Teresa Alejo

Background/Objectives: Lipid nanoparticles (LNPs) containing ionizable lipids represent the most advanced non-viral delivery vehicles and have become state-of-the-art carriers for RNA therapeutics. However, further improvements in endosomal escape efficiency and biodegradability are still needed, especially for nucleic acids with transient activity such as messenger RNA (mRNA) and transfer RNA (tRNA). Methods: In this study, a novel library of highly biodegradable ionizable lipids featuring thioester groups within the linker region was designed and synthesized, thereby expanding the chemical linker toolbox for future ionizable lipid development. Results: Comprehensive in vivo structure–activity relationship studies led to the identification of CP-LC-1272 as a lead candidate that markedly enhances endosomal escape and exhibits superior in vivo biodegradability, attributed to the high acid-lability of thioester bonds. LNPs containing CP-LC-1272 maintained in vivo activity after six months of storage in lyophilized form and demonstrated superior in vivo efficiency compared to SM-102 in mRNA expression studies, as well as similar protein restoration in a tRNA delivery model targeting premature stop-codon mutations. Conclusions: The rapid biodegradability of these thioester-activated ionizable lipids (TAILs) suggests a reduced risk of accumulation, with the potential to enable safe repeated dosing or high-dosage RNA therapies, positioning TAILs as a versatile and safe platform for next-generation RNA therapeutics.

Background/Objectives: Lipid nanoparticles (LNPs) are leading mRNA delivery vehicles, with ionizable lipids (ILs) as their key component. However, the relationship between the IL structure and LNP endogenous organ-targeting is not well understood. In this study, we developed a novel library of biodegradable ILs featuring beta-propionate linkers, which, when incorporated into a four-component LNP formulation, show excellent extrahepatic selectivity and high protein expression. Methods: We explored the impact of structural modifications in the hydrophobic chains and polar-head groups in the ILs while keeping the linkers unchanged. In vivo results were evaluated to examine how structural changes influence the biodistribution to spleen or lungs. LNP formulations were assessed for their protein expression levels and organ-specific targeting. Additionally, protein corona formation by the best-performing LNPs was examined to provide further mechanistic insights. Results: Organ targeting was significantly influenced by structural changes in the ILs, allowing for precise control of the biodistribution between the spleen and lungs. Branched hydrophobic chains demonstrated a higher propensity for spleen targeting, while modifications in the polar-head group could drastically shift biodistribution from the lung to the spleen. This led to the identification of LNPs’ zeta potential as a key determinant of their extrahepatic targeting properties. Notably, ionizable lipid A3T2C7, also known as CP-LC-1495, displayed strong lung selectivity (97%) and high protein expression in lung tissue (1.21 × 10⁸ p/s). Similarly, several promising candidates for spleen-targeting LNPs displayed protein expression levels exceeding 1 × 10⁷ p/s (selectivity >80%). Conclusions: This study elucidates the structure–function relationships of ILs in passive organ-specific mRNA delivery, highlighting how the fine-tuning of hydrophobic chains, polar-head groups, and surface charge (zeta potential) allows for the precise control of LNP endogenous biodistribution, a mechanism influenced by protein corona formation. These findings enable the rational design of targeted LNP systems, enhancing their therapeutic potential for specific organs, such as the spleen and lungs.

The success of mRNA vaccines against SARS-CoV-2 has prompted interest in mRNA-based pharmaceuticals due to their rapid production, adaptability, and safety. Despite these advantages, the inherent instability of mRNA and its rapid degradation in vivo underscores the need for an encapsulation system for the administration and delivery of RNA-based therapeutics. Lipid nanoparticles (LNPs) have proven the most robust and safest option for in vivo applications. However, the mid- to long-term storage of mRNA-LNPs still requires sub-zero temperatures along the entire chain of supply, highlighting the need to develop alternatives to improve mRNA vaccine stability under non-freezing conditions to facilitate logistics and distribution. Lyophilization presents itself as an effective alternative to prolong the shelf life of mRNA vaccines under refrigeration conditions, although a complex optimization of the process parameters is needed to maintain the integrity of the mRNA-LNPs. Recent studies have demonstrated the feasibility of freeze-drying LNPs, showing that lyophilized mRNA-LNPs retain activity and stability. However, long-term functional data remain limited. Herein, we focus on obtaining an optimized lyophilizable mRNA-LNP formulation through the careful selection of an optimal buffer and cryoprotectant and by tuning freeze-drying parameters. The results demonstrate that our optimized lyophilization process maintains LNP characteristics and functionality for over a year at refrigerated temperatures, offering a viable solution to the logistical hurdles of mRNA vaccine distribution.

The concept of “codon optimisation” involves adjusting the coding sequence of a target protein to account for the inherent codon preferences of a host species and maximise protein expression in that species. However, there is still a lack of consensus on the most effective approach to achieve optimal results. Existing methods typically depend on heuristic combinations of different variables, leaving the user with the final choice of the sequence hit. In this study, we propose a new statistical-physics model for codon optimisation. This model, called the Nearest-Neighbour interaction (NN) model, links the probability of any given codon sequence to the “interactions” between neighbouring codons. We used the model to design codon sequences for different proteins of interest, and we compared our sequences with the predictions of some commercial tools. In order to assess the importance of the pair interactions, we additionally compared the NN model with a simpler method (Ind) that disregards interactions. It was observed that the NN method yielded similar Codon Adaptation Index (CAI) values to those obtained by other commercial algorithms, despite the fact that CAI was not explicitly considered in the algorithm. By utilising both the NN and Ind methods to optimise the reporter protein luciferase, and then analysing the translation performance in human cell lines and in a mouse model, we found that the NN approach yielded the highest protein expression in vivo . Consequently, we propose that the NN model may prove advantageous in biotechnological applications, such as heterologous protein expression or mRNA-based therapies.

In breast cancer, Targeted Axillary Dissection (TAD) allows for the selective excision of the sentinel lymph node (SLN) during primary tumor surgery. TAD consists of the resection of labelled SLNs prior to neoadjuvant chemotherapy (NACT). Numerous clinical and preclinical studies have explored the use of carbon-based colloids for SLN tattooing prior to NACT. However, carbon vectors show varying degrees of inflammatory reactions and, in about one fifth of cases, carbon particles migrate via the lymphatic pathway to other nodes, causing the SLN to mismatch the tattooed node. To overcome these limitations, in this study, we explored the use of melanin as a staining endogenous pigment. We synthesized and characterized melanin-loaded polymeric nanoparticles (Mel-NPs) and used them to tattoo lymph nodes in pig animal models given the similarity in the size of the human and pig nodes. Mel-NPs tattooed lymph nodes showed high identification rates, reaching 83.3% positive identification 16 weeks after tattooing. We did not observe any reduction in the identification as time increased, implying that the colloid is stable in the lymph node tissue. In addition, we performed histological and ultrastructural studies to characterize the biological behavior of the tag. We observed foreign-body-like granulomatous inflammatory responses associated with Mel-NPs, characterized by the formation of multinucleated giant cells. In addition, electron microscopy studies showed that uptake is mainly performed by macrophages, and that macrophages undergo cellular damage associated with particle uptake.

Osteoarthritis (OA) is a common chronic joint pathology that has become a predominant cause of disability worldwide. Even though the origin and evolution of OA rely on different factors that are not yet elucidated nor understood, the development of novel strategies to treat OA has emerged in the last years. Cartilage degradation is the main hallmark of the pathology though alterations in bone and synovial inflammation, among other comorbidities, are also involved during OA progression. From a molecular point of view, a vast amount of signaling pathways are implicated in the progression of the disease, opening up a wide plethora of targets to attenuate or even halt OA. The main purpose of this review is to shed light on the recent strategies published based on nanotechnology for the early diagnosis of the disease as well as the most promising nano‐enabling therapeutic approaches validated in preclinical models. To address the clinical issue, the key pathways involved in OA initiation and progression are described as the main potential targets for OA prevention and early treatment. Furthermore, an overview of current therapeutic strategies is depicted. Finally, to solve the drawbacks of current treatments, nanobiomedicine has shown demonstrated benefits when using drug delivery systems compared with the administration of the equivalent doses of the free drugs and the potential of disease‐modifying OA drugs when using nanosystems. We anticipate that the development of smart and specific bioresponsive and biocompatible nanosystems will provide a solid and promising basis for effective OA early diagnosis and treatment.This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement

Virgin olive oil, the main source of fat in the Mediterranean diet, contains a substantial amount of squalene which possesses natural antioxidant properties. Due to its highly hydrophobic nature, its bioavailability is reduced. In order to increase its delivery and potentiate its actions, squalene has been loaded into PLGA nanoparticles (NPs). The characterization of the resulting nanoparticles was assessed by electron microscopy, dynamic light scattering, zeta potential and high-performance liquid chromatography. Reactive oxygen species (ROS) generation and cell viability assays were carried out in AML12 (alpha mouse liver cell line) and a TXNDC5-deficient AML12 cell line (KO), which was generated by CRISPR/cas9 technology. According to the results, squalene was successfully encapsulated in PLGA NPs, and had rapid and efficient cellular uptake at 30 µM squalene concentration. Squalene reduced ROS in AML12, whereas ROS levels increased in KO cells and improved cell viability in both when subjected to oxidative stress by significant induction of Gpx4. Squalene enhanced cell viability in ER-induced stress by decreasing Ern1 or Eif2ak3 expressions. In conclusion, TXNDC5 shows a crucial role in regulating ER-induced stress through different signaling pathways, and squalene protects mouse hepatocytes from oxidative and endoplasmic reticulum stresses by several molecular mechanisms depending on TXNDC5.

The development of new gene-editing technologies has fostered the need for efficient and safe vectors capable of encapsulating large nucleic acids. In this work we evaluate the synthesis of large-size plasmid-loaded PLGA nanoparticles by double emulsion (considering batch ultrasound and microfluidics-assisted methodologies) and magnetic stirring-based nanoprecipitation synthesis methods. For this purpose, we characterized the nanoparticles and compared the results between the different synthesis processes in terms of encapsulation efficiency, morphology, particle size, polydispersity, zeta potential and structural integrity of loaded pDNA. Our results demonstrate particular sensibility of large pDNA for shear and mechanical stress degradation during double emulsion, the nanoprecipitation method being the only one that preserved plasmid integrity. However, plasmid-loaded PLGA nanoparticles synthesized by nanoprecipitation did not show cell expression in vitro, possibly due to the slow release profile observed in our experimental conditions. Strong electrostatic interactions between the large plasmid and the cationic PLGA used for this synthesis may underlie this release kinetics. Overall, none of the methods evaluated satisfied all the requirements for an efficient non-viral vector when applied to large-size plasmid encapsulation. Further optimization or alternative synthesis methods are thus in current need to adapt PLGA nanoparticles as delivery vectors for gene editing therapeutic technologies.