Intraoperative Indocyanine Green Fluorescence Enables Primary Tumor Localization and Treatment De‐Escalation in <scp>SCCUP</scp> : A Case Report

Head & Neck · 2026-03-26

BACKGROUND: Unknown primary cancer in the head and neck presents a difficult surgical treatment dilemma. Patients with squamous cell carcinoma of unknown primary (SCCUP) typically present with an enlarging neck mass found on biopsy but with no indication of primary site on diagnostic exams such as flexible laryngoscopy, CT, MRI, and/or PET/CT. Failure to identify primary sites eliminates surgical treatment as an option, pushing patients toward definitive chemoradiation with associated side effects. METHODS: Indocyanine green (ICG) has been used to identify primary carcinoma in known oropharyngeal squamous cell carcinoma while using transoral robotic surgery (TORS). In this case, we injected ICG intraoperatively in a patient with SCCUP to help with the real-time localization of ICG in the tumor. RESULTS: ICG fluorescence successfully identified a previously undetected primary lesion within the oropharynx during TORS, which enabled precise surgical excision of the tumor. CONCLUSIONS: This case demonstrates the potential of ICG-guided TORS to localize primary tumors in SCCUP patients, offering a pathway to surgical treatment and potentially reducing reliance on chemoradiation.

Modulation of Cell Signaling Pathways in Silica Nanoparticle-Saturated Macrophages

Pharmaceutics · 2026-03-11

Background/Objectives: Upon systemic delivery, macrophages take up a significant portion of nanoparticles and may become saturated. The saturation of macrophages may pose risks to overall immune function and signaling pathways. While some information is available on the survival and functionality of macrophages upon saturation with nanoparticles, there is limited understanding of the molecular-level changes that can occur and their corresponding influences on macrophage phenotypes, gene expression, and immune signaling pathways. Methods: In this study, RAW 264.7 macrophages were saturated with silica nanoparticles (SNPs) of different sizes (50 and 100 nm), porosities (nonporous, mesoporous), densities (solid, mesoporous, and hollow), and surface compositions (hydrophobicity) at their maximum non-toxic concentrations. The saturated macrophages were evaluated for changes in gene expression and immune signaling pathways by RNA sequencing, weighted gene co-expression network analysis (WGCNA), and Hallmark and KEGG pathway analyses. Results: Our results show that in the range studied, the particle size did not have a significant effect on the gene expression profile. Porous SNPs of comparable sizes resulted in increased and unique changes in the gene expression profile compared to nonporous SNPs. Major immune signaling pathways, including TNF-alpha signaling via NF-κB pathways, mTORC1 signaling, and p53 pathways, were modulated in SNP-saturated macrophages. This modulation depended on the physicochemical properties of the particles. The Th1/Th2 multiplex immunoassay revealed that the uptake of SNPs increases the amount of the TNF-alpha cytokine compared to the nontreated controls, whereas no changes in IL-6 and IL-12p70 pro-inflammatory cytokines were observed. Conclusions: Our results demonstrate that physicochemical properties of SNPs, such as porosity, size, surface functionality, and density, influence the modulation of gene expression and macrophage immune signaling pathways. These results, along with others, can provide guidance on the selection of silica nanoparticles for the safe and effective systemic delivery of bioactive agents.

Macrophage Responses to Silica Nanoparticles: Role of Physicochemical Properties and Surface Modification

Molecular Pharmaceutics · 2026-02-17

Silica nanoparticles are widely studied nanomaterials for biomedical applications owing to their tunable physicochemical properties, such as size, porosity, geometry, and surface modification. Despite their promising potential, concerns regarding their safety continue to limit clinical translation. In this study, we systematically investigated how key physicochemical parameters and surface attachment of poly(ethylene glycol) (PEG) affect the cytotoxicity and immune activation profiles of silica nanoparticles in macrophages. A structurally diverse set of silica nanoparticles (rod, spherical, porous, nonporous, and surface-modified) was synthesized and characterized. RAW 264.7 macrophages were used as a model cell line to evaluate nanoparticle internalization, membrane integrity, apoptosis, cell cycle progression, and macrophage activation. While PEGylation and physicochemical variations significantly influenced both cellular uptake and maximum nontoxic dose, none of the tested nanoparticles impaired macrophage viability or baseline functionality at their respective saturation points. Notably, PEGylated silica nanoparticles approximately 100 nm in diameter and rod-shaped nanoparticles elicited pronounced immune activation, highlighting their distinct immunomodulatory potential despite the preserved cellular integrity.

Enhanced tyrosine kinase inhibitor response on chronic myeloid leukemia cells upon MS4A3 mRNA delivery

Blood · 2025-11-03

Abstract Chronic myeloid leukemia (CML) is caused by BCR::ABL1, a constitutively active tyrosine kinase that induces profound expansion of the myeloid cell compartment, while terminal differentiation is largely maintained. Tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1 are highly effective in CML, reflecting the dependence of CML progenitor cells (LPCs) on BCR::ABL1 kinase activity. However, most patients harbor residual leukemia, suggesting that TKI-resistant CML stem cells (LSCs) survive despite continued inhibition of BCR::ABL1. We have previously shown that membrane spanning four A3 (MS4A3) promotes myeloid differentiation of CML CD34+ cells by enhancing cellular response to IL-3 and GM-CSF. As MS4A3 is downregulated in primitive CML cells in a BCR::ABL1 independent manner, we hypothesize that re-establishing MS4A3 expression may overcome TKI resistance by promoting differentiation of TKI resistant LSCs into TKI-sensitive LPCs [1]. To test this, we have developed a CD34-targeted lipid nanoparticle (LNP) that encapsulates MS4A3 mRNA to increase expression of MS4A3 in LSPCs. We synthesized LNPs by mixing an ionizable lipid, cholesterol, a helper lipid, and a poly(ethylene glycol)-lipid in ethanol and MS4A3 or enhanced green fluorescence protein (EGFP) mRNAs in acidic media with a microfluidic mixing chip. LNPs were characterized for their average diameter, polydispersity index (PDI) and zeta potential using dynamic light scattering (DLS). We determined mRNA loading at different amine to phosphate (N/P) ratios using agarose gel electrophoresis. We next synthesized CD34 targeted LNPs by modifying the surface with thiolated CD34 antibodies through thiol-maleimide conjugation, using isotype-matched nonspecific anti-human IgG as control (termed CD34-LNP and ISO-LNP, respectively). We used the RPMI-8402 B lymphoblastic leukemia cell line (100% CD34+) and CD34+ cells from chronic phase CML patients for in vitro internalization studies. We assessed time dependent internalization of CD34-LNPs using Cy5-tagged LNPs, determined intracellular uptake by flow cytometry, and measured the effect of LNPs on cell viability by CCK8 assay. We analyzed CML cells transfected with EGFP or MS4A3 mRNA encapsulated LNPs for expression of the respective protein by flow cytometry and tested the effect of MS4A3 mRNA with and without added imatinib in colony forming assays of primary human CD34+ CML cells. The average hydrodynamic diameter of LNPs was 90.9 ± 1.0 nm and increased to 129.3 ± 6.3 post-modification with antibodies, indicating LNPs within the desired size range. Approximately 65% of the CD34+ RPMI-8402 cells showed LNP uptake after 2 h incubation with CD34-LNPs, while only 45% had internalized ISO-LNP (p=0.01). Selective uptake was maintained over more prolonged incubation times (up to 24 h). Similarly, CML CD34+ cells demonstrated 3.8-fold higher uptake of CD34-LNPs compared to ISO-LNPs at 2 h (p=0.01). We validated EGFP and MS4A3 protein expression upon mRNA delivery using the CD34-LNPs in RPMI-8402 cells. EGFP expression was first detected after 24 h and showed the highest fluorescence intensity with approximately 100% of cell positivity on day 5. The MS4A3 expression was detectable after 72 h with approximately 40% of cell positivity and a 2-fold increase in expression. Next, we delivered MS4A3 mRNA into CML CD34+ cells. A 2-fold increase of MS4A3 protein expression was detected on day 5 upon mRNA treatment. In the clonogenic assays, MS4A3-LNPs and imatinib (0.5 µM) alone reduced colony formation by 19% and 50%, respectively, while the combination reduced colonies by 82% (p=0.001), consistent with our overarching hypothesis. (i) Synthesis of CD34 targeted LNPs is feasible, with significantly increased uptake compared to non-targeted LNP. (ii) Targeted MS4A3 mRNA delivery increases MS4A3 protein expression in cell lines and primary CML cells. (iii) CD34-LNP-induced MS4A3 expression enhances imatinib toxicity toward primary CML cells. Studies are underway to validate the results using additional endpoints including cell cycle distribution, differentiation and apoptosis. Delivery of MS4A3 mRNA to CML patients may improve TKI response. This project was funded by the National Institutes of Health (NIH) under grant no. R01CA268496. ReferencesZhao, H., et al., MS4A3 promotes differentiation in chronic myeloid leukemia by enhancing common β-chain cytokine receptor endocytosis. 2022. 139(5): p. 761-778.

PEGylation of Propofol Reduces Its Adsorption to Extracorporeal Membrane Oxygenator (ECMO) Components

Pharmaceutical Research · 2025-06-05

Transdermal delivery of ultradeformable cationic liposomes complexed with miR211–5p (UCL-211) stabilizes BRAFV600E+ melanocytic nevi

Journal of Controlled Release · 2025-03-01 · 6 citations

An in vitro study on macrophage toxicity of allotrope silver nanoparticles

Journal of Nanoparticle Research · 2025-05-01 · 4 citations

Effects of silica nanoparticles with varied physicochemical properties on the survival and functionality of saturated macrophages

Journal of Controlled Release · 2025-03-17 · 5 citations

Reducing hydrophobic drug adsorption in an in-vitro extracorporeal membrane oxygenation model

European Journal of Pharmaceutics and Biopharmaceutics · 2024 · 1 citations

• Chemistry
• Chromatography
• Pharmacology

Transdermal Delivery of Ultradeformable Cationic Liposomes Complexed with miR211-5p (UCL-211) Stabilizes BRAFV600E+ Melanocytic Nevi

bioRxiv (Cold Spring Harbor Laboratory) · 2024-10-21

Abstract Small non-coding RNAs (e.g. siRNA, miRNA) are involved in a variety of melanocyte-associated skin conditions and act as drivers for alterations in gene expression within melanocytes. These molecular changes can potentially affect the cellular stability of melanocytes and promote their oncogenic transformation. Thus, small RNAs can be considered as therapeutic targets for these conditions, however, their topical delivery to the melanocytes through the epidermal barrier is challenging. We synthesized and extensively evaluated ultradeformable cationic liposome (UCLs) carriers complexed with synthetic microRNAs (miR211-5p; UCL-211) for transdermal delivery to melanocytes. UCL-211 complexes were characterized for their physicochemical properties, encapsulation efficiency, and deformability, which revealed a significant advantage over conventional liposomal carriers. Increased expression of miR211-5p stabilizes melanocytic nevi and keeps them in growth-arrested state. We did a comprehensive assessment of cellular delivery, and biological activity of the miR211-5p carried by UCL-211 in vitro and their permeation through the epidermis of intact skin using ex vivo human skin tissue explants. We also demonstrated, in vivo , that topical delivery of miR211-5p by UCL-211 stabilized BRAFV600E+ nevi melanocytes in a benign nevi state.