Science

In Vitro Studies

Our in vitro studies provide the foundational evidence supporting the biological rationale of our nanomedicine platform. Using well-established cell culture models, we examined how our technology interacts with key cell types involved in the inflammatory and wound healing cascade, including macrophages and fibroblasts assessing its effects on inflammatory mediator expression, macrophage phenotype transition, and extracellular matrix regulation. These controlled experiments allow us to isolate and interrogate specific biological mechanisms, establishing a rigorous preclinical basis for the therapeutic effects observed. The results presented here represent a critical first step in translating the science of macrophage modulation into a viable, evidence-based approach to reducing pathological scarring and supporting healthier tissue repair.

Cellular Uptake

Fluorescence Microscopy Visualization of CYTONECT^™ nanoparticle Interactions with Different Cell Models

To evaluate cellular internalization of our nanoparticles, uptake assays were performed across multiple cell lines using flow cytometry. Mean Fluorescence Intensity (MFI) was measured as a quantitative proxy for intracellular nanoparticle accumulation, allowing direct comparison of uptake efficiency between cell types. THP-1 cells (human monocytic leukemia-derived) a macrophage model central to our therapeutic target demonstrated significantly higher nanoparticle uptake compared to HEK293 (human embryonic kidney cells) and HPD6C7 (human pancreatic epithelial cells) cells, confirming preferential internalization by macrophage-lineage cells. This selectivity is a critical feature of our platform, supporting the premise that our nanoparticles are designed to engage the immune cells most responsible for driving dysregulated inflammation, precisely where therapeutic intervention is needed most.

Fluorescence microscopy was used to examine the cellular association and distribution of CYTONECT^™ nanoparticles across different cell models. Green fluorescence identifies the nanoparticle-associated signal, while DAPI staining highlights cell nuclei. The merged images provide a comparative visualization of nanoparticle fluorescence in relation to cellular structures.

Cellular Uptake Assay

Flow Cytometry Analysis

To assess the cell-selectivity of CYTONECT^™ nanoparticles, a preferential uptake assay was conducted using flow cytometry across three distinct cell lines: THP-1 cells (a monocyte-derived macrophage model), HEK293 cells (human embryonic kidney), and HPD6C7 cells (human pancreatic epithelial cells). Mean Fluorescence Intensity, used as a quantitative measure of nanoparticle internalization, was significantly higher in THP-1 cells compared to both HEK293 (p < 0.0001) and HPD6C7 cells (p < 0.0001). These findings demonstrate that CYTONECT^™ nanoparticles are preferentially internalized by macrophage-lineage cells, supporting the targeted mechanism of action central to our platform. This cell-type selectivity is a defining characteristic of CYTONECT^™ and underpins its potential to modulate macrophage behavior precisely at the site of dysregulated inflammation.

Pro-Inflammatory Gene Expression

Reduced LPS-Induced Pro-Inflammatory Gene Expression in THP-1 Cells

In vitro studies using THP-1 cells evaluated the effects of CYTONECT^™ nanomedicine on the expression of key pro-inflammatory genes following lipopolysaccharide (LPS) stimulation. Across the two experimental replicates shown, LPS stimulation significantly increased TNF-α and IL-1β gene expression compared with untreated cells. Treatment with the nanomedicine significantly reduced the expression of both inflammatory markers relative to LPS-stimulated cells. For IL-6, LPS stimulation did not produce a statistically significant increase compared with untreated cells; however, nanomedicine treatment significantly reduced IL-6 expression relative to the LPS group. Collectively, these findings demonstrate the nanomedicine's ability to attenuate the expression of key pro-inflammatory genes in this in vitro THP-1 cell model, supporting further investigation of its immune-modulating activity.

Pro-Inflammatory Cytokine Secretion

Reduced LPS-Induced Pro-Inflammatory Cytokine Secretion in THP-1 Cells

LPS stimulation of THP-1 macrophages produced a marked increase in secretion of TNF-α and IL-1β, two principal mediators of the pro-inflammatory cascade central to dysregulated wound healing. Treatment with CYTONECT^™ nanomedicine significantly reduced TNF-α and IL-1β concentrations to levels comparable to untreated controls (p < 0.0001 and p < 0.001, respectively), demonstrating its ability to actively resolve macrophage-driven inflammation at the cytokine level. These results position CYTONECT^™ as a potent modulator of the innate immune response, targeting the upstream inflammatory signals that sustain chronic inflammation and impair tissue repair.

Pro-healing Gene expression

Increased Pro-Healing Gene Expression in HDF/THP-1 Co-Culture model

In vitro studies using an HDF/THP-1 co-culture model evaluated the effects of CYTONECT^™ treatment on the expression of two genes associated with tissue repair: IL-10 and VEGFA. The results show a marked increase in both gene expression levels in the LPS+CYTONECT^™ treatment group compared with the control group. IL-10, an important immunoregulatory cytokine involved in resolving inflammation, showed a pronounced increase, while VEGFA, a key regulator of angiogenesis and vascular repair, was also significantly upregulated. LPS alone did not produce a statistically significant change relative to the control for either marker. These findings demonstrate CYTONECT^™-associated modulation of gene expression linked to inflammatory regulation and tissue repair, supporting further investigation of its role in promoting a more favorable wound-healing environment.

Wound Environment on a Cellular Level

THP-1 and HaCaT Co-culture Model

To replicate the complexity of the inflammatory wound microenvironment, THP-1 monocyte-derived macrophages were co-cultured with HaCaT keratinocytes using a transwell membrane system. This configuration allows soluble inflammatory mediators secreted by macrophages to act on the keratinocyte monolayer below, without direct cell-to-cell contact, closely mimicking the paracrine signaling dynamics observed in inflamed wound tissue in vivo. A scratch assay was subsequently performed on the HaCaT monolayer to quantify keratinocyte migration and wound closure capacity under these inflammatory conditions. This model provides a physiologically relevant platform for evaluating the ability of CYTONECT^™ to restore normal healing dynamics in the presence of macrophage-driven inflammation.

In untreated controls, the scratch wound closed progressively over 24 hours, while LPS-stimulated conditions significantly impaired this process, reducing closure to less than 50% at 24 hours, consistent with the well-documented barrier that chronic inflammation places on tissue repair. Strikingly, treatment with CYTONECT^™ nanomedicine under the same inflammatory conditions resulted in near-complete wound closure by 12 hours, a response statistically superior to both the LPS and untreated groups (p < 0.0001).

These results demonstrate that CYTONECT^™ does not merely counteract inflammation, it actively restores and accelerates the cellular machinery of healing, even in a hostile inflammatory microenvironment.

This is the core promise of our platform: not to suppress the immune response, but to redirect it toward recovery.

Reduced Myofibroblast Activation

Immunofluorescence staining for α-SMA, a marker of myofibroblast activation and a key driver of scar formation, revealed significantly elevated expression and prominent stress fiber formation in fibroblasts exposed to LPS-induced inflammation.

Treatment with CYTONECT^™ nanomedicine reduced α-SMA signal intensity by approximately 60% per nucleus compared to LPS alone, demonstrating its capacity to suppress the pathological fibroblast response responsible for excessive collagen deposition and fibrosis.