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Article

Topical Delivery of CNP-miR146a via a Pluronic Lecithin Organogel Enhances Diabetic Wound Healing

by
Bailey D. Lyttle
1,*,
James Bardill
1,
Alyssa E. Vaughn
1,
Anisha Apte
2,
Alyssa San Agustin
2,
Elayaraja Kolanthai
3,
Sudipta Seal
3,
David M. Jackson
4,
Kenneth W. Liechty
2,4 and
Carlos Zgheib
2,4
1
Laboratory for Fetal and Regenerative Biology, Children’s Hospital Colorado, University of Colorado School of Medicine, Aurora, CO 80045, USA
2
Laboratory for Fetal and Regenerative Biology, Department of Surgery, Banner Children’s at Diamond Children’s Medical Center, University of Arizona Tucson College of Medicine, Tucson, AZ 85721, USA
3
Advanced Materials Processing and Analysis Center, Nanoscience Technology Center, University of Central Florida, Orlando, FL 32826, USA
4
Ceria Therapeutics, Inc., Tucson, AZ 85721, USA
*
Author to whom correspondence should be addressed.
Pharmaceutics 2026, 18(2), 248; https://doi.org/10.3390/pharmaceutics18020248
Submission received: 30 November 2025 / Revised: 20 December 2025 / Accepted: 25 December 2025 / Published: 17 February 2026
(This article belongs to the Special Issue Nanosystems for Advanced Diagnostics and Therapy)
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Abstract

Background: Diabetes mellitus is common and associated with numerous complications including diabetic foot ulcers (DFU), which affect a third of patients and are associated with high morbidity and mortality. There are limited pharmacologic treatment options available with mixed efficacy. We have developed a novel therapeutic targeting inflammation and oxidative stress by conjugating microRNA-146a to cerium oxide nanoparticles to create CNP-miR146a and have found that injectable CNP-miR146a is associated with improved wound healing in a diabetic murine model. We hypothesized that a topical formulation of CNP-miR146a would be associated with equivalent improvements in wound healing. Methods: Release tests of CNP conjugated to fluorescein isothiocyanate were performed to determine the optimal gel base for sustained release. Diabetic (db/db) mice were cutaneously wounded and treated with topical CNP-miR146a, empty gel, injectable CNP-miR146a, or injectable phosphate-buffered saline (PBS). Wound healing over time was compared between groups. Histological samples were collected and analyzed for CD45 and CD31 positivity at multiple timepoints. Results: CNP-miR146a in a topical pluronic lecithin organogel (PLO) base was associated with significantly improved wound healing compared to empty gel or injected PBS and equivalent to injected CNP-miR146a. Treatment with CNP-miR146a was also associated with decreased CD45 positivity and increased CD31 positivity, suggesting decreased inflammation and improved angiogenesis. Conclusions: Topical delivery of CNP-miR46a in a PLO base holds significant promise as a potential therapeutic for DFU and may improve patient compliance due to ease of delivery.

1. Introduction

Diabetes mellitus is one of the most common diseases worldwide, currently affecting nearly 10% of the global population and almost half a billion individuals [1,2]. Chronic wounds such as diabetic foot ulcers (DFU) are highly prevalent in patients with diabetes, affecting up to third of patients with diabetes within their lifetime [3]. DFUs are associated with significant morbidity, with DFU preceding nearly 80% of all lower extremity amputations [4]. The 5-year mortality following any amputation in the setting of DFU is approximately 30% and over 70% in the setting of major amputations [5]. Given the need for frequent hospitalizations and costly interventions, diabetic foot ulcers are associated with tremendous costs to the healthcare systems, amounting to more than 13 billion dollars annually in the United States alone [6,7].
There are numerous biologic processes that have been implicated in the impaired wound healing associated with diabetes, including an upregulated inflammatory response, increased levels of oxidative stress, and impaired angiogenesis [8]. The inflammatory response is regulated in part by microRNAs (miRNA), which are small non-coding RNAs that regulate gene expression and have been implicated in the pathogenesis of multiple disease processes including diabetes and impaired wound healing [9,10,11,12]. MicroRNA-146a (miR146a) specifically regulates the nuclear factor κB (NF-κB) pathway, acting as a molecular break to prevent the release of pro-inflammatory cytokines [13]. Our previous studies have demonstrated that diabetic wounds are specifically deficient in miR146a which may contribute to the increased inflammatory state seen in diabetes [14]. Cerium oxide nanoparticles (CNPs) are rare earth metal nanoparticles with an oxidation state that allows them to act as free radical scavengers targeting oxidative stress, and in recent years have been utilized as an antioxidative-based drug delivery system [15,16]. Additionally, CNPs have also been found to induce hypoxia inducible factor (HIF)-1α expression with subsequent upregulation of pro-angiogenic vascular endothelial growth factor (VEGF), contributing to improved angiogenesis [17]. We therefore conjugated CNPs to mir146a to develop CNP-miR146a, a novel conjugate nanoparticle that targets both inflammation and oxidative stress while promoting angiogenesis [18]. We have shown that not only is CNP-miR146a stable as a conjugated particle, but that CNP appeared to have a protective effect on miR146a preventing oxidative damage and degradation [18,19]. Further, we have studied CNP and miR146a independently to evaluate their effects on wound healing and found that the conjugate nanoparticle CNP-miR146a outperformed its individual components [20]. We have previously studied CNP-miR146a as an injectable therapeutic in murine models of diabetic wound healing and demonstrated its efficacy with associated improvement in rates of healing and correction of the previously discussed pathophysiologic derangements seen in diabetic wounds [20,21,22]. We have also specifically demonstrated that injectable CNP-miR146a is associated with both decreased inflammation and improved angiogenesis with histological analysis of CD31 and CD45, respectively, as CD45 is a pan-leukocyte marker that stains white blood cells and acts as a marker of inflammation, while CD31, also known as platelet endothelial cell adhesion molecule-1 (PECAM-1), is involved in cellular endothelial interactions and acts as a marker of angiogenesis. However, the translation of an injectable therapeutic to a clinical setting can be challenging for patients due to pain and need for in-office visits that may result in poor compliance, which could be resolved by developing a topical formulation to deliver CNP-miR146a.
There are currently numerous options to consider for topical delivery of therapeutics. Hydroxyethylcellulose gels (HCG) are commonly used for drug delivery due to its ease of preparation, versatility, and biocompatibility [23]. Pluronic lecithin organogels (PLO) are biomedical polymer-based gels composed of both hydrophilic and hydrophobic components that self-assemble into micelles and act as effective drug carriers that release their contents in a slow, controlled manner [24]. When selecting topical delivery mechanisms, one must also consider the composition and function of the skin barrier. The stratum corneum is composed of protein-rich corneocytes embedded in a lipid matrix that provides a physical barrier to chemical transport of therapeutics through the skin to be pharmacologically efficacious, which poses the challenge to identifying effective topical delivery mechanisms [25,26,27]. The objective of the present study is to identify the optimal topical formulation of CNP-miR146a in a murine model of diabetic wound healing. We hypothesized that topical sustained delivery of CNP-miR146a would improve diabetic wound healing in a murine model, equivalent to intradermal CNP-miR146a, specifically by targeting inflammation and improving angiogenesis.

2. Materials and Methods

2.1. Synthesis of CNP-miR146a

CNPs were synthesized in the previously published method via chemical hydrolysis [28,29]. CNPs were then conjugated to miR146a using a chemical crosslinker 1,1′-carbonyldiimidazole (CDI) to activate the functional hydroxyl groups, instigating an imidazole intermediate state formation that subsequently reacts with the amine group in miR146a and resulting in the conjugate nanoparticle CNP-miR146a [19]. The conjugate nanoparticles were dialyzed in DNAse/RNAse free water and stored in aliquots at −20 °C until used. The physicochemical properties of CNP-miR146a have been previously described [18].

2.2. Hydroxycellulose Gel Formulation

We prepared a batch of 15 mL of HCG according to previously described formulations and preliminary experiments [30]. Briefly, propylene glycol (3% w/w, 0.45 g), glycerin (5% w/w, 0.75 g), methyl paraben (0.17% w/w, 0.0255 g), and propyl paraben (0.02% w/w, 0.003 g) were mixed in a glass scintillation vial. In a separate vial, ethylenediaminetetraacetic acid (EDTA) (0.05%, 0.005 g), monobasic sodium phosphate (0.263%, 0.026 g), dibasic sodium phosphate (0.044%, 0.0075 g), and D-mannitol (0.05%, 0.0075 g) were mixed. The two mixtures were combined and mixed. Hydroxycellulose (Mv~1,300,000; 2% w/w, 0.3 g) was added to the solution. The solution was then diluted to a total volume of 15 mL using sterile water and combined using a stir bar. Before the gel turned viscous, 1 mL aliquots were added to 2 mL centrifuge tubes. To prepare the HCG for the release test as detailed below, 200 µL of fluorescein isothiocyanate (FITC)-tagged CNP (FITC-CNP) was added to the 1 mL aliquot prior to complete gelling and mixed until viscous. HCG for animal studies was not prepared based off the results of the release test as described below to reduce animal and resource waste.

2.3. Pluronic Lecithin Organogel Formulation

We prepared 5 g batches of PLO (Transderma PLO Kit, Xenex Labs, Coquitlam, BC, Canada) according to the manufacturer instructions. Briefly, 3.75 g of the hydrophilic component was combined with 1.25 g of the organic component in a 50 mL sterile beaker and whipped with a sterile spatula until thickened. To prepare the PLO for the release test, 1 mL of FITC-CNP was added to the organic component and mixed prior to the addition of the hydrophilic component. To prepare PLO + CNP-miR146a, 1 mL of FITC-CNP was added to the organic component and mixed prior to the addition of the hydrophilic component. Once mixed, PLO and PLO + CNP-miR146a were aspirated into sterile 1 mL syringes for application to the murine wounds.

2.4. Fluorescein Isothiocyanate-Tagged CNP Gel Release Test

CNPs were conjugated to FITC via the hydrolysis method as described in previously published studies [31,32]. Briefly, A 10 mL aliquot of 5 mM water-based CNP solution was taken in a beaker and combined with 0.5 mL of 2 mg/mL FITC solution prepared in dimethyl sulfoxide (DMSO). The mixture was stirred at room temperature. Then, 10 mL of sodium bicarbonate buffer was added to the FITC-CNP mixture to maintain a stable pH throughout the reaction. The mixture was stirred continuously for one day under dark conditions and then dialyzed with deionized water using a 3.5 kDa dialysis tube for five days to remove any unbound FITC or CNP. FITC-CNP was then incorporated into HCG and PLO as detailed above. Release tests were performed using 24-well plates with Transwell insert membranes. 50 μL of PLO with FITC-CNP (n = 5) or HCG with FITC-CNP (n = 5) were added to the top of the insert membrane. 600 μL of phosphate-buffered saline (PBS) solution was added to the bottom wells. The inserts were placed in the 24-well plate to expose the membrane to the PBS and incubated at 37 °C. Release of FITC-CNP into the PBS was assessed at 15 min, 30 min, one hour, two hours, four hours, six hours, and then daily for seven days. Fluorescence was measured using a BioTek Synergy (Winooski, VT, USA) H1 modular multimode microplate reader (Agilent Technologies, Santa Clara, CA, USA) and plotted as ng released over time.

2.5. Animals

The Institutional Animal Care and Use Committee at University of Colorado Denver Anschutz Medical Campus approved all experimental protocols, which were all compliant with the NIH Guide for the Care and Use of Laboratory Animals. Twelve-week-old female mice homozygous for the Leprdb mutation (db/db) were used for all experiments (BKS.Cg-Dock7m+/+Leprdb/J, strain No. 000642, Jackson Laboratory). Animals were cared for by trained veterinarians and technicians, maintained in standard housing with exposure to 12 h alternating day-night cycles, and fed ad libitum.

2.6. Diabetic Murine Wound Healing Model

Mice were anesthetized using inhaled isoflurane and placed in the prone position. The dorsal skin of each mouse was prepared with depilatory cream which was removed with normal saline and then cleansed with alcohol. An 8 mm punch biopsy (Miltex Inc., York, PA, USA) was used to create a single full-thickness dorsal wound, including the panniculus carnosus. The wounds were then treated with 50 μL of injected PBS, injected CNP-miR146a, empty PLO, or PLO with 1 ng CNP-miR146a (n = 5–6 animals per group). The wounds were covered with Tegaderm (3M, St. Paul, MN, USA) sterile dressings which were removed two days after wounding. After the procedure, each mouse received a weight-based injection of buprenorphine (Schering-Plough Animal Health Corp., Whitehouse Station, NJ, USA) for post-operative analgesia. The mice were observed post-operatively until they had returned to their previous level of activity. The above study was conducted thrice to include three separate timepoints for wound measurements and immunohistochemical analysis as follows: 3 days post-wounding, 7 days post-wounding, and full closure. A total of 70 animals were included in the study to complete the above experiments and no animals were excluded. Animals were randomly selected for treatment groups and individually housed after wounding to prevent confounding.

2.7. Wound Measurement Protocol

Photographs of the wounds were taken every other day for the first week and then daily until fully closed, using a ruler for measurement and scale. The wounds were then analyzed by a blinded observer using ImageJ software (Version 1.53, National Institutes of Health, Bethesda, MD, USA) to calculate the wound area as a percentage of the original wound size. The wound area percentage was plotted over time to calculate closure rates and average day of final closure between groups.

2.8. Immunohistochemical Analysis

Wounds from each treatment group (n = 5–6 per group) were harvested at three days, seven days and full closure. Separate groups of mice were used for each timepoint. The wounds were then immersion fixed in 10% formalin solution for 24 h at room temperature. The tissue was then dehydrated in 70% ethanol prior to embedding in paraffin and sectioning at 5 µm. The sections were mounted on positively charged slides and deparaffinized. The Biocare Medical Decloaker (Pacheco, CA, USA) was used for heat-induced epitope retrieval, and slides were then stained using Leica’s Bond Rx Instrument (Wetzlar, Germany). Slides were treated with primary CD45 and CD31 antibodies (1:50 solution, BD Biosciences, San Jose, CA, USA) followed by development with Vectastain Elite ABC Kit (Vector Laboratories, Newark, CA, USA). Random images along the wound edges of ten high-powered fields (HPF) at 20× magnification were collected for each sample. A blinded observer then quantified the number of CD45- and CD31-positive cells per HPF using an automated counting algorithm on NIS Elements—Advanced Research imaging software (Version 5.42.01, Nikon Instruments, Melville, NY, USA). The ten random images per sample were then averaged and compared between groups.

2.9. Statistical Analysis

Statistical analyses were performed using GraphPad Prism 9.5.1. Outliers were identified and removed using the ROUT method. One-way analysis of variance (ANOVA) with multiple comparisons using post hoc Tukey’s Honestly Significant Difference (HSD) test was used for quantitative data. Two-way ANOVA with post hoc Tukey’s HSD test was used for to study differences in wound closure across time between groups. The following pairwise comparisons: PBS vs. PLO, PBS vs. CNP-miR146a, PBS vs. PLO + CNP-miR146a, PLO vs. CNP-miR146a, PLO vs. PLO + CNP-miR146a, and CNP-miR146a vs. PLO + CNP-miR146a. An alpha value of <0.05 was considered statistically significant.

3. Results

3.1. PLO Undergoes Sustained Release of FITC-CNP

Release of FITC-CNP was used as a representation of how CNP-miR146a would be expected to be released from both HCG and PLO. HCG was found to undergo rapid release, with nearly all of the FITC-CNP released within the first two days of the release test. Conversely, PLO demonstrated a slow, sustained release of FITC-CNP, with complete release not occurring until nearly two weeks after the start of the release test (Figure 1). Based on these results, we selected PLO as the base for our topical formulation to be used in animal studies evaluating the topical application of CNP-miR146a in a diabetic murine model.

3.2. Single Application of CNP-miR146a Accelerates Diabetic Wound Healing

Diabetic murine wounds treated with CNP-miR146a, both as an injectable and as a topical therapeutic delivered via PLO, demonstrated significantly faster healing than those treated with injected PBS or empty PLO. Representative photos of healing wounds from each treatment group are included in Figure 2.
Overall, there were significant differences in wound areas between groups from day 4 until day 15, with mice treated with both injectable and topical CNP-miR146a demonstrating smaller wound areas compared to PBS and empty PLO (Figure 3A). From Days 6 to 9, PLO + CNP-miR146a significantly reduced wound size compared with PBS: Day 6 (85% vs. 125%, p = 0.01), Day 8 (75% vs. 127%, p = 0.003), and Day 9 (68% vs. 106%, p = 0.02). By Day 10, PBS and PLO wounds remained large (85% and 92%), whereas CNP-miR146a and PLO + CNP-miR146a wounds were markedly smaller (53% and 45%), with significant differences versus PLO (p = 0.001 and p < 0.0001) and PBS (p = 0.001 and p < 0.0001). This trend persisted through Day 11 (PLO + CNP-miR146a: 27%; CNP-miR146a: 35%; PLO: 63.7%; PBS: 63.2%), with PLO + CNP-miR146a outperforming both PLO (p = 0.002) and PBS (p = 0.0002) and CNP-miR146a outperforming PBS (p = 0.008). On Day 12, PLO + CNP-miR146a and CNP-miR146a outperformed PBS (p = 0.005 and p = 0.02, respectively; PLO + CNP-miR146a: 27%; CNP-miR146a: 35%; PLO: 42.3%; PBS: 44.4%). This trend persisted on Day 13 for both PLO + CNP-miR146a (8.4%, p = 0.005) and CNP-miR146a (12.7%, p = 0.02) compared to PBS (28.9%). By Day 14, wound size was nearly resolved in both CNP-miR146a–treated groups (injectable: 6%, p = 0.02; topical: 10%, p = 0.01) compared to PBS (19%). When looking at average day to full wound closure, the groups treated with CNP-miR146a closed significantly faster than the control groups, with both injectable and topical CNP-miR146a closing on average by day 14, while empty PG and PBS closed on days 16 and 17, respectively (Figure 3B). Day of final closure was significantly different when comparing PLO + CNP-miR146a to PBS (p = 0.001) and empty PLO (p = 0.01) and when comparing injectable CNP-miR146a to PBS (p = 0.001) and empty PLO (p = 0.01). Injectable and topical application of CNP-miR146a were not significantly different, nor were PBS and empty PLO.

3.3. CNP-miR146a Is Associated with Reduced Inflammation and Improved Angiogenesis

When evaluating CD45 positivity as a marker of inflammation (Figure 4A), groups treated with both injectable and topical CNP-miR146a had significantly less CD45 positivity, indicating lower levels of inflammation, when compared to both PBS and empty PG at three days, seven days, and full closure. On Day 3, PBS and empty PLO-treated wounds demonstrated 143 and 111 CD45+ cells per 20× HPF, while injectable CNP-miR146a and PLO + CNP-miR146a-treated wounds both demonstrated 41 CD45+ cells per 20× HPF. PLO + CNP-miR146a-treated wounds demonstrated significantly lower CD45+ counts compared to both PBS (p < 0.0001) and empty PG (p = 0.001). Injectable CNP-miR146a-treated wounds also demonstrated significantly lower CD45+ counts compared to both PBS (p < 0.0001) and empty PG (p = 0.001) (Figure 4B). On Day 7, PBS and empty PLO-treated wounds demonstrated 141 and 96 CD45+ cells per 20× HPF, while injectable CNP-miR146a and PLO + CNP-miR146a-treated wounds demonstrated 37 and 40 CD45+ cells per 20× HPF, respectively. PLO + CNP-miR146a-treated wounds demonstrated significantly lower CD45+ counts compared to both PBS (p < 0.0001) and empty PG (p = 0.01). Injectable CNP-miR146a-treated wounds also demonstrated significantly lower CD45+ counts compared to both PBS (p < 0.0001) and empty PG (p = 0.008) (Figure 4B. On day of final closure, PBS and empty PLO-treated wounds demonstrated 124 and 110 CD45+ cells per 20× HPF, while injectable CNP-miR146a and PLO + CNP-miR146a-treated wounds demonstrated 20 and 23 CD45+ cells per 20× HPF, respectively. PLO + CNP-miR146a-treated wounds demonstrated significantly lower CD45+ counts compared to both PBS (p = 0.007) and empty PG (p = 0.02). Injectable CNP-miR146a-treated wounds also demonstrated significantly lower CD45+ counts compared to both PBS (p = 0.006) and empty PG (p = 0.02) (Figure 4B).
When evaluating CD31 positivity as a marker of angiogenesis (Figure 5A), groups treated with both injectable and topical CNP-miR146a had significantly higher CD31 positivity, indicating increased angiogenesis, when compared to both PBS and empty PG at three and seven days. On Day 3, PBS and empty PLO-treated wounds both demonstrated 22 CD31+ cells per 20× HPF, while injectable CNP-miR146a and PLO + CNP-miR146a-treated wounds demonstrated 36 and 42 CD31+ cells per 20× HPF. PLO + CNP-miR146a-treated wounds demonstrated significantly higher CD431+ counts compared to both PBS (p = 0.0004) and empty PG (p = 0.0007). Injectable CNP-miR146a-treated wounds also demonstrated significantly higher CD31+ counts compared to both PBS (p = 0.01) and empty PG (p = 0.02) (Figure 5B). On Day 7, PBS and empty PLO-treated wounds demonstrated 26 and 36 CD31+ cells per 20× HPF, while injectable CNP-miR146a and PLO + CNP-miR146a-treated wounds demonstrated 75 and 78 CD31+ cells per 20× HPF, respectively. PLO + CNP-miR146a-treated wounds demonstrated significantly higher CD31+ counts compared to both PBS (p = 0.0004) and empty PG (p = 0.0007). Injectable CNP-miR146a-treated wounds also demonstrated significantly higher CD31+ counts compared to both PBS (p = 0.01) and empty PG (p = 0.02) (Figure 5B. On day of final closure, PBS and empty PLO-treated wounds demonstrated 23 and 34 CD31+ cells per 20× HPF, while injectable CNP-miR146a and PLO + CNP-miR146a-treated wounds demonstrated 42 and 63 CD31+ cells per 20× HPF, respectively. PLO + CNP-miR146a-treated wounds demonstrated significantly higher CD31+ counts compared to PBS (p = 0.007) but there were no other significant differences between groups on day of final closure (Figure 5B).

4. Discussion

In this study, we demonstrate that a single topical application of CNP-miR146a in a PLO base is associated with accelerated wound healing in a diabetic murine model. Subjects treated with both injectable and topical CNP-miR146a healed significantly faster than those treated with injectable PBS or empty PLO. Additionally, subjects treated with both injectable and topical CNP-miR146a demonstrated reduced inflammation as evidenced by reduced CD45 positivity and increased angiogenesis as evidenced by increased CD31 positivity. Finally, there were no differences between injected and topically administrated CNP-miR146a, suggesting that the two are equivalent in their ability to deliver CNP-miR146a in diabetic wounds.
When selecting a vehicle for the topical delivery of CNP-miR146a, our goal was to identify a gel that could be easily formulated, applied, and maintained on the wound bed with subsequent continuous release into the wound to allow ongoing therapeutic activity between applications. We have also explored the use of a topical delivery mechanism using nanosilk, a biomaterial derived from silk fibroin, which was associated with improved wound healing, reduced inflammation, and improved collagen deposition [33], though in this study we wished to explore the use of other gels that are readily available for use in a clinical setting. We therefore considered both HCG and PLO for our formulation, as both are already commonly used for topical delivery of therapeutics. HCG was an appealing option due to its biocompatibility, low cost, and ease of formulation. HCG has demonstrated promising results for the as delivery mechanisms for therapeutics used in wounds such as burns, and most notably, HCG is the base for the only current pharmaceutical topical treatment for DFU [34,35,36,37]. However, HCG can be challenging to maintain on the wound’s surface due to its high solubility, and treatments are released rapidly from HCG so frequent re-applications are necessary. This is consistent with our findings from the gel release test, showing that HCG released CNP almost immediately whereas PLO released CNP in a sustained fashion. Pluronic gel, or Pluronic F127, a non-ionic polymer composed of a polyoxypropylene group flanked on either side by polyoxyethylene groups and with a particularly high concentration of ethylene oxide that makes Pluronic F127 more hydrophilic [24]. Pluronic F127 exists as monomolecular micelles that aggregate into multimolecular collections as their concentration increases, acting as effective carriers of therapeutics. PLO is formed when Pluronic F127 is combined with the organic solvent lecithin to create a micelle-based microemulsion with both an oil phase and an aqueous phase [38]. One challenge of topical administration is the ability to permeate and deliver therapeutics through the dermal and epidermal layers, particularly the stratum corneum, a selective permeability layer that is responsible for preventing water loss in mammals [39]. Due to its amphiphilic nature, PLO is able to permeate the skin barrier by increasing the fluidity of the stratum corneum, leading to an enhanced ability to deliver therapeutics [24]. Other studies have demonstrated that PLO holds promise as a topical delivery mechanism in the setting of skin disease in general and particularly wound healing, with associated accelerations in wound closure and re-epithelization in animal models [40,41]. Further, Pluronic F127, a component of PLO, has been shown to have anti-inflammatory and pro-angiogenic properties when evaluating wound healing in a rodent model, with stimulation of both VEGF and transforming growth factor-beta1 (TGF-β1) [42]. In addition to our confirmatory findings that PLO releases therapeutics in a slow and sustained manner allowing for continued delivery into the wound, the added benefit of inherent anti-inflammatory and pro-angiogenic properties makes PLO an ideal selection for topical administration of CNP-miR146a. Further developments of organogel technology offer interesting future directions for the application of CNP-miR146a. Similar to pluronic gels, Janus hydrogels are composed of a hydrophilic and hydrophobic component that creates a simultaneous barrier while promoting wound healing with promising capabilities in drug delivery [43]. Nanozyme technology, a class of biomaterials with enzyme-like catalytic activity, have also been recently employed specifically in the field of diabetes to target inflammation with promising results [44]. Another gel has been developed also employing nanozyme technology that is specifically activated by glucose levels, and when combined with a ROS-responsive hydrogel, resulted in significant improvements in wound healing with anti-inflammatory and pro-angiogenic activity [45]. The combination of CNP-miR146a with developing gel technologies may provide even more robust effects regarding improved diabetic wound healing and is a direction for future studies.
The identification of suitable topical treatments for DFU has been a long-standing goal within the scientific and medical community, but safe and effective options have been limited [46,47,48]. Currently, the only United States Food and Drug Administration (FDA)-approved topical treatment for DFU is Regranex®, or becaplermin, which is a topical formulation of recombinant human platelet-derived growth factor (PDGF) in a carboxymethylcellulose gel base that was found to result in a significant increase in complete or >90% wound healing of DFU in human participants during phase II clinical trial [37,49]. However, the efficacy of becaplermin has not been well demonstrated in actual clinical practice, and there have been concerns about its association with malignancy for which it previously had a black box warning, though this was removed in 2018 [49,50,51]. The utilization of becaplermin may also be cost prohibitive for many patients, as a single tube currently costs over $1300, and numerous tubes may be needed even for a partial healing response [49,52]. Further, there has not been any additional topical therapeutics that have been approved by the FDA since the approval of becaplermin over 25 years ago [53]. Given the rising incidence of diabetes and associated comorbidities including DFU and the associated cost burden on the medical system, it is imperative to identify alternative treatments. We have previously identified that CNP-miR146a is an affective therapeutic for the treatment of diabetic wounds in a murine model when administered as an injection [20,22]. Further, we have demonstrated the utility of CNP-miR146a in numerous other disease processes that are driven by inflammation, including multiple etiologies of lung injury and colitis [31,54,55,56,57,58]. By developing a therapeutic with benefit across numerous disease processes using easily accessible materials, we aim to create a safe, effective, and financially reasonable option for patients with DFU.
One of the challenges of developing suitable therapeutics in wound healing is the sheer number of potential targets that are involved in the wound healing process [53]. Even in normal wound healing, there is a complex milieu of cellular events that must be tightly coordinated as the wound progresses through hemostatic, inflammatory, proliferative, and remodeling stages of healing [59]. Previous studies have demonstrated the marked alterations in the diabetic inflammatory response during wound healing, including upregulation of pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α as downstream products of the NF-κB pathway, as well as alterations of the cellular phenotype present in healing diabetic wounds including macrophages and fibroblasts [8,60,61,62]. Inflammation in diabetic wounds is further driven by oxidative stress, which is upregulated by hyperglycemia and characterized by abnormally high levels of reactive oxygen species (ROS) that are released from inflammatory cells such as macrophages, also modulated in part by NF-κB signaling [63,64]. Finally, downregulation of angiogenesis further exacerbates poor wound healing in diabetes, driven in part by both oxidative stress and dysfunctional macrophages leading to suppression of VEGF [65,66]. Our conjugate nanoparticle CNP-miR146a was specifically designed with these mechanisms in mind to holistically target the biochemical derangements that drive impaired wound healing in diabetes. There has been a surge in research related to MiRNAs in general due to their ability target and regulate gene expression in a wide range of biologic processes. We specifically selected miR146a due to its role as a molecular brake in the NF-κB pathway and identified deficiencies in diabetic wounds, but studies have also implicated that miR146a plays an additional role by targeting and upregulating angiogenesis via the HIF-1α and VEGF pathways [14,22,67]. The use of CNPs as the carrier for miR146a serves a dual purpose. First, CNPs allow the delivery of miRNAs directly into cellular cytoplasm, which would otherwise be rapidly degraded with poor intracellular uptake in the setting of their negative charge [68,69,70]. Second, CNPs themselves are characterized by dual valency 3+ redox active and 4+ oxidation states which allow the particles to act as effective free radical scavengers [71]. The 3+ state of CNPs also further promotes angiogenesis via modulation of reactive oxygen species and stabilization of endogenous HIF-1α [17]. CNP-miR146a therefore functions as a multi-mechanistic therapeutic that targets the numerous deranged biochemical pathways throughout the entire wound healing process, suggesting that it would offer some benefit regardless of the stage of wound healing at which it was applied.
We have found that CNP-miR146a delivered through a topical PLO gel is associated with significantly improved wound healing with reduced inflammation and increased angiogenesis and is equivalent to intradermally injected CNP-miR146a. This topical delivery strategy may improve patient adoption and compliance by decreasing patient concerns about pain associated with injection, making this more clinically relevant. Topically delivered CNP-miR146a holds significant promise as a potential therapeutic for the treatment of diabetic wounds.

Author Contributions

Conceptualization, B.D.L., K.W.L. and C.Z.; methodology, B.D.L., J.B., A.E.V., A.A., A.S.A., E.K., S.S., K.W.L. and C.Z.; formal analysis, B.D.L. and J.B.; investigation, B.D.L. and J.B.; resources, K.W.L. and C.Z.; writing—original draft preparation, B.D.L.; writing—review and editing, B.D.L., J.B., A.E.V., A.A., A.S.A. S.S., E.K., D.M.J., K.W.L. and C.Z.; supervision, K.W.L. and C.Z.; funding acquisition, B.D.L., D.M.J., K.W.L. and C.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partially funded by T32 training grant 5T32AR007411-35 as well as internal grants awarded to Ken Liechty and Carlos Zgheib from the Department of Surgery at the University of Arizona.

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Review Board of the University of Colorado (protocol code 1149, approved 28 September 2020).

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors upon request.

Acknowledgments

Thank you to Kerri York for administrative support and S. Christopher Derderian for providing laboratory space and resources at the University of Colorado.

Conflicts of Interest

Authors David M. Jackson, Kenneth W. Liechty, and Carlos Zgheib are employed by the company Ceria Therapeutics, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

DFUDiabetic foot ulcer
PBSPhosphate-buffered saline
PLOPluronic lecithin organogel
miRNAMicroRNA
miR146aMicroRNA-146a
NF-κBNuclear factor κB
CNPCerium oxide nanoparticle
HIF-1αHypoxia inducible factor-1α
VEGFVascular endothelial growth factor
HCGHydroxyethylcellulose
CDICarbonyldiimidazole
EDTAEthylenediaminetetraacetic acid
FITCFluorescein isothiocyanate
PECAM-1Platelet endothelial cell adhesion molecule-1
HPFHigh-powered field
ANOVAAnalysis of variance
TGF-β1transforming growth factor-beta1
FDAFood and Drug Administration
PDGFplatelet-derived growth factor
ILinterleukin
TNFtumor necrosis factor
ROSreactive oxygen species

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Pharmaceutics 18 00248 g001
Figure 1. Hydroxyethyl cellulose gel (HCG) is represented by blue circles while pluronic lecithin organogel (PLO) is represented by red squares. Release was measured by fluorescence of FITC-CNP using a fluorescent plate reader. The raw data were used to calculate cumulative nanograms released and plotted over time. HCG released FITC-CNP rapidly over the course of the first day of the test while PLO released FITC-CNP in a slower and sustained manner.
Figure 1. Hydroxyethyl cellulose gel (HCG) is represented by blue circles while pluronic lecithin organogel (PLO) is represented by red squares. Release was measured by fluorescence of FITC-CNP using a fluorescent plate reader. The raw data were used to calculate cumulative nanograms released and plotted over time. HCG released FITC-CNP rapidly over the course of the first day of the test while PLO released FITC-CNP in a slower and sustained manner.
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Figure 2. Diabetic murine wounds were treated once with either PBS, PLO, CNP-miR146a, or PLO + CNP-miR146a and photographed every other day for the first week and then daily until final closure. Representative images from a single subject from each group are displayed here.
Figure 2. Diabetic murine wounds were treated once with either PBS, PLO, CNP-miR146a, or PLO + CNP-miR146a and photographed every other day for the first week and then daily until final closure. Representative images from a single subject from each group are displayed here.
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Figure 3. (A) Diabetic wounds were measured as a percentage of the original wound area and plotted over time, with significant differences occurring between treatment groups from Day 4 until Day 15. (B) Day of final closure was calculated as an average within each group and compared across treatment groups. Wounds treated with CNP-miR146a, both topical and injectable, closed significantly faster than those treated with PBS or empty PLO. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
Figure 3. (A) Diabetic wounds were measured as a percentage of the original wound area and plotted over time, with significant differences occurring between treatment groups from Day 4 until Day 15. (B) Day of final closure was calculated as an average within each group and compared across treatment groups. Wounds treated with CNP-miR146a, both topical and injectable, closed significantly faster than those treated with PBS or empty PLO. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
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Figure 4. Wounds were isolated from diabetic mice at three days, seven days, and full closure, stained with anti-CD45 to evaluate inflammation. (A) Representative slides evaluated at 20× magnification from Day 7 for each treatment group are included. (B) CD45+ and cells were counted in 10 HPFs per sample for each timepoint, averaged, and compared between groups. * = p < 0.05, ** = p < 0.01, **** = p < 0.0001.
Figure 4. Wounds were isolated from diabetic mice at three days, seven days, and full closure, stained with anti-CD45 to evaluate inflammation. (A) Representative slides evaluated at 20× magnification from Day 7 for each treatment group are included. (B) CD45+ and cells were counted in 10 HPFs per sample for each timepoint, averaged, and compared between groups. * = p < 0.05, ** = p < 0.01, **** = p < 0.0001.
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Figure 5. Wounds were isolated from diabetic mice at three days, seven days, and full closure, stained with anti-CD31 to evaluate for angiogenesis. (A) Representative slides evaluated at 20× magnification from Day 7 for each treatment group are included. (B) CD31+ and cells were counted in 10 HPFs per sample for each timepoint, averaged, and compared between groups. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
Figure 5. Wounds were isolated from diabetic mice at three days, seven days, and full closure, stained with anti-CD31 to evaluate for angiogenesis. (A) Representative slides evaluated at 20× magnification from Day 7 for each treatment group are included. (B) CD31+ and cells were counted in 10 HPFs per sample for each timepoint, averaged, and compared between groups. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
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Lyttle, B.D.; Bardill, J.; Vaughn, A.E.; Apte, A.; San Agustin, A.; Kolanthai, E.; Seal, S.; Jackson, D.M.; Liechty, K.W.; Zgheib, C. Topical Delivery of CNP-miR146a via a Pluronic Lecithin Organogel Enhances Diabetic Wound Healing. Pharmaceutics 2026, 18, 248. https://doi.org/10.3390/pharmaceutics18020248

AMA Style

Lyttle BD, Bardill J, Vaughn AE, Apte A, San Agustin A, Kolanthai E, Seal S, Jackson DM, Liechty KW, Zgheib C. Topical Delivery of CNP-miR146a via a Pluronic Lecithin Organogel Enhances Diabetic Wound Healing. Pharmaceutics. 2026; 18(2):248. https://doi.org/10.3390/pharmaceutics18020248

Chicago/Turabian Style

Lyttle, Bailey D., James Bardill, Alyssa E. Vaughn, Anisha Apte, Alyssa San Agustin, Elayaraja Kolanthai, Sudipta Seal, David M. Jackson, Kenneth W. Liechty, and Carlos Zgheib. 2026. "Topical Delivery of CNP-miR146a via a Pluronic Lecithin Organogel Enhances Diabetic Wound Healing" Pharmaceutics 18, no. 2: 248. https://doi.org/10.3390/pharmaceutics18020248

APA Style

Lyttle, B. D., Bardill, J., Vaughn, A. E., Apte, A., San Agustin, A., Kolanthai, E., Seal, S., Jackson, D. M., Liechty, K. W., & Zgheib, C. (2026). Topical Delivery of CNP-miR146a via a Pluronic Lecithin Organogel Enhances Diabetic Wound Healing. Pharmaceutics, 18(2), 248. https://doi.org/10.3390/pharmaceutics18020248

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