrid magnetic nanoparticles (from SP1 and selected in SP4) in causing hyperthermia was evaluated in vitro by measuring temperature elevation induced by an AC electric fields. The kinetics of temperature increase during irradiation were collected for all the hybrid systems and the temperature increase as function of time was evaluated for different sample concentrations.
The hyperthermic efficiency of hybrid samples based on SA3300 magnetite inorganic core seems to be, by far, the most efficient (even at low concentration of inorganic phase) with respect to other inorganic cores. In particular, hybrid BlockM (and its derivatives, developed by INSTM-UniPI) and NBRh13 (COLORITA) show enhanced hyperthermic efficiencies. Samples of maghemite loaded HNP-PEG-b-PBLG-NT002 and HNP-PTMC-b-PGA-NT008 (provided by LCPO) instead, showed a very low (or even none) hyperthermic efficiency, even at high concentrations of inorganic phase in the range of frequencies 90-250 kHz. Nevertheless, the obtained results can be explained to the frequency adopted for the LCPO particles, which should be exposed to higher frequencies (around 800kHz).
The device herein and optimized hybrid not-loaded NPs (NBRh13) were delivered to TAU for the in vitro evaluation of hyperthermia efficacy (M22). At the end of the experimental session held in TAU (M34), the whole apparatus was sent to Leitat in order to accomplish SP5 scheduled activities.
Concerning Task 4.2.3 Preliminary evaluation in vivo COLORITA has asked to move this activity to SP5, in particular WP 5.5. The correspondent deliverable is D 5.5.1 (Report on evaluation of effect of drug unloaded and loaded targeted and not targeted hybrid NPs on selected in vivo tumoral cell models in vivo according to the last amendment proposed in September 2011). Even though SP4 and SP5 foresee different partners involved (PHMAR should be in charge of D 4.2.4 while LEITAT is responsible of D.5.5.1) PHMAR agreed not to perform the activity since it was no longer in its priority due to accumulated delays in the availability of the thermotherapy equipment. COLORITA accepted this decision because the in vivo tests in SP4 and SP5 meant on the same hybrid NPs compounds were redundant; LEITAT also accepted that task in replacement of DPK. This issue was recognised by the SPLs and the project coordinator who accepted the agreement between the partners and the merging of task 4.2.3 with task 5.5.1.
WP4.3 Therapeutic evaluation of organic and hybrid targeting nanoparticles
The activity related to WP 4.3 was mostly affected by the withdrawn of DPK and the consequent late availability of the antibodies needed for the development of targeted nanoparticles. Despite the engagement of Leitat on the targeting topic and its role in the production of EGFR antibodies, the targeting activity was carried on several other models and later (last 6 months) was performed also on the anti-EGFR antibodies provided by Leitat and anti-Her2 mAb (trastuzumab isolated from Herceptin®) by LCPO, Targeted nanoparticles with either antibodies or folic acid moieties were prepared.
WP4.4 Scale-up of the selected nanosystems
Argus focused on the synthesis of PTMC-b-PGA and PEG-b-PBLG copolymers from LCPO and ISB19b from INSTM, Pisa. The syntheses were optimized using the right conditions in order to achieve an effiecient scale-up. Although several problems occurred during the scale-up of PTMC-b-PGA copolymer, a final batch of 440g was finnaly synthesized and the material validated by NMR and GPC analyses, in agreement with LCPO 's specifications. Furthermore, one batch of 400g PEG-b-PBLG was also synthesized and validated. With respect to the copolymer ISB19b, originally coming from INSTM R.U. of Pisa, several tests were performed in order to convert the bulk conditions applied at INSTM into a process performed in solution. At the end, the product batch 115/15 (50 g), validated by NMR and GPC, was tested by INSTM and the corresponding nanoparticles were stable. Regarding scale-up of NPs formulations, ARGUS was able to scale-up the preparation of PTMC-b-PGA and PEG-b-PBLG organic nanoparticles on a 30 g scale.
WP5.1 Characterisation of magnetic, hyperthermic and relaxometric properties
The complete magnetic and dielectric characterisations of available and new magnetic products coming from SP1 and SP4 have been accomplished in a wide range of experimental condition.
Among all the samples investigated, two inorganic samples (cobalt ferrite d0 = 6,1nm and magnetite d0 = 12nm) have been selected as magnetic cores for hybrid ferromagnetic/organic NPs preparation, thanks to their promising magnetic, hyperthermic and relaxometric characteristics (both their hyperthermia and relaxometry were found enhanced with respect to commercial/literature-reported products). Thanks to the feedback provided by SP5 several hybrid samples were prepared afterwards in SP1 and optimized in SP4; they have all been characterized as well and in all cases the main properties of the naked core were found to be nearly unaffected by the embedding processes, thus confirming the goodness of these systems as theranostics devices.
In the last period a new batch of magnetite core, produced by COLORITA, has been characterized and compared with the old one. A good compatibility with the two cores was observed, so the second batch was therefore validated as magnetic core for hybrid NPs preparation. The relevant technical information is described in Deliverable D.5.1.1 and in D.5.1.2.
WP5.1 Characterisation of magnetic, hyperthermic and radio properties
Available not-loaded hybrid NPs were already characterised in task 5.1.1 and task 5.1.2. Promising compounds showing both good enhanced magnetic and hyperthermic properties were already selected. The technical details are collected and reported in the Deliverable Report D.5.1.1 and D.5.1.2. The magnetic characterization of hybrid samples NBRh1 and NBRh13 radio labelled by TEIA have been performed starting from M12. A batch of NBRh13_hEGFR NPs was received by TEIA (sent by COLORITA) and successfully radiolabeled. When radioactivity expired, this batch was send to INSTM-UniFi. Its magnetic characterization was accomplished and the magnetic behaviour of this assembly was compared to that displayed by the not-targeted counterpart. The agreement of cobalt ferrite-based sample (NBRh1) is quite perfect, while a small difference in magnetization values for sample NBRh13 containing magnetite was observed. This can be ascribed to an underestimate of the magnetic material concentration and, partially, to the different interparticle interaction or surface spin contribution due to the different dispersion medium or NPs aggregation state. In conclusion, the radio-labelling process does not affect the main magnetic properties of the samples, which can then be considered as promising candidates for theranostics involving PET /SPECT and MFH applications. Technical results are described in deliverable report D.5.2.1 and D.5.2.2.
WP5.3 Characterisation of radio labelled organic NPs
After receiving a lot of organic and hybrid NPs from the partners, TEIA radiolabeled them by applying the developed protocol, thus having a radiochemical yield near to 95%. Radiolabeled samples were injected in small mice and biodistribution studies in SPECT mode were carried out 1h and 24h post injection. In all cases, imaging studies confirmed biodistribution results. NPs were concentrated in liver, spleen and bladder. The concentration in tumor was not significant since no targeting agent was present. Therefore, Block-PTX-FA NPs were provided to TEIA (by INSTM) and successfully radiolabeled using the standard direct protocol; the yield was greater than95%. Stability tests were also carried out. This system, has been then tested in tumour bearing mice, but no accumulation was observed. It was subsequently confirmed that folic acid was not ideal targeting regarding HT-29 cancer cells. This system has hence been tested in mice bearing tumour of a different cell lines (U87MG - glioma brain cells) and an accumulation up to 6% was found.
WP5.4 Therapeutic effect of hybrid NPs and organic NPs in vitro
During this task, COLORITA has optimised an analytical method for paclitaxel detection by means of HPLC, for confirming paclitaxel presence inside organic/hybrid matrix and for optimizing its loading. A method for paclitaxel extraction from the organic matrix has been also developed, in order to evaluate total paclitaxel content (loaded and not loaded). Due to the low stability of the paclitaxel-loaded hybrid NBRh13 during the purification step, paclitaxel was discarted and efforts were focused on loading of doxorubicin. Nevertheless, due to the extreme sensitivity of doxorubicin to pH and to buffer properties, free and encapsulated doxorubicin undergo rapid degradation in phosphate buffer. For this reason COLORITA focused its effort with a new hybrid drug-loaded material (BlockM-PTX-FA from INSTM). This sample has been characterized in terms of drug delivery triggered by hyperthermia and due to its promising features it was selected for the final tasks of the project.
The aim of this task was to verify the therapeutic effect of selected radiolabeled NPs on small tumor bearing mice. The SPECT imaging protocol described in D.3.3.1 has been used in all studies. In agreement with the other partners, only the system 15_BlockM-PTX-FA was selected for this study. 15_BlockM-PTX-FA has been then successfully radiolabeled by TEIA using 99mTc. Imaging studies and biodistributions were carried out on HT-29 bearing tumor mice provided by LEITAT. SPECT images have shown high concentration in liver and relatively low NPs accumulation (~6%) in the tumor. For this reason and taking into account the limited number of HT-29 mice that were available in the project, direct tumor injection was also assessed. In this way, only tumor was depicted and no other organs were visible in SPECT images. Also transaxial slices of the tumor area were very clear (see D.5.5.3). Mice were imaged using PET before hyperthermia session and after this, since PET can image the viability of cancer cells. A cold area in the tumor region, which was not observed before hyperthermia and could suggest therapeutic effect has been observed.
By using the same set of mice provided by LEITAT (see Task 5.5.3) in vivo imaging studies have been carried out by TEIA. NBRh13-hEGFR was successfully radiolabeled using Tc99m and the direct labelling protocol. Imaging studies and biodistributions were carried out in mice provided by LEITAT. Unfortunatelly tumors were not shown both in images as well as in biodistribution studies. Nevertheless, assays carried out with other tumour lines (U87MG, glioma brain cells, which express EGFR) showed noticeable accumulation in the interested tissues up to 2h p.i although it was possible to test only one mouse, thus the protocol was not optimized. For this reason, only intravenous protocol was assessed due to the low number of animals for this pilot study. These preliminary encouraging results promote the evaluation of different tumoral models if necessary. According to the common agreements taken in the last meeting, no more studies have involved hybrid NBRh13_hEGFR and efforts have been focused on 15_Block-M-PTX-FA(115/15) only.
SP6 Biodistribution and Efficacy (GAIKER)
WP6.1 Interaction of NPs with blood components in vitro and intracellular fate
This task was performed using protocols validated by the Nanotechnology Characterisation Laboratory. It consisted on the incubation of the nanoparticles with blood serum, and the quantification and analysis of the proteins adsorbed to the nanoparticles recovered after several centrifugation steps. Our results suggest that the interaction of the tested nanoparticles (all of them without targeting moieties) with plasma proteins is not significant, normally below 0.1 %. In the cases where sufficient interaction was observed, the proteins involved consisted mainly on apolipoproteins, inmunoglobulins and lipid transport related proteins. Consistent with the nature of the polymers used, most of them bearing a lipidic-like nature. This task is relevant for later in vivo experimentation, as it may give hints on the NP behavior when in contact with physiological fluids. Experiments were performed using 10 individuals pooled fractions of plasma to avoid inter-individual bias.
The BT474 (human breast carcinoma) and HT-29 (human colon adenocarcinoma) cell lines were selected and used to investigate, by means of fluorescent microscopy, the internalization and the intracellular localization of fluorescently labeled nanoparticles.
After cell exposure to nanoparticles, and as means to identify the uptake and the intracellular fate of nanoparticles, cells were stained using specific antibodies. This staining allowed the identification of specific intracellular organelles that could be targeted by the internalized nanoparticles. In particular, the colocalization of nanoparticless with early endosomes, late endosomes and lysosomes was investigated. We could conclude that both Block_FITC_IS19b and FITC-PTMC-b-PGA based nanoparticles could be internalized by BT474 and HT-29 cells. However, although being internalized, no colocalization with the endosomal (early and late) compartment or with the lysosomes was observed. It is important to refer that these assays were performed using living cells. In addition, NPs were observed forming aggregates/agglomerates which have adhered on the cell membrane, and apparently were not able to be internalized, or have adhered to the glass slides where cells were cultivated. As a result, the amount of NPs taken up by BT474 and HT-29 cells was very low. The adsorption of the nanoparticles to cells, at 4°C, was minimal and similar to the control cells. While there was almost no uptake by the HT29 cells of the three hybrid NPs, at 4h post incubation, after 24h a significant increase was observed. HT-29 cells could internalize non-hybrid PTMC-PGA based nanoparticles to a higher extent, with significant uptake observed after 4h incubation. It can be assumed that the addition of the maghemite to the PTMC-PGA nanoparticles could cause a reduction/delay in the uptake into the cells.
The NPs were located inside the cell and at the cell periphery, showing a higher uptake at the longer time incubation (24h). These results are in agreement with the previous findings where NPs were attached to the cell membrane.
NPs showed colocalization with the endosomal compartments at both 4 and 24h: Rab5 (early endosome), Rab7 (24h not 4 h) (late endosome) and Catepsin D (lysosome); Rab11 (recycling endosome) compartment showed no colocalization. These experiments were performed with fixed cells and this may be the reason why these results are in contradiction with the assays performed in living cells.
We have also observed that slight modifications of the surface of the nanosystem can make it behave in very different manners. FITC and RhoB addition generated quite different results so this point must also be taken into account. All systems seemed to internalize, each of them with different kinetics.
WP6.2 Biodistribution
The goal of this study was to analyze the biodistribution of three different untargeted nanoparticles (PTMC-PGA, PEG-PBLG and PEG-Hya) developed by LCPO, in normal mice and to identity their fate once intravenously injected. The nanoparticles were tagged with a fluorescent dye allowing its location by an in vivo imaging system. To do so, two approaches were defined: the acute setting; administration and read out after 24h, and the chronic setting; administration every two days for one week and read out after 3 doses of nanoparticles. The conclusion obtained from those studies was that all nanoparticles were widely distributed in the animal body, but mainly accumulated in spleen and liver.
The biodistribution nanoparticles loaded with taxol (15_Block-P-FA; IS19b) was evaluated and compared with a Cremophor formulation (Paclitaxel injection, Mylan). Each formulation (1 mg/kg of taxol) were administered by single intravenous injections to male ICR. Concentrations of taxol were evaluated in plasma and in selected organs by LC/MS/MS method. The pharmacokinetic properties of the two taxol formulations were similar. Generally, taxol rapidly disappeared from the plasma within 6 hours post admnistration. Taxol loaded into IS19b-NPs presented lower maximum plasma concentration, but its levels decreased slower during the first hour post admnistration. Taxol showed similar affinity, in both tested formulations to, the following organs: liver greater than kidney greater than lung = spleen. The distribution into the tissues revealed decreasing taxol contents over time, up to 10-16 hours in all tissues. Taxol levels in these organs were higher than its simultaneous plasma concentration for both formulations. The results did not reveal substantial differences between free and IS19b-NP forms of taxol, relevant for target transport of this anti-cancer drug.
The biodistribution of PEG-DY700-PTMC-PGA nanoparticles and grafted with trastuzumab antibody HER-PEG-DY700-PTMC-PGA was determined by the evaluation of DY700 presence in the blood and in vital organs of healthy ICR mice. Nanoparticles remained in systemic circulation for a relative short time (up to 3 h), with a correspondent circulation half-life time of less than 1.5 h. The naked nanoparticles showed lower Cmax and AUCs, what suggests that trastuzumab grafted nanoparticles are more effectively distributed. Biodistribution data in mice revealed high initial organ uptake of both used nanoparticle formulations. Naked and trastuzumab grafted nanoparticles showed different tissue uptakes in various organs, they decreased in the following order: liver greater than kidney greater than blood ~ lung greater than spleen ~ heart and brain. In vivo organ exposure to trastuzumab grafted nanoparticles was higher and elimination slower than for the naked nanoparticles. The relatively high content in the cardiac area and in the brain, at early time points post-dosing, indicates higher blood circulation for trastuzumab-grafted nanoparticles. In summary, the results from this study indicate greater exposures (expressed as Cmax and AUCs) in the vital organs, longer mean residence times and elimination half-lives in the liver for trastuzumab grafted in comparison to naked nanoparticles.
The goal of the present study was to analyze the biodistribution of two nanoparticle formulations (PTMC-DY700 and PTMC-DY700-mAb EGFR), developed by LCPO, in tumor bearing mice. More specifically, if by means of the targeting moieties the targeted nanoparticles could reach the tumor differentially, as well as to assess their biodistribution in other organs. Both nanoparticles were tagged with a fluorescent dye allowing its location by an in vivo imaging system. To do so, two approaches were defined: the acute setting; administration and read out after 24h, and the chronic setting; administration every two days for one week and read out after 3 doses of nanoparticles. Significant increased tumor targeting (30% higher) was observed for the antibody grafted formulation for both settings.
The biodistribution profile of paclitaxel (taxol) loaded - folic acid targeted Block-MP-FA (IS19b) nanoparticles was evaluated in breast cancer bearing mice, presenting high expression of folate receptor (FR), as means to demonstrate taht targeted nanoparticles could transport taxol to solid tumors with high FR expression. The distribution of taxol in vital organs and breast cancer tissue was as follows: liver, kidney, spleen lung, tumor and poor in heart. Taxol was not detected in the brain. Pharmacokinetic analysis indicated that the terminal half-life in tumor was longer than in liver or in the rest of vital organs, corresponding to a longer mean residence time. Elimination half-life of taxol in tumors was 4.73 h and mean residence time of 7.43 h. In contrast to the rest of the vital organs, small amounts of taxol were still present in the liver and tumor at 16 h; with longer mean residence time (7 h) and elimination half-life (4 h) in the tumors. Content of taxol in the tumor tissues reached a small peak followed by its gradual elimination until 16 h; however, these results demonstrated a significant increase in tumor residence time and a subsequent 1.5 - fold prolongation of elimination half-life than in liver and up to 5 - times than in the rest of the vital organs.
The pharmacokinetic parameters in plasma and the distribution into several organs as well as into tumors were evaluated after single intravenous administration of different formulations of Aplidin to mice bearing MRI-H-121 (renal) and HT29 (colon) xenografted tumors. The nanoparticles formulation studied were:
- Aplidin loaded nanoparticles of PTMC-b-PGA
- Aplidin loaded nanoparticles of PEG-b-PBLG
- Aplidin loaded EGFR targeted PTMC-b-PGA nanoparticles
- Aplidin in CEW: Cremophor:Ethanol:Water formulation
Blood, tumor, kidney, liver, femur, breast, intestine, spleen and brain tissue samples were harvested from each animal at 0.5 1, 2, 4, 8 and 24 hours post-administration. Samples were analyzed by a supported-liquid extraction (SLE) followed by a gradient reversed phase chromatography and detection by positive ion electrospray tandem mass spectrometry (ESI/MS/MS) using multiple reaction monitoring (MRM).
Results showed similar pharmacokinetic behavior and tumor distribution of Aplidin regardless of the formulation administered or the tumor model. In other tissues, no major differences were seen with any formulation, except in the bone assayed: femur. Approximately 2-fold higher Cmax and AUC0-24 and largest t1/2 (3.3-fold) were observed after the administration of 0.25 mg/kg of Aplidin formulated in PTMC-b-PGA nanoparticles, than in PEG-b-PBLG nanoparticles. No differences were observed between targeted and not targeted nanoparticles.
WP6.3 Efficacy in vitro
Several nanoparticle formulations were studied concerning vitro efficacy. The test performed included mostly basic cytotoxicity and endpoint assays that were estimated to be the most representative or relevant. Different antibodies were used to test if targeting could be achieved. The tested drugs consisted on Aplidin, taxol and siRNA
Performed MTT assays indicate that drug loaded nanosystems are more toxic than those unloaded. Targeted nanosystems are more toxic than non-targeted, in HT29 cell line and similar in LS174T cell line. PTMC nanoparticles seemed to be the most versatile system, as it can be be loaded with both Aplidin and siRNA.
Aplidin assays indicate that although usual Aplidin formulation performs much better than encapsulated Aplidin, the toxic effect of the adjuvant needed to administer Aplidin must be also part of the equation mostly paying attention to the different values of toxicity. Studied typical toxicity endpoints showed little differences between antibody+ and antibody- formulations, indicating that maybe the toxic effect of these drugs is not mediated by apoptosis or ROS, but by other parameters. Also, variability of the batched is also something to be taken into account and quite difficult to solve, as it seems to be a problem of nanotechnology on itself.
siRNA efficacy was evaluated upon encapsulation into several types of nanoparticles. The siHIF encapsulated into the polymeric nanoparticles was able to block HIF expression in different types of cancer cells in vitro. Furthermore, nanoparticles grafted with anti-HER2mAb selectively bind breast cancer cells that overexpress the HER2 receptor, supporting the specific and efficient targeting of our approach in vitro. Similar results were obtained when nanoparticles grafted with anti-EGFRmAb were used in colon cancer cells, although specificity and efficacy were lower. Also, nanoparticles loaded with siHIF were shown to reduce tumour growth in vivo. In addition, the in vitro efficacy of doxorubicin loaded nanoparticles nanoparticles was confirmed. By loading a combination of doxorubicin and maghemite nanoparticles within the polymeric vesicles we have shown that upon exposure to a high frequency magnetic field a boost in drug release and subsequent increased toxic effect could be obtained.
The non-hybrid NP is not toxic to HT-29 cells after 24h incubation, however after 72h incubation the non-hybrid NPs exert a slight toxicity at higher concentrations.
Hybrid NPs seem to be more toxic than non-hybrid NPs. At higher concentrations a 50-60% viability decrease was observed, after 72 h incubation with HT-29 cells.
Three different PTMC hybrid NPs were tested in HT-29 cells that overexpress the EGFR, namely: naked, trastuzumab and anti-EGFR mAb grafted Nps. No significant differences in terms of toxicity were observed, contrarily to the initial expectations where anti-EGFR mAb grafted Nps shoeld exert higher tocity, due to increased cell specific internalization.
The results show that the irradiation of NBRh13 nanoparticles could affect HT-29 cells viability at both temperatures, 38 and 42 ºC, but to higher extent at 42 ºC. Similarly, the effect of the irradiation of NBRh13 hEGFR was significantly higher than incubating the cells at the same temperature, 42 or 38 ºC (30% compared to 25% ). However by targeting the NPs with hEFGR did not show any additive value for the efficacy of the hyperthermic effect. On the contrary, it reduced the toxicity from 70% (NBRh13) to 30% (NBRh13-EGFR) at 42 ºC, and from 30% to 25 % at 38 ºC, respectively.
WP6.4 Efficacy in vivo
The aim of this study was to determine and compare the efficacy of Paclitaxel loaded organic nanoparticle (IS19b) targeted to cancer cells using Folic Acid, as well as its toxic profile. Paclitaxel cytostatic was used reference compound. Both formulations were administered by intravenous (iv) route to a nude mice strain bearing a orthotopic mammary tumor. Animals administered with the nanoparticles without paclitaxel were employed as control group. Tumor efficacy studies demonstrated similar results for both formulations. It can be concluded that the coupling of Paclitaxel to the IS19b-M-FA did not improve the efficacy of Paclitaxel on the tested tumor model. On the contrary, the activity profile of nanoparticle-coupled paclitaxel regarding animal body weight reinforces the toxicity of this cytostatic compound.
Antitumoral efficacy evaluation of aplidin loaded PEG-PBLG nanoparticles, aplidin loaded PTMC-PGA nanoparticles and aplidin loaded EGFR targeted PTMC-PGA nanoparticles was evaluated and compared with aplidin convencional formulation (cremophor solution CEW). Nanoparticles were administered intravenously to athimic nude mice bearing subcutaneous tumors induced by human colon adenocarcinoma HT-29 cells.
The animals were injected by iv route five daily doses schedule for 2 consecutives cycles (days 0-4 and 9-13). The administered concentration was 0.25 mg/kg/day of aplidin. For targeting evaluation a more intensive treatment (ten daily doses schedule, day 0 to day 9) was decided taking into account the results obtained with no targeted nanoparticles.
Results demonstrated that Aplidin nanoparticles were well tolerated by tumor bearing animals. Aplidin, formulated in Cremophor or loaded in targeted or non-targeted nanoparticles formulations resulted in no antitumor effect in the HT-29 model with a minimal T/C of 67.6 78 and 67.1 % respectively.
WP6.5 Preparation of preclinical nanoparticle batches for therapeutic evaluation
These two tasks accounted for nanosystems preparation. Several batches were prepared and distributed to the different partners involved. The same batches were sent to several partners at the same time, as means to reduce in vivo variability. It was clear from previous in vivo assays that the batch-to-batch variability could affect the final results. All partners received the nanosystems on time and there was no delay in this task
Potential Impact:
- Reinforcing competitiveness and associated economic impact
Targeted diagnostics and therapy in solid cancer is defined as one of the most promising tools for cancer treatment. NANOTHER has proposed a leading research partially based on partners achievements and successful preliminary results aiming at a nanosystems platform capable of binding targeting moieties, enhanced contrast agents for imaging, therapeutic agents and intracellular drug delivery, for solid cancer with a proof of concept for breast and colon cancer. The realization of this project effectively contributes, to the transformation of the European medicine, pharmacology, medical imaging and nanotechnology related industries from a resource-intensive to a knowledge-intensive phase, which further contributes to the EU WW position in these fields. This project is part of the chain in this transformation, introducing high added value technologies and products in a sustainable manner. The outputs of the project has increased European scientific and technological qualities, and at the same time has created industrial and employment growth within Europe.
- Reinforcing competitiveness - World versus European nanotech market
The nanotechnology in Europe is gaining exponential interest in the past decade. The market in nanotech is expected to increase in about a 23 % by 2016. Companies and SMEs started some time ago to develop technologies where nanomaterials are an important part. Nanotech market in Europe is growing fast, still not at the same pace as US market, but it must be taken into account that Europe entered nanotechnology some years behind US and Japan. Nevertheless, the increasing efforts made by European countries to reach a similar nanotechnology research status as US or Japan has made market evolve at a very high speed. European market is demanding nanotech products but with the warranty that they are safe and provide added value to industry and society.
- Economic impact and health market towards new methods of cancer diagnosis and therapy
The performance of NANOTHER platform could improve imaging thus enabling earlier detection, more accurate staging and post-therapy monitoring. Furthermore, NPs and HyNps selective and potent inhibition, which blocks tumor metastasis, will be a key therapeutic approach for colon and breast, thus having a tremendous impact on the European citizens' health care and quality of life.
Epidemiological data change for different types of tumors and for geographical area: in the next picture the incidence data for different tumors are listed for the three main geographical area of north America, Europe and Japan
The scenario for the antitumoral market is in rapid evolution due to the relevant innovation degree . The medium level of increasing from 2006 up to now has been about 18%but only in USA it has been about 50%.
For the next 5 years an increase of 7% is expected due to the medium increase of the desease. Notwithstanding, the lunch of different produts, the competition of generic drugs and the cytotoxic and hormone therapy will mantain a dominant position, expecially in the form of combined therapies.
Antitumoral global market in Billions USD (font IMS)
2006 2007 2008 2009 2010 2011 2012
Products in total 48,7 55,8 61,7 67,3 72,3 73,9 74,3
Products in development 0,6 1,7 2,9 4,5 6,1 7,9 9,8
Associated Community societal objectives
- Health and improving everyday life or quality of life of EU citizens
Nanotechnology advances have not progressed at the same pace as regulations had. All scientific outcomes form NANOTHER project have some impact in the future possibility of better cancer therapies. The possibility of encapsulating drugs at high levels opens a pathway where more research is needed but improved therapies may be feasible to obtain. One of the major problems of current therapies is the secondary effects. Those can only be corrected by three ways; improving adjuvants used for drug delivery and distribution decreasing their toxicity by chemical modification, using nanoparticles with stealth properties to hide the drug from (water based) body fluids and discovering new drugs which do not need any adjuvant to be administered. We have shown in this project that the second method is possible.
As mentioned above, regulation is a handicap in terms of commercializing nanomaterials based compounds. Furthermore, nanomedicine by itself requires detailed clinical phases not needed for other nanomaterials. As long as there is no progress in this area of regulation, placing nanomaterial based drugs or therapies will be quite difficult. In this project only one step further has been gone to accelerate the entrance of these nano-based drugs in the market. There is still a long way ahead but the in vitro assays here performed have opened the door for further development. As an example, some SMEs have been contacted to pulse the interest in continuing the research done in NANOTHER showing, some of them, clear interest in finding commercial exploitation for several nanocarriers for cancer therapy (CENIX Biosciences, Germany).
- Societal issues acceptance of nanomedicine applications
This is one of the most critical problems of nanotechnology based products. The effect on society that is causing nanomaterial applications either applied to medicine or other industrial uses is quite similar to the effect caused in the past by genetically modified foods. The low amount of information regarding toxicity of nanomaterials generates fear in society. This is something that cannot be avoided and only information, dissemination and clear explanation of what nanomaterials are and what they can do inside the human body is a must. Nowadays, some topics in Seventh Framework Programme (FP7) have been created for proposals to work in regulations and standards. Also some ideas are arising to organize expert committees or centers of competence to gather all information and make it accessible to the public and to the industrial companies.
The case of nanomedical products or devices can be better accepted by society as these products, in some cases, become an alternative to current therapies that are not working or even if they work, provide a low quality of life. Industry and regulatory agents are the opposite as the require further toxicity assays than they would for a normal chemical product due to the low information available. The main function of these regulatory bodies is to preserve the wellbeing of citizens and stop any harmful product to be released in the market.
The scientific improvements presented in this project are still far away from the market and society sees them as products to increase the hopes in science to better tackle diseases like cancer.
List of Websites:
http://www.nanother.eu/
The NANOTHER project was aimed to provide further development in nanotechnology applied to nanomedicine. In particular, the focus was to enhance and improve cancer therapies and diagnostics using these new tools that have arisen in the early twenty first century. The objectives of the project were i) to synthesize, based on previously existing methods, new nanoparticles (NPs) capable of interacting with living tissues, non-toxic and able to carry molecular drugs and other therapeutic molecules (siRNA), and ii) to make a proof of concept that efficacy of NPs with such properties would be better than the current therapies and could be potentially applicable to several types of cancer treatment.
Project Context and Objectives:
Over the last few decades, healthcare costs have been increasing due to a number of factors such as improved diagnostics, generalised screening, improved treatments and the general aging of the European population, which puts ever increasing pressures on healthcare budgets. There is significant pressure on the diagnostic and pharmaceutical industries to maintain innovation and product enhancement in the face of relatively restricted market growth. Nanomedicine and theranostics are emerging markets in which developing technologies and capabilities in nanotechnology, and diagnostic and therapeutic sectors are increasingly converging to improve efficiency and competitiveness of the discovery, development and marketing of diagnostics and therapeutics for various diseases such as cancer.
Concept
Two of the most important advances in nanotechnology rely on the development of novel technologies for new and effective medical treatments and diagnostic methods. Nanodiagnostics aims to identify disease at the earliest stages - at the cellular level, and perform drug delivery using nanoparticles (NPs) which will enable reduced toxicity of therapeutic drugs by delivering them to the disease site, and the consequent activation of the particles when they reach their target.
Overall NANOTHER objectives
NANOTHER intersects biomedical, health and nano industries, and R &D sits at the interface of chemical, biological and physical sciences and engineering. The main NANOTHER objective is therefore based on the integration of 5 key elements of current technology:
a) NP functionalisation technology,
b) fluorescence / contrast agent and specific antibody diagnostic techniques and imaging equipment,
c) novel drug-delivery and activation systems
d) new uses for electromagnetic based technology and medical equipment.
e) Another important innovation is RNAi technology, and the objective is to investigate the successful formulation and application of nanocarriers including siRNA as the therapeutic agent.
Characteristics and main research routes of the proposed project:
Functional nanocarrier = NANOTHER will focus on two of the most promising applications for cancer treatment:
i) polymer micelle core-shell NPs and
ii) magnetic NPs T
They were to be functionalised by linking several antibodies or ligands that bind distinctive targeted cancer antigens, and by linking therapeutic agents such as anticancer molecules and siRNA.
DIAGNOSIS = An important part of the NANOTHER project was the synthesis of NPs loaded with contrast agents or fluorescent labels to localise and detect the cancerous cells to be visualised by imaging systems such as MRI, SPECT, PET or fluorescence based imaging.
THERAPY = Hyperthermia was used to trigger drug release from hybrid magnetic NPs. For polymer micelle core-shell NPs, release mode is via biodegradable, bioerodible or pH sensitive systems. Pharmaceutical partners provided drugs already used, such as Doxorubicin, Taxol, Camptothecin (and derivatives). One new drug compound (from Pharmamar) in clinical trials Aplidin® NPs was also to be functionalised with siRNA to investigate nano delivery for the silencing approach. These investigations of different therapeutic agents on NPs for these specific applications contributed to the originality of this project.
THERANOSTICS = NANOTHER equally investigated a combined diagnostic and therapeutic approach. Initially, magnetic nanoparticles, properly functionalised and drug loaded, or furthermore, used only for their hyperthermic behaviour, were introduced in the body with an antibody specific for a chosen type of cancer. If a tumour is present, it will be automatically detected and the particles accumulate in the tumour area, at first by the passive enhanced permeability retention (EPR) process, followed by more specific binding through the appropriate antibodies to tumour cells. By applying an electromagnetic field, a hyperthermic effect is induced which will a) directly kill the tumour cells and b) trigger drug release of drug loaded NP simultaniously assisting with temperature increase, and thus with destroying tumour cells. This is the NANOTHER theranostic approach as there is a positive diagnostic (identification of tumour presence) and therapy by drug molecules, or hyperthermia, or both. An essential part of NANOTHER is the selection of drugs, biological determination of nanocarrier efficiency and determination of toxicology at different stages of the project
Project Results:
SP1: NP Functional specifications and selection (LCPO)
Sub-Project 1 was dedicated to the development of the new polymeric and hybrid (organic/inorganic) nanovectors. The first step in this work was the elaboration of polymeric and magnetic inorganic materials that constitute the scaffold of the proposed nanoparticule drug delivery systems. The idea was to synthesize advanced polymers with properties that will provide multifunctionality to the new drug carriers, such as thermo and/or pH responsiveness (for controlled release of chemotherapeutics), or ability to complex nucleic acid (for optimal siRNAs loading). Then, starting from these materials, a large panel of processes was investigated for the preparation of nano-objects with a focus on reproducibility and scalability. A third aspect that was studied in this SP is the drug loading, aiming at the higher loading contents and loading efficiencies to provide systems that can be realistically transferred to the industrial field. Finally, a last work package was devoted to preliminary studies of particles functionalization (for active cancer targeting) and an initial selection of the best systems based on objective criteria such as stability and toxicity.
WP1.1 Synthesis and characterisation of pH and/or temperature responsive polymers and block copolymers
A wide range of polymers was synthesized as potential material for nanoparticles preparation, mainly by INSTM and LCPO. INSTM explored many polymer families such as poly(amido amine)s, poly(ester)s, poly(ether-ester urethane)s, hemiesters of poly(maleic anhydride-co-n,butylvinyl ether)s, phosphonate polysaccharides and poly(hydroxypropylmethacrylamide-co-methacryloylglycylglycine-OH). Focusing on poly(carbonate)s, poly(amino acid)s and poly(saccharide)s blocks, LCPO performed the controlled synthesis of hydrophobic homopolymers and amphiphilic diblock copolymers. From this vast pool of biocompatible polymers, the selected materials were mainly amphiphilic copolymers with a diblock, multiblock or grafted structure.All the synthesized polymers have been extensively characterized, aiming to establish a relation between molecular properties and self-assembly behaviour.
These polymers were considered to be the most interesting for the preparation of novel nanotherapeutics.
Polymer name and chemical structure Developer Synthetic path Main properties
WP1.2 NPs formulation and characterisation
Methods explored for the preparation of polymer and hybrid nano-objects included supercritical CO2 processes (RESS/RESSAS, anti-solvent spraying), co-precipitation and nanoprecipitation (with or without the addition of stabiliser). Supercritical CO2 techniques developed by FEYECON produced particles in the range 200-400nm for some of the tested polymers but the cold powders obtained after spraying attracted moisture, hampering the harvesting step and causing particles agglomeration. The preferred method was nanoprecipitation, which allows the preparation of drug-loaded nanoparticles in one step with a good control over size and size dispersity with reasonable reproducibility and scalability (as demonstrated by ARGUS in SP4). Hybrid particles were also elaborated by this process, starting from the same polymer materials and magnetic NPs (mainly magnetite NPs provided by COLORITA).
WP1.3 Polymer and hybrid nanoparticles drug loading and characterisation
(drug, peptide, siRNA)
Anti-cancer drugs of various natures (small chemotherapeutic, peptide, siRNA) were successfully loaded in both polymer and hybrid nanoparticles. For each drug, the particles that showed the higher loading capability (usually one or two systems) were selected for further optimization (performed in SP4). Interestingly, some nanoparticles, like PTMC-PGA vesicles were able to load drugs of different natures.
Characterization of the new nanomedicines included classical physical-chemistry methods such as DLS (for hydrodynamic size determination), TEM, SEM, AFM (morphology and structure study) as well as chemical assays, spectroscopic and chromatographic methods for the quantification of loaded species. Specific extraction and quantification protocols have been set up for each drug and particle system.
In addition to magnetic NPs (that are used as negative MRI contrast agents), fluorescent dyes have been included in the proposed nanosystems, to serve as labels for either in vitro experiments (FITC, Rhodamine) or for in vivo biodistribution studies (near infrared dye).
WP1.4 Functionalization and selection of the 'best ' system
Functionalization strategies for ligand (folate, antibodies) grafting were developed in relation with polymer chemistry. Folate targeted PCL-PEG was prepared in bulk by INSTM and targeted particles were obtained directly from the functional polymer. For antibody grafting, amphiphilic polymers (PCL-PEG, PLGA-PEG, PTMC-PGA) were first modified using PEG heterobifunctional linkers. Antibodies were linked to the surface in aqueous medium after particles synthesis. Although requiring several steps, this strategy presents the advantage of being highly versatile since potentially any antibody, protein or peptide could be linked. Optimization of the most promising targeted systems has been performed in SP4.
For the selection of these 'best ' systems, objective criteria, listed hereafter, have been agreed upon by all partners and evaluated thanks to the work performed in different SPs: toxicity (SP2), stability in biological media (SP6), drug loading (SP1), scale up (SP4), magnetic properties (SP3, SP5), novelty.
Four nanoparticles selected for in vitro and in vivo testings: typical size, potential payloads and surface properties. All these particles are prepared by the nanoprecipitation method using either acetone or DMSO as organic solvent.
siRNA
Targeting Yes (folate, antibodies) Yes (antibodies) no Yes (antibodies)
Other functions Fluorescent labelling (FITC) Fluorescent labelling Fluorescent labelling (FITC, near infrared dye) Fluorescent labelling (FITC, Rhodamine, near infrared dye)
SP1 has provided a large supply of new nanosystems with promising features for cancer therapy and imaging (high loading capability, good stability, incorporation of imaging agents, targeting properties, stimuli responsiveness), that have been after a first pre-selection transferred to the other SPs for optimization and biological evaluation.
SP2 NP Toxicology and Biocompatibility (JRC)
PEG-PBLG unloaded NPs: CFE has shown that NPs have induced low cytotoxicity in all the cell lines examined, with the exception of MDCK where 100% toxicity (no colony formation) has been observed. No toxicity or very low toxicity has been measured by Alamar Blue in A549, HepG2, HaCaT, TK6 and Caco-2 exposed to PEG-PBLG unloaded NPs. No apoptosis/necrosis and no oxidative stress have been detected up to 72h exposure and in A549 cells PEG-PBLG unloaded NPs have induced significant secretion of the pro-inflammatory cytokines IL-6 and IL-8. In addition, MTT and Alamar Blue assays have not detected cytotoxicity induced by PEG-PBLG unloaded NPs in HSOC, as well as no apoptosis/necrosis has been measured.
PTMC-PGA unloaded NPs: CFE has not shown any cytotoxic effect exerted by PTMC-PGA unloaded NPs in any of the cell lines examined. PTMC-PGA unloaded NPs have not induced apoptosis/necrosis but, in contrast, they have significantly induced oxidative stress in TK6 and HepG2 cells and moderately in Caco-2 and A549 cells. Further studies have shown that the oxidative stress induced in TK6 and HepG2 was dose-dependent. In HSOC PTMC-PGA unloaded NPs has moderately induced cytotoxicity and cell death. In HaCaT, Caco-2, A549 and HepG2 PTMC-PGA unloaded NPs have induced inflammation as observed by detecting secretion of IL-6, IL-8 and TNF-alpha cytokines.
Magnetic PEG-PBLG NPs: CFE has shown high toxicity in all the cell lines investigated after 72h exposure to magnetic PEG-PBLG NPs. In contrast, Alamar Blue has not shown toxicity induced by these NPs in Balb/3T3 and MDCK cells.
Magnetic PTMC-PGA NPs: Alamar Blue has not shown toxicity induced by these NPs in Balb/3T3 and MDCK cells. In contrast, low cytotoxicity following 72h exposure to magnetic PTMC-PGA NPs has been observed by CFE assay, with the exception of HaCaT, where particles were highly toxic (100% toxicity).
NBRh13 NPs: CFE results have shown that NBRh13 NPs are highly cytotoxic for all the cell lines investigated; in particular they have induced 100% toxicity in Balb/3T3 and Caco-2 cells. By MTT and Alamar Blue has been shown that NBRh13 NPs have not induced toxicity, with the exception of Caco-2 where NPs resulted highly toxic already after 24h exposure. No cell death, detected as apoptosis and necrosis, has been observed, but NBRh13 NPs have induced significant oxidative stress in TK6, HaCaT, HepG2, Balb/3T3, Caco-2, A549 and MDCK cells. Since NBRh13 shows magnetic properties, it has been irradiated and the long-lived toxic intermediated have been investigated in vitro in Caco-2 and TK6 cells. Irradiated NPs have induced a reduction in the Caco-2 and TK6 viability. Reduction in Caco-2 survival has been measured also when short-lived toxic intermediates have been investigated NpsBlock_IS19b NPs: with the only exception of MDCK, where NpsBlock_IS19b NPs have induced high toxicity, low cytotoxicity has been observed in all the cell lines investigated by CFE following exposure to NpsBlock_IS19b NPs. Alamar Blue has shown that NpsBlock_IS19b NPs is toxic in Balb/3T3 cells, but not in Caco-2 and moderately toxic in TK6 cells. In addition, after 24 exposure NpsBlock_IS19b NPs were not toxic, did not induced cell death even after UVB irradiation and did not stimulated the secretion of pro-inflammatory cytokines in HSOC.
Block-M IS19b NPs: no cytotoxicity, investigated by MTT and Alamar Blue assays, has been observed in HaCaT cell line. Alamar blue performed on Caco-2 exposed to Block-M IS19b has shown that NPs are highly toxic and in TK6 cells.
MS60 NPs: CFE assay has shown that MS60 NPs are cytotoxic in all the cell lines investigated but not in A549 cells. Alamar Blue has shown that MS60 is not toxic in Caco-2 and low toxicity and oxidative stress has been detected in TK6 cells. No apoptosis or necrosis has been measured in all cell lines exposed to MS60 NPs, with the exception of HepG2 cells. In HSOC MS60 NPs resulted non toxic (MTT and Alamar Blue assays) and did not induce apoptosis. Following exposure to MS60 low risk of inflammation has been observed in HSOC. No oxidative stress has been observed in all the cell lines investigated following exposure to MS60, with the exception of TK6 exposed to MS60-batch6.
NBRh14 NPs: MTT and Alamar blue assay have shown that NBRh14 NPs do not induce cytotoxicity in Caco-2 cells but, in contrast, NBRh14 NPs impair the viability of TK6 cells.
VAM41-PEG NPs: CFE has shown that VAM41-PEG NPs are not cytotoxic in all the cell lines investigated. By Alamar Blue assay VAM41-PEG NPs resulted cytotoxic in Caco-2 and TK6 cells, but not in A549 and HepG2. In addition, no apoptosis and/or necrosis have been induced following exposure to VAM41-PEG NPs.
PBLG-b-Hyalunorate NPs: Alamar Blue assay has shown that PBLG-b-Hyalunorate NPs are not cytotoxic in A549, TK6, MDCK, Balb/3T3 and HepG2 cells. PBLG-b-Hyalunorate NPs, in addition, do not induce apoptosis and necrosis in A549, HaCaT, TK6 and Caco-2 cells.
PTMC-b-PGA-hybrid NPs: NPs have inhibited the viability of HT-29 and BT-474 cells already after 24h exposure and more severely after 72h, revealing thus a time- and a dose-dependent cytotoxic effect. Irradiating PTMC-b-PGA-hybrid NPs and analyzing the short-lived toxic intermediates in HT-29 cells has not shown any sigh of cytotoxicity, suggesting that these cells did not underwent to local heating during irradiation.
In vivo, the toxicological profiles of selected NPs have been investigated in CD1 mice via the Maximum Tolerated Dose (MTD) after single bolus administration and after multiple intravenous administrations (MTMD). Formulations have been selected taking into consideration the in vitro toxicity profiles and the future applications of the particles (investigated in other SP).
SP3 Targeting and Diagnostics (VICOMTEC)
Subproject 3 aimed at investigating and evaluating the selected nanosystems in terms of targeting and diagnosis. The first step in this work package was the production and attachment of antibodies and other targeting ligands to nanoparticles, and then the selection and the optimization of the best functionalization procedures. A task was thus dedicated to the nanoparticles characterization in terms of size, stability, etc. The second step in this work package was the in vivo preclinical evaluation of the functionalized NPs using MRI and SPECT/PET imaging. The functionalized NPs were evaluated from the point of view of their physical properties in the conditions of MR and SPECT/PET imaging. Then their targeting efficacy was tested first in vitro, and in vivo in mice, in both planar and tomographic modes.
WP3.1 Functionalization of NPs for targeting
This task has been confronted in its early stage to the large number of nanoparticles investigated and to the absence of a source of antibodies within the consortium, accentuated by the withdrawal of DKP.
In an initial phase, prior to the selection of the cellular targets and corresponding antibodies, various approaches have been developed for nanoparticle functionalization; they were optimized using model human antibodies and fusion proteins, with a minimal set of nanoparticles from LCPO, INSTM and Colorobbia, selected on the basis of toxicity evaluation.
WP3.2 Preclinical evaluation of the NPs in vivo using MR
The first thing to do in this work package was to establish the magnetic properties of the designed nanoparticles to verify which were suitable or not for MR imaging. To do so, measurement of longitudinal and transverse relaxivities (R1 and R2) of encapsulated nanoparticles provided by LCPO and of bare nanoparticles immersed in liquid DEG provided by COLORITA has been carried out. These systems were first loaded with magnetic core by their respective providers (Fe2O3 for LCPO NPs and CoFe2O4 or Fe3O4 for the COLORITA NPs). The experiment at high field were carried out by RMSB at 4.7T (200 MHz operating frequency) on a Bruker Biospec system, where transverse relaxivity measurement was made using a multiple spin-echo 2D imaging sequence and the longitudinal relaxivity measurement using an inversion-recovery 2D imaging sequence. The experiment at low filed were carried out by INSTM by means of two spectrometers, the using a saturation recovery sequence 90º -90º to measure the longitudinal relaxation time and the Carr-Purcell-Meiboom-Gill sequence for the measure of transverse relaxation time.
WP3.3 Preclinical evaluation of the NPs in vivo using SPECT/PET
In order to evaluate the selected NPs using SPECT/PET imaging systems, a first step was the design of a radiolabelling protocol that will be used through the project. TEIA initially received NPs produced by COLOROBBIA, and managed to successfully design a labelling protocol that preserves the nanoparticles stability. This protocol is a direct 99mTc labelling In which, typically 100 µl of the pertechnetate generator eluate is reduced with 40 μl of SnCl2 and the pH adjusted to 7-8 using NaHCO3 prior to the addition of 0.5ml nanoparticles solution and incubation at room temperature for 30min.
SP4 Pro-therapy (INSTM)
SP4 subproject aimed at the optimization of the drug loaded organic and hybrid nanoparticle formulations, being targeted or not, as well as at the NPs production scale-up. Objectives of SP4 included the preliminary in vitro and in vivo screening of the organic, hybrid and targeted, drug loaded nanoparticles; the satisfactory achievement of the objectives has guided the selection of the best formulation candidates employed in the biodistribution and efficacy tests performed in SP5 and SP6.
The main achievements of SP4 are summarised hereafter:
- 5 types of therapeutic agents, including three chemotherapeutics (Doxorubicin, Paclitaxel, Aplidin®), one biotherapeutic agent (siRNAs directed against the hypoxia inducible factor 1a) and two inorganic cores (magnetite NPs and maghemite Nanoparticles aimed at cancer treatment by hyperthermia and as MRI diagnosis tools, were loaded into the nanocarriers
- 3 different targeting ligands (1. anti-EGFR mAb for colon cancer model; 2. anti-Her2 mAb for breast cancer models; 3. Folate for breast cancer and general cancer targeting) were investigated and grafted to nanoparticles carrying the appropriate therapeutic agent for each cancer model.
- The production of polymeric materials, inorganic cores and their assembly into nanoparticles was performed under scale-up conditions.
- An in vitro and in vivo preliminary screening was performed in order to allow the selection of nanocarriers submitted to SP5 and SP6 studies. The characterizations performed pointed out the high potential of the developed nanovectors according to their chemical-physical, magnetic and relaxivity properties. The targeting ability using mAb was also demonstrated in vitro.
The activities performed within SP4 were divided into four Workpackages and are described, in the following paragraps, by each Partner role.
WP4.1 Optimisation of drug loaded polymeric nanoparticles and preliminary in vitro and in vivo screening.
Preliminary experiments were performed by loading retinoic acid (RA) as a hydrophobic model drug, later substituted by the loading of paclitaxel (PTX). Several polymeric matrices were screened and among those, the most successfully employed were the 2-methoxyethanol hemiester of poly(maleic anhydride-alt-butyl vinyl ether) 5% pegylated (VAM41-PEG), the poly(caprolactone)-block-poly(ethylene glycol) (PEG-b-P BLG) (IS19b) block copolymer, and novel polyammidoamines.
The applied processes were highly reproducible and the obtained nanoparticles had a monomodal diameter distribution with a mean diameter of 60-140 nm, depending on the material and the preparation method. The diameter distributions were assessed by dynamic light scattering technique (DLS) and confirmed by scanning electron microscopy (SEM), atomic force microscopy (AFM), and/or (TEM) Transmission electron microscopy. VAM41-PEG PTX-loaded nanoparticules formulation presented a 18% of loading and 77% of encapsulation efficiency. IS19b PTX-loaded nanoparticles formulation (Block-PTX) presented a 2.5 % of loading and an encapsulation efficiency of 86%. IS19b doxorubicin loaded NPs (Block-D) with average diameter of 140-160nm were obtained and the loading of the doxorubicin resulted in a 3.5% wt. Additionally, novel cyclodextrin /PAA cross-linked resins containing different amounts of ß- and cyclodextrin were prepared by copolymerization in aqueous media, at pH12, and converted into aqueous nano-suspensions by the High Pressure Homogenization technique (average sizes of about 320-370 nm).
WP4.2 Preliminary therapeutic evaluation of nanoparticles for hyperthermia and thermal assisted drug release
Efforts were devoted to refine the synthesis of two hybrid products coming from COLORITA's preexisting know-how (NBRh1 and NBRh13), which were produced with 10 and 5 liter-scale processes respectively. Products NCh14 and NCh15 have instead been produced with 400 mL-scale processes. In each case no significant stability changes occur by varying the synthesis scale.
According to the results came out from SP1, preliminary trials of drug loading on hybrid systems were carried out. Commercial paclitaxel (from Discovery Chemicals) was tested for encapsulation. Aiming to define the efficiency of loading, preliminary trials on the polymeric shell alone were investigated (NBRh13-blank; block-copolymer PLGA-b-PEG). The trials revealed that the polymer/paclitaxel weight ratio can be pushed up to 5/1 when the total amount of paclitaxel is maintained lower than 2.5 mg/mL. The maximum PTX loading capability found for hybrid NBRh13 was LCh = 1.78%, corresponding to polymer/PTX ratio = 4/1 and polymer/MAG ratio = 1/1. Doxorubicin loading was investigated as well, and according to the latest results the highest Doxo loading contents were assessed around 10% in hybrid systems containing 50% of magnetite as well Hybrid organic/magnetic nanoparticles were prepared by using the VAM41-PEG polymer and magnetite NPs provided by COLORITA. Human Serum Albumin and 0-glycidyl-2,3-0-isopropylidenglycerol-cyclodextrin were used as cofactors and coprecipitation method was applied. The prepared nanoparticles displayed a monomodal diameter distribution in suspension, with a mean diameter of 160nm. Paclitaxel loaded hybrid NPs (Block-MP) were also prepared under optimised protocols, without affecting the loading capability of the system and displaying preserved magnetic, relaxivity and hyperthermia properties.
Characterization of hybrid NPs.
The magnetic and relaxometric characterization of the provided nanoparticles was performed considering the main parameters commonly used to describe a magnetic system (i.e. blocking temperature, coercive field, remnant and saturation). The magnetic properties of the magnetite and/or maghemite core were well preserved after polymeric coating and functionalization (targeting moieties grafting and drug loading). In some cases, small differences in the coercivity and ZFC shape were observed, associated to a modification of the surface spin adjustment during the functionalization procedures. However, they were not so important and to produce a significant variation of the hyperthermic efficacy. The difference in the saturation magnetization has to be ascribed to the accuracy of evaluation of the magnetic material concentration, which was often very low in the case of the hybrid samples.
The optimisation of the experimental set-up using a coil suitable for future in vivo tests (f 10 cm) was accomplished. The new experimental conditions were adapted for applied field intensity and frequency of 22 kA/m and 186 kHz, respectively.
The efficacy of different synthesised hybrid magnetic nanoparticles (from SP1 and selected in SP4) in causing hyperthermia was evaluated in vitro by measuring temperature elevation induced by an AC electric fields. The kinetics of temperature increase during irradiation were collected for all the hybrid systems and the temperature increase as function of time was evaluated for different sample concentrations.
The hyperthermic efficiency of hybrid samples based on SA3300 magnetite inorganic core seems to be, by far, the most efficient (even at low concentration of inorganic phase) with respect to other inorganic cores. In particular, hybrid BlockM (and its derivatives, developed by INSTM-UniPI) and NBRh13 (COLORITA) show enhanced hyperthermic efficiencies. Samples of maghemite loaded HNP-PEG-b-PBLG-NT002 and HNP-PTMC-b-PGA-NT008 (provided by LCPO) instead, showed a very low (or even none) hyperthermic efficiency, even at high concentrations of inorganic phase in the range of frequencies 90-250 kHz. Nevertheless, the obtained results can be explained to the frequency adopted for the LCPO particles, which should be exposed to higher frequencies (around 800kHz).
The device herein and optimized hybrid not-loaded NPs (NBRh13) were delivered to TAU for the in vitro evaluation of hyperthermia efficacy (M22). At the end of the experimental session held in TAU (M34), the whole apparatus was sent to Leitat in order to accomplish SP5 scheduled activities.
Concerning Task 4.2.3 Preliminary evaluation in vivo COLORITA has asked to move this activity to SP5, in particular WP 5.5. The correspondent deliverable is D 5.5.1 (Report on evaluation of effect of drug unloaded and loaded targeted and not targeted hybrid NPs on selected in vivo tumoral cell models in vivo according to the last amendment proposed in September 2011). Even though SP4 and SP5 foresee different partners involved (PHMAR should be in charge of D 4.2.4 while LEITAT is responsible of D.5.5.1) PHMAR agreed not to perform the activity since it was no longer in its priority due to accumulated delays in the availability of the thermotherapy equipment. COLORITA accepted this decision because the in vivo tests in SP4 and SP5 meant on the same hybrid NPs compounds were redundant; LEITAT also accepted that task in replacement of DPK. This issue was recognised by the SPLs and the project coordinator who accepted the agreement between the partners and the merging of task 4.2.3 with task 5.5.1.
WP4.3 Therapeutic evaluation of organic and hybrid targeting nanoparticles
The activity related to WP 4.3 was mostly affected by the withdrawn of DPK and the consequent late availability of the antibodies needed for the development of targeted nanoparticles. Despite the engagement of Leitat on the targeting topic and its role in the production of EGFR antibodies, the targeting activity was carried on several other models and later (last 6 months) was performed also on the anti-EGFR antibodies provided by Leitat and anti-Her2 mAb (trastuzumab isolated from Herceptin®) by LCPO, Targeted nanoparticles with either antibodies or folic acid moieties were prepared.
WP4.4 Scale-up of the selected nanosystems
Argus focused on the synthesis of PTMC-b-PGA and PEG-b-PBLG copolymers from LCPO and ISB19b from INSTM, Pisa. The syntheses were optimized using the right conditions in order to achieve an effiecient scale-up. Although several problems occurred during the scale-up of PTMC-b-PGA copolymer, a final batch of 440g was finnaly synthesized and the material validated by NMR and GPC analyses, in agreement with LCPO 's specifications. Furthermore, one batch of 400g PEG-b-PBLG was also synthesized and validated. With respect to the copolymer ISB19b, originally coming from INSTM R.U. of Pisa, several tests were performed in order to convert the bulk conditions applied at INSTM into a process performed in solution. At the end, the product batch 115/15 (50 g), validated by NMR and GPC, was tested by INSTM and the corresponding nanoparticles were stable. Regarding scale-up of NPs formulations, ARGUS was able to scale-up the preparation of PTMC-b-PGA and PEG-b-PBLG organic nanoparticles on a 30 g scale.
WP5.1 Characterisation of magnetic, hyperthermic and relaxometric properties
The complete magnetic and dielectric characterisations of available and new magnetic products coming from SP1 and SP4 have been accomplished in a wide range of experimental condition.
Among all the samples investigated, two inorganic samples (cobalt ferrite d0 = 6,1nm and magnetite d0 = 12nm) have been selected as magnetic cores for hybrid ferromagnetic/organic NPs preparation, thanks to their promising magnetic, hyperthermic and relaxometric characteristics (both their hyperthermia and relaxometry were found enhanced with respect to commercial/literature-reported products). Thanks to the feedback provided by SP5 several hybrid samples were prepared afterwards in SP1 and optimized in SP4; they have all been characterized as well and in all cases the main properties of the naked core were found to be nearly unaffected by the embedding processes, thus confirming the goodness of these systems as theranostics devices.
In the last period a new batch of magnetite core, produced by COLORITA, has been characterized and compared with the old one. A good compatibility with the two cores was observed, so the second batch was therefore validated as magnetic core for hybrid NPs preparation. The relevant technical information is described in Deliverable D.5.1.1 and in D.5.1.2.
WP5.1 Characterisation of magnetic, hyperthermic and radio properties
Available not-loaded hybrid NPs were already characterised in task 5.1.1 and task 5.1.2. Promising compounds showing both good enhanced magnetic and hyperthermic properties were already selected. The technical details are collected and reported in the Deliverable Report D.5.1.1 and D.5.1.2. The magnetic characterization of hybrid samples NBRh1 and NBRh13 radio labelled by TEIA have been performed starting from M12. A batch of NBRh13_hEGFR NPs was received by TEIA (sent by COLORITA) and successfully radiolabeled. When radioactivity expired, this batch was send to INSTM-UniFi. Its magnetic characterization was accomplished and the magnetic behaviour of this assembly was compared to that displayed by the not-targeted counterpart. The agreement of cobalt ferrite-based sample (NBRh1) is quite perfect, while a small difference in magnetization values for sample NBRh13 containing magnetite was observed. This can be ascribed to an underestimate of the magnetic material concentration and, partially, to the different interparticle interaction or surface spin contribution due to the different dispersion medium or NPs aggregation state. In conclusion, the radio-labelling process does not affect the main magnetic properties of the samples, which can then be considered as promising candidates for theranostics involving PET /SPECT and MFH applications. Technical results are described in deliverable report D.5.2.1 and D.5.2.2.
WP5.3 Characterisation of radio labelled organic NPs
After receiving a lot of organic and hybrid NPs from the partners, TEIA radiolabeled them by applying the developed protocol, thus having a radiochemical yield near to 95%. Radiolabeled samples were injected in small mice and biodistribution studies in SPECT mode were carried out 1h and 24h post injection. In all cases, imaging studies confirmed biodistribution results. NPs were concentrated in liver, spleen and bladder. The concentration in tumor was not significant since no targeting agent was present. Therefore, Block-PTX-FA NPs were provided to TEIA (by INSTM) and successfully radiolabeled using the standard direct protocol; the yield was greater than95%. Stability tests were also carried out. This system, has been then tested in tumour bearing mice, but no accumulation was observed. It was subsequently confirmed that folic acid was not ideal targeting regarding HT-29 cancer cells. This system has hence been tested in mice bearing tumour of a different cell lines (U87MG - glioma brain cells) and an accumulation up to 6% was found.
WP5.4 Therapeutic effect of hybrid NPs and organic NPs in vitro
During this task, COLORITA has optimised an analytical method for paclitaxel detection by means of HPLC, for confirming paclitaxel presence inside organic/hybrid matrix and for optimizing its loading. A method for paclitaxel extraction from the organic matrix has been also developed, in order to evaluate total paclitaxel content (loaded and not loaded). Due to the low stability of the paclitaxel-loaded hybrid NBRh13 during the purification step, paclitaxel was discarted and efforts were focused on loading of doxorubicin. Nevertheless, due to the extreme sensitivity of doxorubicin to pH and to buffer properties, free and encapsulated doxorubicin undergo rapid degradation in phosphate buffer. For this reason COLORITA focused its effort with a new hybrid drug-loaded material (BlockM-PTX-FA from INSTM). This sample has been characterized in terms of drug delivery triggered by hyperthermia and due to its promising features it was selected for the final tasks of the project.
The aim of this task was to verify the therapeutic effect of selected radiolabeled NPs on small tumor bearing mice. The SPECT imaging protocol described in D.3.3.1 has been used in all studies. In agreement with the other partners, only the system 15_BlockM-PTX-FA was selected for this study. 15_BlockM-PTX-FA has been then successfully radiolabeled by TEIA using 99mTc. Imaging studies and biodistributions were carried out on HT-29 bearing tumor mice provided by LEITAT. SPECT images have shown high concentration in liver and relatively low NPs accumulation (~6%) in the tumor. For this reason and taking into account the limited number of HT-29 mice that were available in the project, direct tumor injection was also assessed. In this way, only tumor was depicted and no other organs were visible in SPECT images. Also transaxial slices of the tumor area were very clear (see D.5.5.3). Mice were imaged using PET before hyperthermia session and after this, since PET can image the viability of cancer cells. A cold area in the tumor region, which was not observed before hyperthermia and could suggest therapeutic effect has been observed.
By using the same set of mice provided by LEITAT (see Task 5.5.3) in vivo imaging studies have been carried out by TEIA. NBRh13-hEGFR was successfully radiolabeled using Tc99m and the direct labelling protocol. Imaging studies and biodistributions were carried out in mice provided by LEITAT. Unfortunatelly tumors were not shown both in images as well as in biodistribution studies. Nevertheless, assays carried out with other tumour lines (U87MG, glioma brain cells, which express EGFR) showed noticeable accumulation in the interested tissues up to 2h p.i although it was possible to test only one mouse, thus the protocol was not optimized. For this reason, only intravenous protocol was assessed due to the low number of animals for this pilot study. These preliminary encouraging results promote the evaluation of different tumoral models if necessary. According to the common agreements taken in the last meeting, no more studies have involved hybrid NBRh13_hEGFR and efforts have been focused on 15_Block-M-PTX-FA(115/15) only.
SP6 Biodistribution and Efficacy (GAIKER)
WP6.1 Interaction of NPs with blood components in vitro and intracellular fate
This task was performed using protocols validated by the Nanotechnology Characterisation Laboratory. It consisted on the incubation of the nanoparticles with blood serum, and the quantification and analysis of the proteins adsorbed to the nanoparticles recovered after several centrifugation steps. Our results suggest that the interaction of the tested nanoparticles (all of them without targeting moieties) with plasma proteins is not significant, normally below 0.1 %. In the cases where sufficient interaction was observed, the proteins involved consisted mainly on apolipoproteins, inmunoglobulins and lipid transport related proteins. Consistent with the nature of the polymers used, most of them bearing a lipidic-like nature. This task is relevant for later in vivo experimentation, as it may give hints on the NP behavior when in contact with physiological fluids. Experiments were performed using 10 individuals pooled fractions of plasma to avoid inter-individual bias.
The BT474 (human breast carcinoma) and HT-29 (human colon adenocarcinoma) cell lines were selected and used to investigate, by means of fluorescent microscopy, the internalization and the intracellular localization of fluorescently labeled nanoparticles.
After cell exposure to nanoparticles, and as means to identify the uptake and the intracellular fate of nanoparticles, cells were stained using specific antibodies. This staining allowed the identification of specific intracellular organelles that could be targeted by the internalized nanoparticles. In particular, the colocalization of nanoparticless with early endosomes, late endosomes and lysosomes was investigated. We could conclude that both Block_FITC_IS19b and FITC-PTMC-b-PGA based nanoparticles could be internalized by BT474 and HT-29 cells. However, although being internalized, no colocalization with the endosomal (early and late) compartment or with the lysosomes was observed. It is important to refer that these assays were performed using living cells. In addition, NPs were observed forming aggregates/agglomerates which have adhered on the cell membrane, and apparently were not able to be internalized, or have adhered to the glass slides where cells were cultivated. As a result, the amount of NPs taken up by BT474 and HT-29 cells was very low. The adsorption of the nanoparticles to cells, at 4°C, was minimal and similar to the control cells. While there was almost no uptake by the HT29 cells of the three hybrid NPs, at 4h post incubation, after 24h a significant increase was observed. HT-29 cells could internalize non-hybrid PTMC-PGA based nanoparticles to a higher extent, with significant uptake observed after 4h incubation. It can be assumed that the addition of the maghemite to the PTMC-PGA nanoparticles could cause a reduction/delay in the uptake into the cells.
The NPs were located inside the cell and at the cell periphery, showing a higher uptake at the longer time incubation (24h). These results are in agreement with the previous findings where NPs were attached to the cell membrane.
NPs showed colocalization with the endosomal compartments at both 4 and 24h: Rab5 (early endosome), Rab7 (24h not 4 h) (late endosome) and Catepsin D (lysosome); Rab11 (recycling endosome) compartment showed no colocalization. These experiments were performed with fixed cells and this may be the reason why these results are in contradiction with the assays performed in living cells.
We have also observed that slight modifications of the surface of the nanosystem can make it behave in very different manners. FITC and RhoB addition generated quite different results so this point must also be taken into account. All systems seemed to internalize, each of them with different kinetics.
WP6.2 Biodistribution
The goal of this study was to analyze the biodistribution of three different untargeted nanoparticles (PTMC-PGA, PEG-PBLG and PEG-Hya) developed by LCPO, in normal mice and to identity their fate once intravenously injected. The nanoparticles were tagged with a fluorescent dye allowing its location by an in vivo imaging system. To do so, two approaches were defined: the acute setting; administration and read out after 24h, and the chronic setting; administration every two days for one week and read out after 3 doses of nanoparticles. The conclusion obtained from those studies was that all nanoparticles were widely distributed in the animal body, but mainly accumulated in spleen and liver.
The biodistribution nanoparticles loaded with taxol (15_Block-P-FA; IS19b) was evaluated and compared with a Cremophor formulation (Paclitaxel injection, Mylan). Each formulation (1 mg/kg of taxol) were administered by single intravenous injections to male ICR. Concentrations of taxol were evaluated in plasma and in selected organs by LC/MS/MS method. The pharmacokinetic properties of the two taxol formulations were similar. Generally, taxol rapidly disappeared from the plasma within 6 hours post admnistration. Taxol loaded into IS19b-NPs presented lower maximum plasma concentration, but its levels decreased slower during the first hour post admnistration. Taxol showed similar affinity, in both tested formulations to, the following organs: liver greater than kidney greater than lung = spleen. The distribution into the tissues revealed decreasing taxol contents over time, up to 10-16 hours in all tissues. Taxol levels in these organs were higher than its simultaneous plasma concentration for both formulations. The results did not reveal substantial differences between free and IS19b-NP forms of taxol, relevant for target transport of this anti-cancer drug.
The biodistribution of PEG-DY700-PTMC-PGA nanoparticles and grafted with trastuzumab antibody HER-PEG-DY700-PTMC-PGA was determined by the evaluation of DY700 presence in the blood and in vital organs of healthy ICR mice. Nanoparticles remained in systemic circulation for a relative short time (up to 3 h), with a correspondent circulation half-life time of less than 1.5 h. The naked nanoparticles showed lower Cmax and AUCs, what suggests that trastuzumab grafted nanoparticles are more effectively distributed. Biodistribution data in mice revealed high initial organ uptake of both used nanoparticle formulations. Naked and trastuzumab grafted nanoparticles showed different tissue uptakes in various organs, they decreased in the following order: liver greater than kidney greater than blood ~ lung greater than spleen ~ heart and brain. In vivo organ exposure to trastuzumab grafted nanoparticles was higher and elimination slower than for the naked nanoparticles. The relatively high content in the cardiac area and in the brain, at early time points post-dosing, indicates higher blood circulation for trastuzumab-grafted nanoparticles. In summary, the results from this study indicate greater exposures (expressed as Cmax and AUCs) in the vital organs, longer mean residence times and elimination half-lives in the liver for trastuzumab grafted in comparison to naked nanoparticles.
The goal of the present study was to analyze the biodistribution of two nanoparticle formulations (PTMC-DY700 and PTMC-DY700-mAb EGFR), developed by LCPO, in tumor bearing mice. More specifically, if by means of the targeting moieties the targeted nanoparticles could reach the tumor differentially, as well as to assess their biodistribution in other organs. Both nanoparticles were tagged with a fluorescent dye allowing its location by an in vivo imaging system. To do so, two approaches were defined: the acute setting; administration and read out after 24h, and the chronic setting; administration every two days for one week and read out after 3 doses of nanoparticles. Significant increased tumor targeting (30% higher) was observed for the antibody grafted formulation for both settings.
The biodistribution profile of paclitaxel (taxol) loaded - folic acid targeted Block-MP-FA (IS19b) nanoparticles was evaluated in breast cancer bearing mice, presenting high expression of folate receptor (FR), as means to demonstrate taht targeted nanoparticles could transport taxol to solid tumors with high FR expression. The distribution of taxol in vital organs and breast cancer tissue was as follows: liver, kidney, spleen lung, tumor and poor in heart. Taxol was not detected in the brain. Pharmacokinetic analysis indicated that the terminal half-life in tumor was longer than in liver or in the rest of vital organs, corresponding to a longer mean residence time. Elimination half-life of taxol in tumors was 4.73 h and mean residence time of 7.43 h. In contrast to the rest of the vital organs, small amounts of taxol were still present in the liver and tumor at 16 h; with longer mean residence time (7 h) and elimination half-life (4 h) in the tumors. Content of taxol in the tumor tissues reached a small peak followed by its gradual elimination until 16 h; however, these results demonstrated a significant increase in tumor residence time and a subsequent 1.5 - fold prolongation of elimination half-life than in liver and up to 5 - times than in the rest of the vital organs.
The pharmacokinetic parameters in plasma and the distribution into several organs as well as into tumors were evaluated after single intravenous administration of different formulations of Aplidin to mice bearing MRI-H-121 (renal) and HT29 (colon) xenografted tumors. The nanoparticles formulation studied were:
- Aplidin loaded nanoparticles of PTMC-b-PGA
- Aplidin loaded nanoparticles of PEG-b-PBLG
- Aplidin loaded EGFR targeted PTMC-b-PGA nanoparticles
- Aplidin in CEW: Cremophor:Ethanol:Water formulation
Blood, tumor, kidney, liver, femur, breast, intestine, spleen and brain tissue samples were harvested from each animal at 0.5 1, 2, 4, 8 and 24 hours post-administration. Samples were analyzed by a supported-liquid extraction (SLE) followed by a gradient reversed phase chromatography and detection by positive ion electrospray tandem mass spectrometry (ESI/MS/MS) using multiple reaction monitoring (MRM).
Results showed similar pharmacokinetic behavior and tumor distribution of Aplidin regardless of the formulation administered or the tumor model. In other tissues, no major differences were seen with any formulation, except in the bone assayed: femur. Approximately 2-fold higher Cmax and AUC0-24 and largest t1/2 (3.3-fold) were observed after the administration of 0.25 mg/kg of Aplidin formulated in PTMC-b-PGA nanoparticles, than in PEG-b-PBLG nanoparticles. No differences were observed between targeted and not targeted nanoparticles.
WP6.3 Efficacy in vitro
Several nanoparticle formulations were studied concerning vitro efficacy. The test performed included mostly basic cytotoxicity and endpoint assays that were estimated to be the most representative or relevant. Different antibodies were used to test if targeting could be achieved. The tested drugs consisted on Aplidin, taxol and siRNA
Performed MTT assays indicate that drug loaded nanosystems are more toxic than those unloaded. Targeted nanosystems are more toxic than non-targeted, in HT29 cell line and similar in LS174T cell line. PTMC nanoparticles seemed to be the most versatile system, as it can be be loaded with both Aplidin and siRNA.
Aplidin assays indicate that although usual Aplidin formulation performs much better than encapsulated Aplidin, the toxic effect of the adjuvant needed to administer Aplidin must be also part of the equation mostly paying attention to the different values of toxicity. Studied typical toxicity endpoints showed little differences between antibody+ and antibody- formulations, indicating that maybe the toxic effect of these drugs is not mediated by apoptosis or ROS, but by other parameters. Also, variability of the batched is also something to be taken into account and quite difficult to solve, as it seems to be a problem of nanotechnology on itself.
siRNA efficacy was evaluated upon encapsulation into several types of nanoparticles. The siHIF encapsulated into the polymeric nanoparticles was able to block HIF expression in different types of cancer cells in vitro. Furthermore, nanoparticles grafted with anti-HER2mAb selectively bind breast cancer cells that overexpress the HER2 receptor, supporting the specific and efficient targeting of our approach in vitro. Similar results were obtained when nanoparticles grafted with anti-EGFRmAb were used in colon cancer cells, although specificity and efficacy were lower. Also, nanoparticles loaded with siHIF were shown to reduce tumour growth in vivo. In addition, the in vitro efficacy of doxorubicin loaded nanoparticles nanoparticles was confirmed. By loading a combination of doxorubicin and maghemite nanoparticles within the polymeric vesicles we have shown that upon exposure to a high frequency magnetic field a boost in drug release and subsequent increased toxic effect could be obtained.
The non-hybrid NP is not toxic to HT-29 cells after 24h incubation, however after 72h incubation the non-hybrid NPs exert a slight toxicity at higher concentrations.
Hybrid NPs seem to be more toxic than non-hybrid NPs. At higher concentrations a 50-60% viability decrease was observed, after 72 h incubation with HT-29 cells.
Three different PTMC hybrid NPs were tested in HT-29 cells that overexpress the EGFR, namely: naked, trastuzumab and anti-EGFR mAb grafted Nps. No significant differences in terms of toxicity were observed, contrarily to the initial expectations where anti-EGFR mAb grafted Nps shoeld exert higher tocity, due to increased cell specific internalization.
The results show that the irradiation of NBRh13 nanoparticles could affect HT-29 cells viability at both temperatures, 38 and 42 ºC, but to higher extent at 42 ºC. Similarly, the effect of the irradiation of NBRh13 hEGFR was significantly higher than incubating the cells at the same temperature, 42 or 38 ºC (30% compared to 25% ). However by targeting the NPs with hEFGR did not show any additive value for the efficacy of the hyperthermic effect. On the contrary, it reduced the toxicity from 70% (NBRh13) to 30% (NBRh13-EGFR) at 42 ºC, and from 30% to 25 % at 38 ºC, respectively.
WP6.4 Efficacy in vivo
The aim of this study was to determine and compare the efficacy of Paclitaxel loaded organic nanoparticle (IS19b) targeted to cancer cells using Folic Acid, as well as its toxic profile. Paclitaxel cytostatic was used reference compound. Both formulations were administered by intravenous (iv) route to a nude mice strain bearing a orthotopic mammary tumor. Animals administered with the nanoparticles without paclitaxel were employed as control group. Tumor efficacy studies demonstrated similar results for both formulations. It can be concluded that the coupling of Paclitaxel to the IS19b-M-FA did not improve the efficacy of Paclitaxel on the tested tumor model. On the contrary, the activity profile of nanoparticle-coupled paclitaxel regarding animal body weight reinforces the toxicity of this cytostatic compound.
Antitumoral efficacy evaluation of aplidin loaded PEG-PBLG nanoparticles, aplidin loaded PTMC-PGA nanoparticles and aplidin loaded EGFR targeted PTMC-PGA nanoparticles was evaluated and compared with aplidin convencional formulation (cremophor solution CEW). Nanoparticles were administered intravenously to athimic nude mice bearing subcutaneous tumors induced by human colon adenocarcinoma HT-29 cells.
The animals were injected by iv route five daily doses schedule for 2 consecutives cycles (days 0-4 and 9-13). The administered concentration was 0.25 mg/kg/day of aplidin. For targeting evaluation a more intensive treatment (ten daily doses schedule, day 0 to day 9) was decided taking into account the results obtained with no targeted nanoparticles.
Results demonstrated that Aplidin nanoparticles were well tolerated by tumor bearing animals. Aplidin, formulated in Cremophor or loaded in targeted or non-targeted nanoparticles formulations resulted in no antitumor effect in the HT-29 model with a minimal T/C of 67.6 78 and 67.1 % respectively.
WP6.5 Preparation of preclinical nanoparticle batches for therapeutic evaluation
These two tasks accounted for nanosystems preparation. Several batches were prepared and distributed to the different partners involved. The same batches were sent to several partners at the same time, as means to reduce in vivo variability. It was clear from previous in vivo assays that the batch-to-batch variability could affect the final results. All partners received the nanosystems on time and there was no delay in this task
Potential Impact:
- Reinforcing competitiveness and associated economic impact
Targeted diagnostics and therapy in solid cancer is defined as one of the most promising tools for cancer treatment. NANOTHER has proposed a leading research partially based on partners achievements and successful preliminary results aiming at a nanosystems platform capable of binding targeting moieties, enhanced contrast agents for imaging, therapeutic agents and intracellular drug delivery, for solid cancer with a proof of concept for breast and colon cancer. The realization of this project effectively contributes, to the transformation of the European medicine, pharmacology, medical imaging and nanotechnology related industries from a resource-intensive to a knowledge-intensive phase, which further contributes to the EU WW position in these fields. This project is part of the chain in this transformation, introducing high added value technologies and products in a sustainable manner. The outputs of the project has increased European scientific and technological qualities, and at the same time has created industrial and employment growth within Europe.
- Reinforcing competitiveness - World versus European nanotech market
The nanotechnology in Europe is gaining exponential interest in the past decade. The market in nanotech is expected to increase in about a 23 % by 2016. Companies and SMEs started some time ago to develop technologies where nanomaterials are an important part. Nanotech market in Europe is growing fast, still not at the same pace as US market, but it must be taken into account that Europe entered nanotechnology some years behind US and Japan. Nevertheless, the increasing efforts made by European countries to reach a similar nanotechnology research status as US or Japan has made market evolve at a very high speed. European market is demanding nanotech products but with the warranty that they are safe and provide added value to industry and society.
- Economic impact and health market towards new methods of cancer diagnosis and therapy
The performance of NANOTHER platform could improve imaging thus enabling earlier detection, more accurate staging and post-therapy monitoring. Furthermore, NPs and HyNps selective and potent inhibition, which blocks tumor metastasis, will be a key therapeutic approach for colon and breast, thus having a tremendous impact on the European citizens' health care and quality of life.
Epidemiological data change for different types of tumors and for geographical area: in the next picture the incidence data for different tumors are listed for the three main geographical area of north America, Europe and Japan
The scenario for the antitumoral market is in rapid evolution due to the relevant innovation degree . The medium level of increasing from 2006 up to now has been about 18%but only in USA it has been about 50%.
For the next 5 years an increase of 7% is expected due to the medium increase of the desease. Notwithstanding, the lunch of different produts, the competition of generic drugs and the cytotoxic and hormone therapy will mantain a dominant position, expecially in the form of combined therapies.
Antitumoral global market in Billions USD (font IMS)
2006 2007 2008 2009 2010 2011 2012
Products in total 48,7 55,8 61,7 67,3 72,3 73,9 74,3
Products in development 0,6 1,7 2,9 4,5 6,1 7,9 9,8
Associated Community societal objectives
- Health and improving everyday life or quality of life of EU citizens
Nanotechnology advances have not progressed at the same pace as regulations had. All scientific outcomes form NANOTHER project have some impact in the future possibility of better cancer therapies. The possibility of encapsulating drugs at high levels opens a pathway where more research is needed but improved therapies may be feasible to obtain. One of the major problems of current therapies is the secondary effects. Those can only be corrected by three ways; improving adjuvants used for drug delivery and distribution decreasing their toxicity by chemical modification, using nanoparticles with stealth properties to hide the drug from (water based) body fluids and discovering new drugs which do not need any adjuvant to be administered. We have shown in this project that the second method is possible.
As mentioned above, regulation is a handicap in terms of commercializing nanomaterials based compounds. Furthermore, nanomedicine by itself requires detailed clinical phases not needed for other nanomaterials. As long as there is no progress in this area of regulation, placing nanomaterial based drugs or therapies will be quite difficult. In this project only one step further has been gone to accelerate the entrance of these nano-based drugs in the market. There is still a long way ahead but the in vitro assays here performed have opened the door for further development. As an example, some SMEs have been contacted to pulse the interest in continuing the research done in NANOTHER showing, some of them, clear interest in finding commercial exploitation for several nanocarriers for cancer therapy (CENIX Biosciences, Germany).
- Societal issues acceptance of nanomedicine applications
This is one of the most critical problems of nanotechnology based products. The effect on society that is causing nanomaterial applications either applied to medicine or other industrial uses is quite similar to the effect caused in the past by genetically modified foods. The low amount of information regarding toxicity of nanomaterials generates fear in society. This is something that cannot be avoided and only information, dissemination and clear explanation of what nanomaterials are and what they can do inside the human body is a must. Nowadays, some topics in Seventh Framework Programme (FP7) have been created for proposals to work in regulations and standards. Also some ideas are arising to organize expert committees or centers of competence to gather all information and make it accessible to the public and to the industrial companies.
The case of nanomedical products or devices can be better accepted by society as these products, in some cases, become an alternative to current therapies that are not working or even if they work, provide a low quality of life. Industry and regulatory agents are the opposite as the require further toxicity assays than they would for a normal chemical product due to the low information available. The main function of these regulatory bodies is to preserve the wellbeing of citizens and stop any harmful product to be released in the market.
The scientific improvements presented in this project are still far away from the market and society sees them as products to increase the hopes in science to better tackle diseases like cancer.
List of Websites:
http://www.nanother.eu/