A team of researchers from Aptar Pharma, INRAE, Université de Tours, and Lovaltech has now published a comprehensive proof-of-concept study in Frontiers in Drug Delivery that addresses this challenge head-on. The work, led by Gonçalo Farias and Mathieu Epardaud, describes the engineering and validation of a nasal spray device specifically designed for human nasal immunization. The device, known as the BiVax 200 µL intranasal atomiser, was developed to deliver a broad, homogeneous spray confined entirely within the nasal cavity, thereby avoiding spill-over into the lower respiratory tract and minimising any risk of pulmonary exposure that could provoke adverse inflammatory responses.

The scientific rationale behind this engineering effort stems from a fundamental anatomical problem. In classical rodent preclinical models such as mice and hamsters, intranasal instillation with a micropipette results in deposition in both the nasal cavity and the upper lungs, a phenomenon aggravated by parameters such as the volume delivered and the anaesthesia used. This inadvertent pulmonary exposure can artificially enhance immune responses beyond what a simple nasal immune reaction would produce, creating a misleading picture of safety and efficacy. In humans, however, a strictly nasal delivery must be prioritised to avoid potential adverse effects such as uncontrolled chronic pulmonary inflammation or cytokine storm, making the transition from animal models to human application particularly treacherous.

To overcome these limitations, the researchers designed their validation strategy around two complementary approaches. First, they employed in vitro human nasal cavity replicas, known as nasal casts, which were constructed from computed tomography images of a plastinated head model and previously validated as predictive tools for nasal aerosol deposition. Second, they utilised an in vivo rabbit model, a species widely accepted by regulatory agencies for preclinical vaccine testing and already approved for regulatory toxicity assays. This dual validation framework was deliberately chosen to bridge the gap between conventional rodent models, which suffer from significant anatomical and physiological differences compared to human nasal structure and airflow dynamics, and the eventual human clinical setting.

The vaccine formulation itself represents an equally sophisticated piece of bioengineering. LVT-001, the drug substance under investigation, is a heteromultimeric recombinant protein encoding both the spike and nucleoprotein of SARS-CoV-2. The vaccine fusion protein is composed of two polypeptide chains that self-assemble into a structure containing six spike proteins and two nucleoproteins, a trimerization strategy shown to enhance both vaccine efficacy and structural stability. This antigen is combined with an original cationic nanocarrier called the muco-excipient, composed of positively charged polymeric sugar matrix particles with an average diameter of 35 to 70 nanometres, at a ratio of three nanocarriers to one antigen within a phosphate-buffered saline formulation.

A critical prerequisite before evaluating any delivery device is confirming that the aerosolisation process itself does not compromise the integrity of the biological product. The researchers subjected the drug product to a rigorous analytical toolkit before and after aerosolisation, including protein quantification by bicinchoninic acid assay, structural assessment by native polyacrylamide gel electrophoresis, particle size distribution measurement by dynamic light scattering, and biological activity evaluation through indirect competitive ELISA targeting both the spike and nucleoprotein domains. Across all analyses, the drug product remained stable post-aerosolisation, with only a minor increase in polydispersity index and Z-average that the authors attribute to analytical and sample variability rather than any genuine degradation of the active component.

The spray characterisation results revealed a droplet size distribution with average Dv10, Dv50, and Dv90 values of 18, 32, and 57 micrometres respectively, with only 1.5 percent of droplets below 10 micrometres. This distribution is significant because droplets smaller than 10 micrometres are more likely to reach the lower respiratory tract, while larger droplets tend to deposit within the nasal cavity. The low fraction of sub-10-micrometre droplets, combined with a broad spray angle of 58 degrees, ensures that the delivered dose remains confined to the nasal structures without pulmonary exposure, a finding confirmed by the complete absence of detectable deposition on a filter placed after the nasal cast model.

Regional deposition studies using the nasal cast model revealed that administration parameters significantly influence where the spray lands within the nasal cavity. A multivariate analysis demonstrated that the insertion angle, insertion depth, and model position all had statistically significant impacts on regional deposition patterns, while airflow did not significantly affect the results. At a fixed insertion depth of 15 millimetres, increasing the administration angle from 30 to 50 degrees led to a marked increase in anterior region deposition from 15 to 46 percent, accompanied by a substantial reduction in posterior deposition, particularly in the lower turbinates where average deposition decreased from 28 to just 4 percent. Conversely, increasing the insertion depth from 2 to 15 millimetres significantly reduced anterior deposition from 73 to 15 percent, with the optimised configuration predominantly localising the dose in the lower and middle turbinates.

The in vivo rabbit experiments provided compelling evidence for the superiority of the BiVax device over conventional micropipette administration. Fifteen minutes post-administration, the total radiant efficiency measured within the regions of interest was approximately 50 percent higher following BiVax delivery compared to micropipette instillation. More importantly, the distribution pattern was dramatically more homogeneous with the spray device, with about 31 percent of the formulation detected in the nasal and maxillary turbinates when delivered with BiVax, compared to only 11 percent with micropipette administration. This unbalanced distribution with the micropipette, characterised by high concentrations in the ethmoid turbinates and pharyngeal area, was attributed to the administration procedure during which the rabbit was held in an inverted position.

The implications of this work extend well beyond a single COVID-19 vaccine candidate. The researchers deliberately limited deposition in the olfactory epithelium and paranasal sinuses, because material in the former raises concerns of central nervous system exposure, while retention in the latter may provoke sinusitis or chronic irritation. This safety consideration is particularly relevant given past clinical failures, such as the withdrawal of the intranasal influenza vaccine Nasalflu following reports of Bell’s palsy, a leading hypothesis for which involved retrograde axonal transport of an E. coli heat-labile enterotoxin adjuvant from the olfactory epithelium to the peripheral facial nerve. By demonstrating negligible olfactory deposition and confirming the absence of neurological effects in rabbit toxicity studies, the team has addressed one of the most persistent safety concerns in the field. The device is now being used in an ongoing Phase I clinical trial, and the researchers argue that this transitory delivery platform offers a scalable, safe, and formulation-compatible solution that could reinforce mucosal immunization strategies across diverse indications, facilitating the translation of nasal vaccines from preclinical research to widespread clinical application.

Subject of Research: Intranasal vaccine delivery device engineering

Article Title: Engineering a medical device for intranasal immunisation: ensuring targeted delivery and formulation stability

Article References: Engineering a medical device for intranasal immunisation: ensuring targeted delivery and formulation stability. (n.d.). https://doi.org/10.3389/fddev.2026.1875011

Image Credits: AI Generated

DOI: 10.3389/fddev.2026.1875011

Keywords: intranasal vaccination, nasal spray device, BiVax, mucosal immunity, NALT, SARS-CoV-2 vaccine, nanocarrier, formulation stability, nasal cast, targeted delivery, aerosolisation, vaccine delivery

The core problem the authors confront is one of specificity. CRISPR-Cas9 can, in principle, rewrite the genome with nucleotide-level precision, disabling oncogenic drivers, restoring tumor suppressor pathways, or reprogramming regulatory noncoding elements. In practice, however, the Cas9 protein and its guide RNA are large, charged biological molecules that do not cross cell membranes on their own, degrade rapidly in the bloodstream, and can trigger off-target edits when they reach the wrong cells. Current viral vectors such as adeno-associated virus, or AAV, offer efficient delivery but raise concerns about insertional mutagenesis, limited cargo capacity, immunogenicity, and the sheer difficulty of restricting their tropism to cancer cells alone. Nonviral alternatives have historically suffered from low efficiency. The review positions aptamer-mediated delivery as a way out of this impasse.

Aptamers are selected through an iterative laboratory process known as SELEX, systematic evolution of ligands by exponential enrichment, which screens enormous random-sequence libraries for strands that bind a chosen target with high affinity. Unlike antibodies, aptamers are chemically synthesized, minimally immunogenic, small enough to penetrate dense tumor tissue, and can be modified at will with fluorescent tags, linker chemistries, or therapeutic payloads. The review catalogs aptamers directed against a who’s-who of tumor-associated surface markers: epithelial cell adhesion molecule (EpCAM) overexpressed on many carcinomas, epidermal growth factor receptor (EGFR) in lung and colon cancers, human epidermal growth factor receptor 2 (HER2) in breast cancer, prostate-specific membrane antigen (PSMA) in prostate cancer, nucleolin and protein tyrosine kinase 7 (PTK7) on a range of malignant cells, and the transferrin receptor, which is abundant on the blood-brain barrier and on glioblastoma cells. Each of these ligands can, in principle, be conjugated to a nanocarrier carrying Cas9 ribonucleoprotein complexes or plasmid DNA, converting a systemically administered particle into a homing missile for tumor cells.

One of the most technically interesting contributions of the review is its emphasis on structure-guided molecular docking as a design tool. Rather than relying purely on trial and error, the authors describe how computational docking and molecular dynamics simulations can predict the binding energetics of an aptamer-receptor pair before any wet-lab work begins. By modeling the three-dimensional folds that aptamers adopt, researchers can rationally introduce mutations that strengthen the binding interface, improve discrimination between the tumor target and closely related proteins on healthy cells, and truncate aptamers to the minimal binding domain, reducing manufacturing cost and immunogenic potential. This computational-first workflow, the review argues, is transforming aptamer engineering from an artisanal craft into a predictable engineering discipline.

Getting the particle bound to the cell surface, however, is only the first hurdle. The review devotes substantial attention to what happens next: receptor-mediated endocytosis, the process by which the cell swallows the bound particle into an endosome, followed by the equally critical step of endosomal escape. Most nanoparticles that successfully enter cells end up trapped and degraded in lysosomes, which is why delivery efficiency is often measured in frustratingly low single digits. The authors survey the escape strategies now being built into aptamer-functionalized carriers, including proton-sponge effects from polyethylenimine-based formulations, pH-responsive membrane-disruptive polymers, and fusogenic lipids that destabilize the endosomal membrane as its interior acidifies. Understanding which endocytic pathway, clathrin-mediated, caveolae-mediated, or macropinocytosis, a given aptamer-receptor pair exploits allows researchers to co-opt the cell’s own trafficking machinery and steer the cargo toward the cytosol and ultimately the nucleus, where Cas9 must arrive to do its work.

The third pillar of the framework is nanocarrier engineering, and here the review offers a sweeping comparison of the leading platforms. Lipid nanoparticles, validated clinically by the mRNA vaccine campaigns, remain the front-runner, with refinements in PEG-lipid architectures, such as cleavable DSPE-PEG anchors and optimized PEG densities, improving colloidal stability while avoiding accelerated blood clearance on repeat dosing. Polymeric carriers built from PLGA and PAMAM dendrimers offer controlled release profiles, while inorganic platforms, including mesoporous silica nanoparticles and gold nanoparticles, provide stimulus-responsive gates that open in the acidic tumor microenvironment. Extracellular vesicles, the natural nanoshuttles secreted by cells themselves, offer innate biocompatibility and the ability to cross biological barriers, and hybrid viral-nonviral platforms attempt to marry viral entry efficiency with the safety and tunability of synthetic materials.

What distinguishes this review from earlier surveys of CRISPR delivery is its insistence that these three elements, targeting ligand, trafficking route, and carrier chemistry, must be co-optimized rather than designed in isolation. A particle decorated with a superb aptamer but lacking endosomal escape capability will never deliver an active nuclease. A carrier with perfect intracellular performance but rapid serum clearance will never reach the tumor. The authors highlight how the dense, fibrotic extracellular matrix of many tumors physically blocks nanoparticle penetration, and how aptamer-mediated binding to receptors expressed deep within tumor spheroids can enhance accumulation in poorly perfused regions. They also discuss spatiotemporally controlled editing, in which carriers release their cargo only in response to tumor-specific cues such as hypoxia, low pH, or externally applied light or ultrasound, limiting genome modification to the tumor microenvironment.

The potential clinical applications span the full breadth of oncology. Targeted knockout of oncogenic drivers such as mutant EGFR in non-small cell lung cancer, disruption of the Wnt pathway via adenomatous polyposis coli-related targets in colorectal cancer, editing of immune checkpoints such as PD-1 to unleash antitumor immunity, and correction of tumor suppressor defects through homology-directed repair are all within the conceptual scope of aptamer-guided delivery. The authors also note synergies with established modalities: aptamer-targeted carriers could deliver Cas9 engineered to knock out drug-resistance genes alongside conventional chemotherapy, or complement CAR T-cell therapy by editing immune cells ex vivo with higher efficiency and lower toxicity than viral transduction.

The authors are admirably candid about the obstacles that remain. In vivo stability of both aptamers and Cas9 cargos in the face of nucleases and proteases, the scalability and reproducibility of GMP-grade nanomanufacturing, the persistent specter of off-target double-strand breaks, and the dearth of rigorous long-term preclinical and clinical validation all stand between the current state of the art and routine bedside use. Aptamer affinity measured in a culture dish does not always translate to four-hour circulation in human plasma, and off-target editing in non-tumor tissue remains the field’s most serious safety concern. Yet the trajectory is unmistakable. By integrating rational aptamer design, mechanistic understanding of intracellular trafficking, and increasingly sophisticated nanocarrier engineering, the field is assembling the complete delivery toolkit that precision genome editing oncology has been waiting for. If the remaining challenges can be met, aptamer-guided CRISPR-Cas9 systems could move genome surgery from the laboratory bench to the cancer clinic, offering patients therapies that edit the disease at its genetic source while sparing healthy tissue entirely.

Subject of Research: Aptamer-mediated CRISPR-Cas9 delivery systems for targeted cancer genome editing

Article Title: Aptamer-mediated CRISPR-Cas9 delivery for precision oncology: integrating molecular targeting, intracellular trafficking, and nanocarrier engineering

Article References: Saffari Natanzi, A., Ahmadi, E., Shahraki, S., Eslamian Koupaei, A., & Haddad Kashani, H. (2026). Aptamer-mediated CRISPR-Cas9 delivery for precision oncology: integrating molecular targeting, intracellular trafficking, and nanocarrier engineering. Advanced Composites and Hybrid Materials. https://doi.org/10.1007/s42114-026-02049-2

Image Credits: AI Generated

DOI: 10.1007/s42114-026-02049-2

Keywords: aptamers, CRISPR-Cas9, precision oncology, targeted delivery, nanocarriers, lipid nanoparticles, gene editing, endosomal escape, molecular docking, SELEX, tumor microenvironment, cancer therapy