Main Research Findings

1) Carbon quantum dots derived from spermidine trihydrochloride were able to be used as nitric oxide donors to promote wound healing

2) Supplementation with spermidine trihydrochloride was shown to have a minimal effect on circulating polyamines, indicating effective homeostatic control and a favorable safety profile.

 

Selected Data
1) This study performed by Tang et al investigated the development and therapeutic potential of nitric oxide-releasing carbon quantum dots (CQDs-NO) synthesized from spermidine trihydrochloride for the treatment of deep partial-thickness burn wounds. The investigators designed a comprehensive experimental strategy that combined nanomaterial synthesis, physicochemical characterization, in vitro cell culture studies, transcriptomic analysis, and an in vivo rat burn model to determine both the biological activity and mechanism of action of the newly developed material. The CQDs-NO nanoparticles were synthesized using a two-step hyperthermia-intermittent ultrasonic method. Briefly, 100 mg of spermidine trihydrochloride was calcined at 275°C for three hours, dissolved in deionized water, subjected to repeated intermittent ultrasonication, centrifuged to remove larger particles, and purified through repeated dialysis using a 1000-Da dialysis membrane. The resulting nanoparticles were characterized using transmission electron microscopy to determine particle size, zeta potential measurements to evaluate colloidal stability, Fourier-transform infrared spectroscopy (FTIR) and mass spectrometry to identify surface functional groups, and X-ray diffraction to determine crystal structure. These characterization techniques confirmed the successful production of nanoscale CQDs with chemical features consistent with nitric oxide-donating functionality [1].
Following nanoparticle synthesis, the investigators examined the biological safety and nitric oxide-releasing properties of CQDs-NO using cultured human umbilical vein endothelial cells (HUVECs). Endothelial cells were maintained in DMEM supplemented with fetal bovine serum and antibiotics under standard cell culture conditions. Nitric oxide release was measured by incubating CQDs-NO alone, CQDs-NO with glutathione peroxidase, or CQDs-NO in the presence of HUVECs. Nitric oxide concentrations were quantified over a 24-hour period using a commercial nitric oxide detection kit. Cytotoxicity was evaluated using both CCK-8 proliferation assays and Live/Dead fluorescence staining after exposure to varying nanoparticle concentrations. To determine whether CQDs-NO influenced endothelial cell migration, scratch wound assays were performed in confluent HUVEC monolayers. Some cultures received the nitric oxide scavenger Carboxy-PITO to determine whether observed effects depended specifically on nitric oxide release. Additional mechanistic studies included Western blot analysis of proteins involved in wound repair, including transforming growth factor-β2 (TGFβ2), heparin-binding epidermal growth factor (HBEGF), and laminin β1 (LAMB1). Protein expression was quantified after 24 hours of nanoparticle treatment with and without nitric oxide inhibition [1].
To further investigate molecular mechanisms, the researchers performed transcriptome sequencing on HUVECs treated with either 1 μg/mL or 5 μg/mL CQDs-NO for 24 hours. Total RNA was isolated, sequencing libraries were prepared using Illumina-compatible methods, and paired-end sequencing was performed. Bioinformatic analyses included Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses to identify biological pathways influenced by CQDs-NO exposure. Genes demonstrating significant differential expression were categorized according to biological processes associated with angiogenesis, extracellular matrix organization, cell adhesion, migration, and wound healing.
The therapeutic efficacy of CQDs-NO was subsequently evaluated using an established rat model of deep partial-thickness burns. Six-week-old male Sprague-Dawley rats were anesthetized, shaved dorsally, and standardized burn injuries were created by applying a copper block heated to 100°C for ten seconds. Animals were randomly assigned to receive either sterile water or topical CQDs-NO solution of 400 μg/mL, which was applied daily with dressing changes throughout the experiment. Digital photographs documented wound closure at 0, 5, 10, 15, and 20 days after injury. At the conclusion of the study, wound tissues were harvested for histological examination using hematoxylin-eosin and Masson’s trichrome staining to evaluate epithelial regeneration and collagen deposition. Immunohistochemical staining for CD31, CD68, and LAMB1 was performed to assess angiogenesis, macrophage infiltration, inflammatory activity, and extracellular matrix remodeling. Statistical analyses were conducted using one-way ANOVA and unpaired Student’s t-tests. This multifaceted experimental design enabled the investigators to comprehensively evaluate nanoparticle biocompatibility, nitric oxide release kinetics, cellular responses, molecular signaling pathways, and functional wound-healing outcomes in vivo [1].
2) This study by Keohane et al was designed as an exploratory randomized, double-blind, placebo-controlled clinical trial to evaluate the safety, tolerability, and effects of a high-purity spermidine trihydrochloride (hpSPD) supplement administered at 40 mg/day in healthy older men. The investigation represented the first human clinical trial of a highly purified spermidine trihydrochloride formulation. The experimental design incorporated two complementary phases: a 7-day randomized crossover phase separated by a washout period of at least two weeks, followed by an additional 21 days of supplementation, creating a total 28-day parallel-group phase. This design allowed investigators to examine both the short-term and longer-term physiological effects of spermidine supplementation while minimizing interindividual variability during the crossover component [2].
Participants consisted of healthy Caucasian men between 50 and 70 years of age with body mass indices ranging from 18.5 to 28 kg/m². Subjects were required to consume relatively low habitual amounts of dietary spermidine of <30 mg/day, as determined using a validated food-frequency questionnaire, and were excluded if they had significant chronic diseases, recent vaccinations, recent antibiotic or steroid use, unstable medication or supplement regimens, chronic anti-inflammatory medication use, smoking history, or allergies to study ingredients. Screening included medical history, physical examination, body weight, body mass index, vital signs, dietary assessment, and review of eligibility criteria. Participants were instructed to maintain their usual diet, exercise habits, and medication use throughout the study while abstaining from alcohol, avoiding vigorous exercise for 24 hours before each study visit, and completing overnight fasts before blood collection. These eligibility criteria were intentionally restrictive to minimize biological variability and improve the sensitivity of safety assessments [2].
Eligible participants were randomly assigned in a 1:1 ratio to receive either hpSPD followed by placebo or placebo followed by hpSPD using a statistician-generated permuted block randomization sequence. Both participants and investigators remained blinded throughout the study. The investigational product consisted of capsules containing 40 mg of spermidine trihydrochloride produced through precision fermentation of an engineered Saccharomyces cerevisiae strain and purified to approximately 86% spermidine trihydrochloride, with the remaining material consisting primarily of sodium chloride and water. Matching placebo capsules contained only inactive excipients but were identical in appearance to preserve blinding. Participants consumed one capsule daily, with or without food, at approximately the same time each morning. Compliance was monitored by capsule counts and participant dosing logs, and consumption rates approaching 100% were required for inclusion in the per-protocol analyses.
A comprehensive panel of clinical, biochemical, and metabolic endpoints was evaluated throughout the intervention. Habitual dietary spermidine intake was quantified using a validated photo-assisted food frequency questionnaire. Blood samples collected after overnight fasting were analyzed for serum spermidine using both enzyme-linked immunosorbent assay and liquid chromatography–mass spectrometry (LC-MS). Additional LC-MS analyses quantified circulating and urinary concentrations of multiple polyamines, including spermidine, spermine, putrescine, ornithine, N-acetyl putrescine, and diaminopropane. Twenty-four-hour urine collections were obtained at each study visit to assess urinary polyamine excretion. Standardized laboratory procedures were used for sample processing, storage, and analysis, including internal standards and calibration curves for LC-MS quantification. Beyond polyamine measurements, investigators assessed body weight, systolic and diastolic blood pressure, fasting blood chemistry, hematology, plasma lipid profiles, and high-sensitivity C-reactive protein (hsCRP) to evaluate systemic safety. All adverse events were documented throughout the study, graded according to severity, and independently evaluated by a study physician for their potential relationship to the intervention [2].
Statistical analyses were performed using SAS software and primarily followed a modified intention-to-treat approach, with additional per-protocol analyses conducted after excluding protocol violations such as poor compliance or prohibited medication use. Continuous outcomes were analyzed using repeated-measures mixed models that accounted for treatment sequence, intervention period, time, and participant-level repeated measurements. Changes in safety laboratory parameters and vital signs were evaluated using Wilcoxon signed-rank tests, while multiple-comparison adjustments were performed using the Benjamini–Hochberg false discovery rate procedure. Sensitivity analyses were conducted after removal of extreme statistical outliers identified using the three-times interquartile range rule. This rigorous methodological framework allowed investigators to comprehensively evaluate the safety profile of high-purity spermidine supplementation while simultaneously examining whether supplementation altered circulating or urinary polyamine homeostasis in healthy older adults [2].

 

Discussion
1) The study completed by the research team of Tang et al demonstrated that the spermidine-derived CQDs-NO nanoparticles possessed favorable physicochemical properties, exhibited minimal toxicity at therapeutic concentrations, continuously released nitric oxide, and significantly enhanced wound healing through multiple complementary biological mechanisms. Characterization studies showed that the synthesized nanoparticles measured approximately 9.9 nm in diameter and exhibited a strongly positive zeta potential, indicating excellent colloidal stability. Transmission electron microscopy confirmed uniform nanoscale morphology, while FTIR and mass spectrometry identified nitrogen-containing functional groups consistent with nitric oxide donor chemistry. X-ray diffraction further demonstrated the amorphous carbon structure expected for carbon quantum dots. Cytotoxicity testing showed that concentrations of 1 and 5 μg/mL produced no measurable toxicity in endothelial cells, whereas exposure to 25 μg/mL significantly inhibited cell proliferation. Live/Dead staining corroborated these findings, indicating that lower concentrations were well tolerated and appropriate for subsequent biological experiments [1].
Nitric oxide release experiments revealed that CQDs-NO functioned as effective sustained-release nitric oxide donors. Only minimal nitric oxide was released when nanoparticles were incubated alone; however, substantially greater nitric oxide production occurred when CQDs-NO were exposed to glutathione peroxidase or co-cultured with HUVECs. During 24 hours of endothelial cell culture, CQDs-NO continuously released nitric oxide in a concentration-dependent manner, indicating that cellular enzymes facilitate gradual nitric oxide liberation from the nanoparticle surface. Functional studies showed that this sustained nitric oxide release significantly accelerated endothelial cell migration during scratch wound assays. Addition of the nitric oxide antagonist Carboxy-PITO markedly reduced this migratory response, demonstrating that nitric oxide release was directly responsible for the enhanced wound closure observed in cultured endothelial cells. Western blot analyses further demonstrated significant upregulation of TGFβ2, HBEGF, and LAMB1 proteins following CQDs-NO treatment, while nitric oxide inhibition largely abolished these increases, confirming that nitric oxide signaling mediated these molecular responses [1].

 

Figure 1: Changes in the concentration of nitric oxide across the various experimental groups.
Transcriptomic analysis provided additional mechanistic insight into the regenerative effects of CQDs-NO. Gene Ontology enrichment analyses demonstrated widespread upregulation of genes involved in angiogenesis, extracellular matrix organization, cell-substrate adhesion, positive regulation of cell migration, and wound healing. Numerous genes central to tissue repair, including TGFB2, HBEGF, LAMB1, FN1, THBS1, COL18A1, ITGAV, SPARC, LOXL2, MMP2, and MCAM, were significantly increased following nanoparticle treatment. These transcriptional changes suggested that CQDs-NO activates multiple coordinated biological pathways rather than a single molecular target. TGFβ2 emerged as one of the most consistently enriched genes across several regenerative pathways, supporting its central role in coordinating extracellular matrix production, angiogenesis, and tissue remodeling. Together, these findings demonstrated that CQDs-NO promotes a broad regenerative transcriptional program capable of enhancing multiple aspects of wound repair simultaneously.
The in vivo experiments confirmed that these molecular and cellular changes translated into substantial improvements in burn wound healing. Rats treated with topical CQDs-NO exhibited faster removal of necrotic tissue, earlier epithelialization, and more rapid overall wound closure compared with control animals. Histological examination demonstrated significantly thicker regenerated epidermis together with increased collagen deposition within the dermis, indicating accelerated tissue reconstruction and extracellular matrix formation. Immunohistochemical analyses further showed significantly increased CD31-positive blood vessels, reflecting enhanced angiogenesis, along with elevated LAMB1 expression, consistent with improved basement membrane formation and cell adhesion [1].
At the same time, CQDs-NO treatment significantly reduced CD68-positive macrophage infiltration, suggesting attenuation of excessive inflammatory responses within healing wounds. Collectively, these findings indicate that CQDs-NO derived from spermidine trihydrochloride accelerates burn wound healing through sustained nitric oxide release that simultaneously promotes vascularization, endothelial migration, extracellular matrix deposition, epithelial regeneration, and controlled inflammatory resolution. The authors concluded that this nanomaterial represents a promising nitric oxide donor with potential therapeutic applications for enhancing healing of deep partial-thickness burns while providing a stable, biocompatible, and easily synthesized platform for regenerative medicine [1].
2) The study by Keohane et al demonstrated that 40 mg/day of hpSPD was safe, well tolerated, and produced minimal effects on circulating polyamine concentrations and systemic clinical biomarkers in healthy older men. A total of 57 individuals were screened, of whom 38 met the eligibility criteria and were randomized into the study. Thirty-seven participants completed the 7-day crossover phase and were included in the modified intention-to-treat analysis, while 37 participants also contributed data to the 28-day parallel phase after one additional withdrawal for personal medical reasons unrelated to the intervention. Participant compliance was exceptionally high, averaging 100.7% during the crossover phase and 99.5% during the parallel phase, indicating that the supplementation regimen was readily tolerated and adhered to throughout the study. Baseline characteristics, including body mass index, body weight, blood pressure, and habitual dietary spermidine intake, were similar between treatment groups before supplementation began [2].
Analysis of serum polyamine concentrations during the 7-day crossover phase showed that hpSPD supplementation produced no statistically significant changes in circulating spermidine concentrations when measured using either enzyme-linked immunosorbent assay or LC-MS. Likewise, serum concentrations of other measured polyamines, including diaminopropane, N-acetyl putrescine, ornithine, putrescine, and spermine, remained largely unchanged compared with placebo. Although a modest reduction in serum spermine was detected in the per-protocol analysis, this finding was not consistently observed in the primary modified intention-to-treat analysis and therefore was not considered evidence of a meaningful alteration in systemic polyamine metabolism. These findings indicated that even relatively high-dose supplementation with purified spermidine did not substantially increase circulating polyamine concentrations over a short-term intervention period, suggesting that endogenous regulatory mechanisms effectively maintain polyamine homeostasis despite increased dietary intake.
Urinary polyamine analyses produced similar findings. Most urinary polyamine concentrations remained unchanged after hpSPD supplementation, whereas several urinary polyamines decreased modestly following placebo administration. A statistically significant interaction was observed for urinary spermine, with concentrations increasing slightly following hpSPD while decreasing after placebo, although the magnitude of this difference remained small. Similarly, urinary spermidine showed a slight tendency to remain stable with hpSPD while declining after placebo. Despite these isolated observations, the overall pattern indicated minimal changes in urinary polyamine excretion, supporting the conclusion that supplementation with purified spermidine did not overwhelm normal metabolic regulation or produce substantial alterations in polyamine turnover. Body weight, systolic and diastolic blood pressure, high-sensitivity C-reactive protein, plasma lipid concentrations, clinical chemistry measurements, and hematological parameters likewise remained unchanged throughout the crossover period, demonstrating no evidence of clinically significant physiological disturbances after one week of supplementation [2].

 

Figure 2: Changes in urine polyamines following supplementation with spermidine trihydrochloride, measured by LC-MS
Results from the 28-day parallel phase reinforced the findings observed during the shorter crossover intervention. After four weeks of supplementation, there were still no significant differences between hpSPD and placebo groups in serum spermidine concentrations measured by either ELISA or LC-MS. Likewise, serum concentrations of the additional polyamines measured by LC-MS remained stable throughout the intervention. Urinary polyamine analyses similarly demonstrated no significant treatment-related changes except for a minor statistical interaction involving urinary spermine that did not correspond to significant within-group differences. Clinical safety assessments again demonstrated remarkable stability across treatment groups. Body weight, blood pressure, inflammatory markers, lipid profiles, blood chemistry, and hematological indices all remained within normal ranges and did not differ between hpSPD and placebo. These findings indicated that prolonged supplementation with purified spermidine did not adversely affect cardiovascular, metabolic, hepatic, renal, inflammatory, or hematologic function in this population of healthy older men.
Evaluation of adverse events further supported the favorable safety profile of hpSPD. Across both intervention phases, 13 adverse events were reported by ten participants, including eight mild and five moderate events. Four events occurred before treatment initiation or during the washout period, and none of the reported adverse events were judged by study physicians to be related to hpSPD supplementation. Only three adverse events occurred while participants were actively receiving hpSPD, and all resolved without discontinuation of treatment. No participant withdrew because of treatment-related side effects. Comparison of ELISA and LC-MS methods further revealed that ELISA generally produced higher estimates of circulating spermidine than LC-MS, although the two analytical methods were poorly correlated, emphasizing methodological differences in polyamine quantification rather than biological changes. Overall, the investigators concluded that 40 mg/day of high-purity spermidine trihydrochloride administered for up to 28 days is safe, well tolerated, and produces minimal disruption of circulating or urinary polyamine homeostasis, supporting continued clinical investigation of purified spermidine as a potential nutritional intervention for healthy aging and related applications [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Tang T, Liu Y, Wang P, et al. Carbon quantum dots as a nitric oxide donor can promote wound healing of deep partial-thickness burns in rats. Eur J Pharm Sci. 2023;183:106394. doi:10.1016/j.ejps.2023.106394

[2] Keohane P, Everett JR, Pereira R, Cook CM, Blonquist TM, Mah E. Supplementation of spermidine at 40 mg/day has minimal effects on circulating polyamines: An exploratory double-blind randomized controlled trial in older men. Nutr Res. 2024;132:1-14. doi:10.1016/j.nutres.2024.09.012

 

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