Toward an international standardisation roadmap for nanomedicine.

The French National Metrology Institute (LNE) initiated a series of events to identify priorities for test methods and their harmonisation that directly address regulatory needs in Nanomedicine. One of these workshops entitled "The International Standardisation Roadmap for Nanomedicine" held in October 2023 (Paris, France) brought together key experts in the characterisation of nanomedicines and medical products containing nanomaterials, including the Joint Research Centre of the European Commission, SINTEF Industry and the metrology institutes of France, the UK, the USA and Canada, two flagship initiatives of the European Commission (PHOENIX and SAFE-n-MEDTECH Open Innovation Test Beds), representatives of a working party on mRNA vaccines at the European Directorate for the Quality of Medicines (EDQM) and members of international standardisation and pre-normative organisations (including CEN, ISO, ASTM, VAMAS). Two take-home message came out from the discussion. First, developing standard test methods and Reference Materials (RMs) for nanomedicines is a key priority for the European Commission and various stakeholders. Furthermore, there was a unanimous recognition of the need for a unified approach between standardisation committees, regulators and the nanomedicine community. At the USA, Canadian and European level, examples of success stories and of future initiative have been discussed. Future perspectives include the creation of a dedicated Working Group under CEN/TC 352 to consolidate efforts and develop a nanomedicine standardisation roadmap.

Understanding the Impact of Polyunsaturated Fatty Acids on Age-Related Macular Degeneration: A Review.

Age-related Macular Degeneration (AMD) is a multifactorial ocular pathology that destroys the photoreceptors of the macula. Two forms are distinguished, dry and wet AMD, with different pathophysiological mechanisms. Although treatments were shown to be effective in wet AMD, they remain a heavy burden for patients and caregivers, resulting in a lack of patient compliance. For dry AMD, no real effective treatment is available in Europe. It is, therefore, essential to look for new approaches. Recently, the use of long-chain and very long-chain polyunsaturated fatty acids was identified as an interesting new therapeutic alternative. Indeed, the levels of these fatty acids, core components of photoreceptors, are significantly decreased in AMD patients. To better understand this pathology and to evaluate the efficacy of various molecules, in vitro and in vivo models reproducing the mechanisms of both types of AMD were developed. This article reviews the anatomy and the physiological aging of the retina and summarizes the clinical aspects, pathophysiological mechanisms of AMD and potential treatment strategies. In vitro and in vivo models of AMD are also presented. Finally, this manuscript focuses on the application of omega-3 fatty acids for the prevention and treatment of both types of AMD.

Unveiling the challenges of engineered protein corona from the proteins' perspective.

It is well known that protein corona affects the "biological identity" of nanoparticles (NPs), which has been seen as both a challenge and an opportunity. Approaches have moved from avoiding protein adsorption to trying to direct it, taking advantage of the formation of a protein corona to favorably modify the pharmacokinetic parameters of NPs. Although promising, the results obtained with engineered NPs still need to be completely understood. While much effort has been put into understanding how the surface of nanomaterials affects protein absorption, less is known about how proteins can affect corona formation due to their specific physicochemical properties. This review addresses this knowledge gap, examining key protein factors influencing corona formation, highlighting current challenges in studying protein-protein interactions, and discussing future perspectives in the field.

Highly Potent Cationic Chitosan Derivatives Coupled to Antimicrobial Peptide Dendrimers to Combat Pseudomonas aeruginosa.

The burden of bacterial wound infections has considerably increased due to antibiotic resistance to most of the currently available antimicrobial drugs. Herein, for the first time, a chemical coupling of two cationic N-aryl (pyridyl and aminocinnamyl) chitosan derivatives to antimicrobial peptide dendrimers (AMPDs) of different generations (first, second, and third) via thioether-haloacetyl reaction is reported. The new chitosan-AMPD conjugates show high selectivity by killing Pseudomonas aeruginosa and very low toxicity toward mammalian cells, as well as extremely low hemolysis to red blood cells. Electron microscopy reveals that the new chitosan derivatives coupled to AMPD destroy both the inner and outer membranes of Gram-negative P. aeruginosa. Moreover, chitosan-AMPD conjugates show synergetic effects within extremely low concentrations. The new chitosan-AMPD conjugates can be used as potent antimicrobial therapeutic agents, to eradicate pathogens such as those present in acute and chronic infected wounds.

Hypoxic glioblastoma-cell-derived extracellular vesicles impair cGAS-STING activity in macrophages.

BACKGROUND: Solid tumors such as glioblastoma (GBM) exhibit hypoxic zones that are associated with poor prognosis and immunosuppression through multiple cell intrinsic mechanisms. However, release of extracellular vesicles (EVs) has the potential to transmit molecular cargos between cells. If hypoxic cancer cells use EVs to suppress functions of macrophages under adequate oxygenation, this could be an important underlying mechanism contributing to the immunosuppressive and immunologically cold tumor microenvironment of tumors such as GBM. METHODS: EVs were isolated by differential ultracentrifugation from GBM cell culture supernatant. EVs were thoroughly characterized by transmission and cryo-electron microscopy, nanoparticle tracking analysis (NTA), and EV marker expression by Western blot and fluorescent NTA. EV uptake by macrophage cells was observed using confocal microscopy. The transfer of miR-25/93 as an EV cargo to macrophages was confirmed by miRNA real-time qPCR. The impact of miR-25/93 on the polarization of recipient macrophages was shown by transcriptional analysis, cytokine secretion and functional assays using co-cultured T cells. RESULTS: We show that indirect effects of hypoxia can have immunosuppressive consequences through an EV and microRNA dependent mechanism active in both murine and human tumor and immune cells. Hypoxia enhanced EV release from GBM cells and upregulated expression of miR-25/93 both in cells and in EV cargos. Hypoxic GBM-derived EVs were taken up by macrophages and the miR-25/93 cargo was transferred, leading to impaired cGAS-STING pathway activation revealed by reduced type I IFN expression and secretion by macrophages. The EV-treated macrophages downregulated expression of M1 polarization-associated genes Cxcl9, Cxcl10 and Il12b, and had reduced capacity to attract activated T cells and to reactivate them to release IFN-γ, key components of an efficacious anti-tumor immune response. CONCLUSIONS: Our findings suggest a mechanism by which immunosuppressive consequences of hypoxia mediated via miRNA-25/93 can be exported from hypoxic GBM cells to normoxic macrophages via EVs, thereby contributing to more widespread T-cell mediated immunosuppression in the tumor microenvironment.

Understanding protein-nanoparticle interactions leading to protein corona formation: In vitro - in vivo correlation study.

Nanoparticles (NPs) in contact with biological fluids form a biomolecular corona through interactions with proteins, lipids, and sugars, acquiring new physicochemical properties. This work explores the interaction between selected proteins (hemoglobin and fetuin-A) that may alter NP circulation time and NPs of different surface charges (neutral, positive, and negative). The interaction with key proteins albumin and transferrin, the two of the most abundant proteins in plasma was also studied. Binding affinity was investigated using quartz crystal microbalance and fluorescence quenching, while circular dichroism assessed potential conformational changes. The data obtained from in vitro experiments were compared to in vivo protein corona data. The results indicate that electrostatic interactions primarily drive protein-NP interactions, and higher binding affinity does not necessarily translate into more significant structural changes. In vitro and single protein-NP studies provide valuable insights that can be correlated with in vivo observations, opening exciting possibilities for future protein corona studies.

Clickable polyethyleneimine incorporated into triblock copolymeric micelles as an efficient platform in the delivery of siRNA to NSCLC cells.

The efficacy of transfection vectors to cross the endosomal membrane into the cytosol is a central aspect in the development of nucleic acid-based therapeutics. The challenge remains the same: Delivery, Delivery, Delivery. Despite a rational and appropriate construct of triblock polymeric micelles, which could serve as an ideal platform for the co-delivery of siRNAs and hydrophobic anticancer drugs, we show here its inability to properly convey oligonucleotides to their final destination. In order to overcome biological barriers, a linear PEI comprising two orthogonal groups was synthesized, holding an appropriate balance between safety and efficacy. Micellar carriers were then formulated with this polymer to enhance endosomal siRNA release. This chemical technology also addresses the two major challenges to consider when developing novel micellar products for siRNA delivery, namely cytotoxicity of polycations and endosomal escape. Herein, we demonstrate successful release of siRNA using a polymer tailoring strategy combined with a relevant in vitro approach, considering STAT3 as a promising target in the treatment of non-small cell lung cancer (NSCLC).

Explosomes: A new modality for DEB-TACE local delivery of sorafenib: In vivo proof of sustained release.

The current medical practice in treating Hepatocellular carcinoma (HCC) using Drug Eluting Transarterial chemoembolization (DEB-TACE) technique is limited only to hydrophilic ionizable drugs, that can be attached ionically to the oppositely charged beads. This limitation has forced physicians to subscribe the more hydrophobic, first treatment option drugs, like sorafenib systemically via the oral route, thus flooding the patient system with a very powerful, non-specific, multiple-receptor tyrosine kinase inhibitor that is associated with notorious side effects. In this paper, a new modality is introduced, where highly charged, drug loaded liposomes are added to oppositely charged DEBs in a manner causing them to "explode" and the drug is eventually attached to the beads in the lipid patches covering their surfaces; therefore we call them "Explosomes". After fully describing the preparation process and in vitro characterization, this manuscript delves into an in vivo pharmacokinetic study over 50 New Zealand rabbits, where explosomal loading is challenged vs oral as well as current practice of emulsifying sorafenib in lipiodol. Over 14 days of follow up, and compared to other groups, explosomal loading of SRF on embolic beads proved to cause a slower release pattern with longer Tmax, lower Cmax and less washout to general circulation in healthy animals. This treatment modality opens a new untapped door for local sustained delivery of hydrophobic drugs in catheterized organs.

Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes.

Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally heterogeneous nanoparticle complexes, which exhibit varying sensitivity to the conditions required for the methodologies available to physicochemically characterize these agents. Currently, the critical quality attributes of iron-carbohydrate complexes have not been fully established. Dynamic light scattering (DLS) has emerged as a fundamental method to determine intact particle size and distribution. However, challenges still remain regarding the standardization of methodologies across laboratories, specific modifications required for individual iron-carbohydrate products, and how the size distribution can be best described. Importantly, the diluent and serial dilutions used must be standardized. The wide variance in approaches for sample preparation and data reporting limit the use of DLS for the comparison of iron-carbohydrate agents. Herein, we detail a robust and easily reproducible protocol to measure the size and size distribution of the iron-carbohydrate complex, iron sucrose, using the Z-average and polydispersity index.

Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles.

The therapeutic efficacy and adverse impacts of nanoparticles (NPs) are strongly dependent on their systemic circulation time. The corona proteins adsorbed on the NPs determine their plasma half-lives, and hence, it is crucial to identify the proteins shortening or extending their circulation time. In this work, the in vivo circulation time and corona composition of superparamagnetic iron oxide nanoparticles (SPIONs) with different surface charges/chemistries were analyzed over time. SPIONs with neutral and positive charges showed the longest and shortest circulation times, respectively. The most striking observation was that corona-coated NPs with similar opsonin/dysopsonin content showed different circulation times, implying these biomolecules are not the only contributing factors. Long-circulating NPs adsorb higher concentrations of osteopontin, lipoprotein lipase, coagulation factor VII, matrix Gla protein, secreted phosphoprotein 24, alpha-2-HS-glycoprotein, and apolipoprotein C-I, while short-circulating NPs adsorb higher amounts of hemoglobin. Therefore, these proteins may be considered to be determining factors governing the NP systemic circulation time.

HDACIs and TKIs combinations and their liposomal delivery for cancer treatment.

Fixed-drug combinations have been used for the treatment of cancer. Current anticancer therapies, however, tend to induce resistance and provoke important toxicity. Therefore, there is still a need for further optimized treatments that would also take into account drug-drug interactions. Tyrosine kinase inhibitors (TKIs) and Histone deacetylase inhibitors (HDACIs) are two anticancer drug classes currently used separately in clinical practice. Those drug classes are currently being investigated in clinical trials. Several findings confirm the safety and tolerability of these treatments accompanied by clinical improvement. The activity and specificity of drug combinations can be further improved by employing appropriate drug delivery systems, such as nanocarrier systems. The different pharmacokinetic profiles of each drug may lead to a loss in synergistic effects, affecting treatment efficiency. Several combinations of either TKI or HDACI with an anticancer drug of another class co-loaded inside liposomal carriers are being investigated in vivo and show promising results in terms of efficacy and safety. In this review we discuss strategies for the delivery of synergistic drug combinations for cancer treatment.

Meeting vaccine formulation challenges in an emergency setting: Towards the development of accessible vaccines.

Vaccination is considered one of the most successful strategies to prevent infectious diseases. In the event of a pandemic or epidemic, the rapid development and distribution of the vaccine to the population is essential to reduce mortality, morbidity and transmission. As seen during the COVID-19 pandemic, the production and distribution of vaccines has been challenging, in particular for resource-constrained settings, essentially slowing down the process of achieving global coverage. Pricing, storage, transportation and delivery requirements of several vaccines developed in high-income countries resulted in limited access for low-and-middle income countries (LMICs). The capacity to manufacture vaccines locally would greatly improve global vaccine access. In particular, for the development of classical subunit vaccines, the access to vaccine adjuvants is a pre-requisite for more equitable access to vaccines. Vaccine adjuvants are agents required to augment or potentiate, and possibly target the specific immune response to such type of vaccine antigens. Openly accessible or locally produced vaccine adjuvants may allow for faster immunization of the global population. For local research and development of adjuvanted vaccines to expand, knowledge on vaccine formulation is of paramount importance. In this review, we aim to discuss the optimal characteristics of a vaccine developed in an emergency setting by focusing on the importance of vaccine formulation, appropriate use of adjuvants and how this may help overcome barriers for vaccine development and production in LMICs, achieve improved vaccine regimens, delivery and storage requirements.

Structure-activity relationship of a peptide permeation enhancer.

The pentapeptide L-R5 has previously been shown to transiently increase the permeability of nasal epithelial cell layers in vitro, allowing paracellular transport of molecules of up to 4 kDa. Protein kinase C zeta (PKC ζ), a member of a family of serine/threonine kinases was shown to be involved in tight junction modulation induced by L-R5. We show here that the ability of L-R5 to modulate tight junctions is comparable to other permeability enhancers such as bilobalide, latrunculin A or C10. Interaction of the peptide with the target protein occurs via electrostatic interaction, with the presence of positive charges being essential for its functionality. L-R5 is myristoylated to allow quick cell entry and onset of activity. While no epithelial cytotoxicity was detected, the hydrophobic myristoyl rest was shown to cause haemolysis. Taken together, these data show that a structural optimization of L-R5 may be possible, both from a toxicological and an efficacy point of view.

Characterization Challenges of Self-Assembled Polymer-SPIONs Nanoparticles: Benefits of Orthogonal Methods.

Size and zeta potential are critical physicochemical properties of nanoparticles (NPs), influencing their biological activity and safety profile. These are essential for further industrial upscale and clinical success. However, the characterization of polydisperse, non-spherical NPs is a challenge for traditional characterization techniques (ex., dynamic light scattering (DLS)). In this paper, superparamagnetic iron oxide nanoparticles (SPIONs) were coated with polyvinyl alcohol (PVAL) exhibiting different terminal groups at their surface, either hydroxyl (OH), carboxyl (COOH) or amino (NH2) end groups. Size, zeta potential and concentration were characterized by orthogonal methods, namely, batch DLS, nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), asymmetric flow field flow fractionation (AF4) coupled to multi-angle light scattering (MALS), UV-Visible and online DLS. Finally, coated SPIONs were incubated with albumin, and size changes were monitored by AF4-MALS-UV-DLS. NTA showed the biggest mean sizes, even though DLS PVAL-COOH SPION graphs presented aggregates in the micrometer range. TRPS detected more NPs in suspension than NTA. Finally, AF4-MALS-UV-DLS could successfully resolve the different sizes of the coated SPION suspensions. The results highlight the importance of combining techniques with different principles for NPs characterization. The advantages and limitations of each method are discussed here.

Impact of peptide permeation enhancer on tight junctions opening cellular mechanisms.

The myristoylated pentapeptide, L-R5, contains an amino acid sequence of the zeta inhibitory peptide (ZIP) portion (pseudosubstrate) of protein kinase C zeta (PKC ζ). As PKC ζ is involved in the modulation of epithelial tight junctions (TJs) through the phosphorylation of TJ proteins, L-R5 was suggested to interact with the enzyme resulting in the enhancement of paracellular permeability. This study shows that L-R5 does not bind to the enzyme but interacts directly with TJ proteins. We show here that the binding of PKC ζ to occludin and its successive phosphorylation is prevented by L-R5, which leads to TJ disruption and enhanced epithelial permeability. Although L-R5 did not show any in vitro cytotoxicity, a proteomics study revealed that L-R5 interferes with other regulatory pathways, e.g., apoptosis and immune response. We suggest that structural modification of the peptide may increase the specificity TJ protein-peptide interaction.

Characterization of the Interaction of Polymeric Micelles with siRNA: A Combined Experimental and Molecular Dynamics Study.

The simulation of large molecular systems remains a daunting challenge, which justifies the exploration of novel methodologies to keep computers as an ideal companion tool for everyday laboratory work. Whole micelles, bigger than 20 nm in size, formed by the self-assembly of hundreds of copolymers containing more than 50 repeating units, have until now rarely been simulated, due to a lack of computational power. Therefore, a flexible amphiphilic triblock copolymer (mPEG45-α-PLL10-PLA25) containing a total of 80 repeating units, has been emulated and synthesized to embody compactified nanoconstructs of over 900 assembled copolymers, sized between 80 and 100 nm, for siRNA complexing purposes. In this study, the tailored triblock copolymers containing a controlled number of amino groups, were used as a support model to address the binding behavior of STAT3-siRNA, in the formation of micelleplexes. Since increasingly complex drug delivery systems require an ever more optimized physicochemical characterization, a converging description has been implemented by a combination of experimentation and computational simulations. The computational data were advantageous in allowing for the assumption of an optimal N/P ratio favoring both conformational rigidifications of STAT3-siRNA with low competitive phenomena at the binding sites of the micellar carriers. These calculations were consistent with the experimental data showing that an N/P ratio of 1.5 resulted in a sufficient amount of complexed STAT3-siRNA with an electrical potential at the slipping plane of the nanopharmaceuticals, close to the charge neutralization.

Synthesis, Formulation and Characterization of Immunotherapeutic Glycosylated Dendrimer/cGAMP Complexes for CD206 Targeted Delivery to M2 Macrophages in Cold Tumors.

Anti-tumor responses can be achieved via the stimulation of the immune system, a therapeutic approach called cancer immunotherapy. Many solid tumor types are characterized by the presence of immune-suppressive tumor-associated macrophage (TAMs) cells within the tumor microenvironment (TME). Moreover, TAM infiltration is strongly associated with poor survival in solid cancer patients and hence a low responsiveness to cancer immunotherapy. Therefore, 2'3' Cyclic GMP-AMP (2'3' cGAMP) was employed for its ability to shift macrophages from pro-tumoral M2-like macrophages (TAM) to anti-tumoral M1. However, cGAMP transfection within macrophages is limited by the molecule's negative charge, poor stability and lack of targeting. To circumvent these barriers, we designed nanocarriers based on poly(amidoamine) dendrimers (PAMAM) grafted with D-glucuronic acid (Glu) for M2 mannose-mediated endocytosis. Two carriers were synthesized based on different dendrimers and complexed with cGAMP at different ratios. Orthogonal techniques were employed for synthesis (NMR, ninhydrin, and gravimetry), size (DLS, NTA, and AF4-DLS), charge (DLS and NTA), complexation (HPLC-UV and AF4-UV) and biocompatibility and toxicity (primary cells and hen egg chorioallantoic membrane model) evaluations in order to evaluate the best cGAMP carrier. The best formulation was selected for its low toxicity, biocompatibility, monodispersed distribution, affinity towards CD206 and ability to increase M1 (STAT1 and NOS2) and decrease M2 marker (MRC1) expression in macrophages.

Translating a Thin-Film Rehydration Method to Microfluidics for the Preparation of a SARS-CoV-2 DNA Vaccine: When Manufacturing Method Matters.

Previous investigations conducted on a liposomal formulation for a SARS-CoV-2 DNA vaccine manufactured using the thin-film layer rehydration method showed promising immunogenicity results in mice. The adaptation of the liposomal formulation to a scalable and reproducible method of manufacture is necessary to continue the investigation of this vaccine candidate. Microfluidics manufacture shows high potential in method translation. The physicochemical characterization of the blank liposomes produced by thin-film layer rehydration or microfluidics were shown to be comparable. However, a difference in lipid nanostructure in the bilayer resulted in a significant difference in the hydration of the thin-film liposomes, ultimately altering their complexation behavior. A study on the complexation of liposomes with the DNA vaccine at various N/P ratios showed different sizes and Zeta-potential values between the two formulations. This difference in the complexation behavior resulted in distinct immunogenicity profiles in mice. The thin-film layer rehydration-manufactured liposomes induced a significantly higher response compared to the microfluidics-manufactured samples. The nanostructural analysis of the two samples revealed the critical importance of understanding the differences between the two formulations that resulted in the different immunogenicity in mice.

Regulatory safety evaluation of nanomedical products: key issues to refine.

Nanotechnologies enable great opportunities for the development and use of innovative (nano)medicines. As is common for scientific and technical developments, recognized safety evaluation methods for regulatory purposes are lagging behind. The specific properties responsible for the desired functioning also hamper the safety evaluation of such products. Pharmacokinetics determination of the active pharmaceutical ingredient as well as the nanomaterial component is crucial. Due to their particulate nature, nanomedicines, similar to all nanomaterials, are primarily removed from the circulation by phagocytizing cells that are part of the immune system. Therefore, the immune system can be potentially a specific target for adverse effects of nanomedicines, and thus needs special attention during the safety evaluation. This DDTR special issue on the results of the REFINE project on a regulatory science framework for nanomedical products presents a highly valuable body of knowledge needed to address regulatory challenges and gaps in currently available testing methods for the safety evaluation of nanomedicines.

Need for Expansion of Pharmacy Education Globally for the Growing Field of Nanomedicine.

The emerging landscape of nanomedicine includes a wide variety of active pharmaceutical ingredients and drug formulations. Their design provides nanomedicines with unique features leading to improved pharmacokinetics and pharmacodynamics. They are manufactured using conventional or biotechnological manufacturing processes. Their physical characteristics are vastly different from traditional small-molecule drugs. Pharmacists are important members of the multi-disciplinary team of scientists involved in their development and clinical application. Consequently, their training should lead to an understanding of the complexities associated with the production and evaluation of nanomedicines. Therefore, student pharmacists, post-doctoral researchers, and trainees should be given more exposure to this rapidly evolving class of therapeutics. This commentary will provide an overview of nanomedicine education within the selection of pharmacy programs globally, discuss the current regulatory challenges, and describe different approaches to incorporate nanomedicine science in pharmacy programs around the world.