Although nanomedicines have long been unknown to the general public, they came to the forefront in 2020 with the development of messenger RNA vaccines to control Covid-19. Nanoparticles are at the heart of this vaccine technology: mRNA is encapsulated in lipid nanoparticles (tiny fat droplets), which protect it from destruction once in the body and can fuse with the cell membrane for safe and effective delivery. This then allows the cells to produce the antigens and their immune response.

Medicine with an eye on the future

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This is the whole principle of nanomedicine. It involves most of the time combining an active ingredient with a nanoscale vehicle, called a 'carrier'. The challenge is to transport the molecule directly to the infected area, to treat it without damaging the healthy cells around it. In other words, to increase the therapeutic effectiveness and reduce the toxicity of treatments. The nanometric size (10-9 m) is indeed the scale of many biological mechanisms in the human body. Nanoparticles and nanomaterials can cross natural barriers and access new drug delivery sites, interact with DNA or small proteins at different levels, whether in the blood or inside organs, tissues and cells. 

However, nanomedicine can no longer be seen as a simple drug delivery system, as the nanomaterials themselves can become the active therapeutic principle. The use of a new class of radiation-enhancing nanoparticles, for example, could be a revolutionary approach to the local treatment of solid tumours treated with radiotherapy. Finally, nanotechnology is also of interest in medical imaging. Some nanoparticles with a luminescent core and specific recognition agents can, for example, detect and 'illuminate' cancerous tumours.

Three generations of nanovectors have been developed ⁽¹⁾ :

• First generation: Identified by the body as foreign bodies, they are captured by the liver, and thus serve to heal it.
• Second generation: These are nanoparticles coated with hydrophilic polymers. As they are less recognised as foreign bodies, they circulate in the bloodstream longer and can be used to treat cancerous tumours.
• Third generation: These are nanoparticles with a more complex structure, which allow the simultaneous delivery of several active ingredients (Vyxeos) or which exploit their structure to modulate the delivery of the active ingredient to the site of action (e.g. messenger RNA vaccines).

A meteoric rise

Until ten years ago, nanomedicine was considered mainly in research laboratories. As explained by the ETPN, it has now already made a concrete difference at the clinical level in the treatment of cancer and other diseases and has reached a new level of maturity ⁽¹⁾. Various formulations are already approved by regulators for cancer treatment, iron replacement therapies, anaesthetics, fungal treatments, macular degeneration and rare genetic diseases. In the last three years alone, the European Medicines Agency (EMA) has approved three new solutions, accelerating the ongoing revolution:

• liposomes of daunorubicin and cytarabine to fight acute myeloid leukaemia;
• lipid-based nanoparticles encapsulating Patisiran (a small double-stranded interfering ribonucleic acid) to treat hereditary amyloidosis;
• Hafnium oxide-based nano-objects that, by providing a heat source to cells, make them more sensitive to the effects of radiotherapy and chemotherapy - a definite plus in the quest for the right dose;
• two vaccines against COVID-19, SPIKEVAX - Moderna ⁽²⁾ and Pfizer-BioNTech COMIRNATY ⁽³⁾

Meanwhile, there are now more than 400 clinical trials around the world focusing on therapy and diagnostics, a sign of the sector's dynamism.

Need for method(s)

However, the LEEM (French Pharmaceutical Companies Association) stresses certain obstacles that need to be overcome, in particular through the creation of a network with qualified methods to answer the quality and safety issues raised by the use of nanotechnologies in medicines.

For their part, researchers feel the need for reliable methods to characterise the physicochemical properties (physical quality attributes) that influence the safety and efficacy of nanotechnology-based medical products. As one of the pioneers of nanomedicine, Patrick Couvreur (Institut Galien Paris-Saclay), explained: ⁽⁴⁾ : « We recently discovered that the shape of nanovectors modifies the distribution of the drug in the body. ».

As for the European Medicines Agency (EMA), it expresses its reservations in the strategy document EMA Regulatory Science to 2025 ⁽⁵⁾ : « The pace of innovation in the field of nanomedicine has accelerated dramatically in recent years and regulators must be prepared to support the development of increasingly complex medicines. [There is a need to] develop and standardise new test methods to assess the quality and safety of nanomedicines. »

To be placed on the market, a nanodrug must meet the same criteria for safety, efficacy and pharmaceutical quality as any other drug. But because of its structural complexity, its evaluation poses substantial analytical challenges, compared to molecular or biological drugs. The EMA and the Food and Drug Administration (FDA/USA) have defined a list of quality attributes considered relevant ⁽⁶⁾: particle size, particle size distribution and polydispersity, surface charge and properties, drug charge and release profile, and complex chemical and physical core-shell structure. However, validated and standardised characterisation methods are needed to measure these accurately and in a comparable way. Only comparable data of high metrological quality can facilitate the regulatory process and the clinical translation to practitioners and patients. praticiens et patients.

To identify which characterisation methods should be developed and standardised, the REFINE European project, funded under the Horizon 2020 programme, compared the information required for nanotechnology-based health products with existing methods and regulatory needs. This work, the results of which were published in 2021 ⁽⁶⁾, will help guide standardisation efforts in response to methodological gaps identified in five main areas:

• surface properties, 
• drug loading and release, 
• kinetic properties in complex biological media,
• ADME parameters (absorption, distribution, metabolism and excretion),
• interaction with blood and the immune system.

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Research is moving forward

Various R&D projects have already led to recommendations for the characterisation of extracellular vesicles (EVs). These are promising for regenerative medicine, because of their ability to cross biological barriers (particularly blood-brain barriers) and to deliver molecules to certain cells to modify their activity. They also have the advantage of low immunogenicity (which avoids an immune response that neutralises the therapeutic activity) and high engineering potential (addition of targeting agents, etc.).

At the end of 2021, the Extracellular Vesicle translatiOn to clinicaL perspectiVEs group - EVOLVE France, thus published a summary document ⁽⁷⁾ on the development of EV-based nanomedicines, with recommendations for manufacturing, quality control, analysis, non-clinical development and clinical trials, in accordance with current European legislation. In particular, it makes recommendations on :

• how to distinguish aggregates from single EVs
• characterisation of impurities, 
• definition of specifications for active substances and products, 
• study of the stability of EV-based products, 
• process control, 
• in vitro, ex vivo and in vivo toxicology and biodistribution studies.  

For their part, the European METVES (2012 - 2015) and METVES II (2019 - 2022) projects, financed under the EMPIR programme organised by EURAMET, have provided initial metrological responses. METVES II, to which LNE is contributing, focuses more specifically on the pre-standardisation of EV concentration measurements, by developing reference materials containing stable particles whose concentrations, fluorescence, refractive indices and sizes are close to those of EVs.

Support and cooperation

In parallel with these R&D initiatives, characterisation infrastructures make it possible to create bridges between regulators, metrologists and standardisation bodies. The result is harmonised and validated test methods to support industry in meeting regulatory requirements and innovation challenges. 

In the United States, the National Cancer Institute's Nanotechnology Characterization Laboratory (NCI-NCL) was the first infrastructure of its kind in 2004, set up in collaboration with the FDA and the NIST (US National Metrology Institute). Europe followed in 2015, with the creation of the European Nanomedicine Characterisation Laboratory (EUNCL), which however did not survive the end of the project that set it up. In addition to providing expertise in nanodrug characterisation, these structures develop numerous Standardised Operational Procedures (SOPs) available to stakeholders.

More recently, in 2021, in order to support dialogue between scientists from industry, regulatory and research backgrounds, the NPL (UK National Metrology Institute) organised a workshop entitled "Advancing Measurement Technologies and Standards for Nanomedicine" ⁽⁹⁾, in collaboration with NIST, JRC (EU) and AstraZeneca, on recent advances and practical challenges on these characterisation issues. This enabled the identification and prioritisation of specific methods and standards needed to move forward in light of new emerging priorities. Lipid-based delivery systems were identified as a priority for standardisation and development of reference materials. These systems play a key role in Covid-19 vaccines and cancer treatment. cancer.

First normative steps

Today, standardisation is already progressing, even if at the European level, the European Committee for Standardization (CEN) has not yet launched anything on nanomedicine. It has however expressed the will to initiate activities through the TC 352 Nanotechnologies, while the European Pharmacopoeia ⁽¹⁰⁾ also considers the subject to be central. This is not the case at the international level where multiple activities are already underway. 

For example, ASTM/E56 Nanotechnologies is working to produce several documents on the analysis of Liposomal Drug Formulations using Multidetector Asymmetrical-Flow Field-Flow Fractionation (WK68060) or the characterisation of Encapsulation, Extraction, and Analysis of RNA in Lipid Nanoparticle Formulations (WK75607). Or it has developed three standards - recently approved - on the quantification of lipids by liquid chromatography coupled to mass spectrometry, evaporative light-scattering or charged aerosol detector.

For its parts, ISO/TC 229 Nanotechnologies is developing a standard on liposome terminology (ISO/AWI TS 4958), and another on RNA-containing nanoemulsions.

Finally, within the VAMAS (Versailles Programme on Advanced Materials and Standards), an international initiative active in the field of pre-standardisation for advanced materials in which LNE represents France on the Steering Committee, the TWA40 Working Group is interested in the physico-chemical profiling of viral-like particles as reference materials for vaccine development and viral particle diagnostics. 

As a participant in the work carried out within ISO/TC 229 and ASTM/E56, as well as in discussions at European Pharmacopoeia level ⁽¹⁰⁾, LNE contributes to standardisation efforts and guidance documents on liposomes and lipid-based formulations. To be continued.

References:

[1] https://www.sciencedirect.com/science/article/pii/S0168365920303825

[2] https://www.ema.europa.eu/en/medicines/human/EPAR/spikevax

[3] https://www.ema.europa.eu/en/medicines/human/EPAR/comirnaty

[4] https://www.universite-paris-saclay.fr/actualites/patrick-couvreur-un-pionnier-du-nanomedicament

[5] https://www.ema.europa.eu/en/documents/regulatory-procedural-guideline/ema-regulatory-science-2025-strategic-reflection_en.pdf

[6] https://www.sciencedirect.com/science/article/pii/S0168365921003035?via%3Dihub

[7] https://www.sciencedirect.com/science/article/pii/S0169409X2100394X?via%3Dihub

[8] https://metpart.eu/health-call-2022

[9] https://www.npl.co.uk/surface-technology/nano-med-webinar

[10] https://www.edqm.eu/en/quality-requirements-for-nanomedicines