Development of a quadruple-conjugated carbon dot nanomodel for targeted glioma therapy

Cilingir, Emel Kirbas; Hettiarachchi, Sajini D.; Rathee, Parth; Zhou, Yiqun; Ferreira, Braulio CLB; Wang, Lukun; Joji, Annu; Gonzalez, Carlos M.; Moreno Hollweg, Maria J.; Shiri, Mehrdad; Wang, Kun; Prabhakar, Rajeev; Vanni, Steven; Leblanc, Roger M.; Graham, Regina M.

doi:10.1038/s42004-026-01900-3

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Published: 30 January 2026

Development of a quadruple-conjugated carbon dot nanomodel for targeted glioma therapy

Communications Chemistry volume 9, Article number: 96 (2026) Cite this article

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Abstract

High-grade gliomas are devastating cancers with dismal prognosis, largely because current chemotherapeutics fail to cross the blood–brain barrier and lack tumor-cell specificity. Nanotechnology aims to overcome these limitations through targeted drug delivery. Here, a quadruple-conjugated nanomodel was synthesized using carbon dots (C-dots) as biocompatible nanocarriers via a one-pot reaction that covalently links two targeting peptides and two anticancer agents. The short peptide (shPep-1) targets the tumor-restricted receptor IL13Rα2, whereas the long peptide (lnPep-1) contains a nuclear localization signal for enhanced intracellular trafficking. Therapeutic cargo consists of epirubicin and the temozolomide metabolite 5-aminoimidazole-4-carboxamide. This nanomodel displays potent cytotoxicity in multiple high-grade glioma cell lines at 50 nM while remaining relatively non-toxic to normal cells (IC₅₀ > 2 µM). Despite a lower drug-loading capacity than single-peptide formulations, it induced greater glioma cell death, underscoring the enhanced therapeutic synergy of its dual-peptide, dual-drug design. Fluorescence studies confirm superior uptake and nuclear delivery, establishing C-dots as a stable, cost-effective, modular platform for next-generation personalized cancer nanotherapies.

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Introduction

Gliomas are the most common primary brain tumors. Patient outcomes with high-grade gliomas, including glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), remain poor. Despite the standard of care consisting of surgery followed by radio- and chemotherapy, the median survival rate of GBM patients is only 14.6 months, with a five-year survival rate of only 10%^1,2. The survival rate for pediatric GBM, which is distinct from adult GBM, ranges from 13 to 73 months with a 5-year survival of less than 20%³. Whereas the median survival DIPG patients is approximately 10 months⁴. Therefore, there is a great need for novel approaches and optimized therapies to target these cancers.

One approach utilizes the incorporation of nanomaterials to develop novel nanoscale strategies for diagnosis and treatment. Although numerous nanoparticle-based drug delivery systems have been investigated, many fail due to the lack of target specificity, larger particle size, short circulation times, and high immune toxicity from the precursors used to synthesize the nanoparticles⁵. Furthermore, it is estimated that only limited amounts (0.7%) of the administrated nanocarriers actually accumulate in solid tumor tissues⁶.

Over the past two decades, carbon dots (C-dots) have emerged as a promising nanoparticle for biomedical applications^7,8. C-dots' unique characteristics include excellent biocompatibility, tunable photoluminescence, excellent water dispersibility, ease of production, and resistance to photobleaching are among the unique characteristics of C-dots^9,10. C-dots have been employed as drug delivery systems (DDS) to enhance drug solubility, increase bioavailability, and accumulation within the tumor, while reducing off-target effects^11,12,13,14. C-dot mediated drug delivery to high-grade gliomas has been demonstrated in both in vitro and in vivo studies^14,15,16,17. Here, we sought to build upon these findings with the goal of developing an advanced C-dot quadruple conjugated nanomodel. Ideally, the C-dot nanomodel should specifically or at least preferentially target tumor cells and efficiently deliver anti-cancer drug(s) into the cell and to the correct intracellular organelle for maximum cancer cell cytotoxicity.

Some of the most effective anti-cancer drugs are anthracyclines such as doxorubicin and epirubicin. Anthracyclines inhibit RNA and DNA synthesis by inserting between adjacent nucleotide base pairs, as well as by forming a tertiary complex with topoisomerase II, thereby preventing double-strand DNA break repair. For optimal cancer cytotoxicity, anthracyclines should be delivered to the cell nucleus, the site of RNA and DNA synthesis, and topoisomerase II activity. An organelle-targeted nanoparticle that can deliver the cargo from the cell membrane to the desired intracellular location would significantly increase the drug effectiveness, thereby reducing the overall therapeutic dose needed and off-target effects.

IL13Rα2 is a tumor-restricted receptor, which is a promising target for high-grade gliomas as well as many other highly aggressive tumors, including melanoma, renal cell carcinoma, and ovarian cancer, among others^18,19,20. Elevated IL13Rα2 expression has been observed in both adult and pediatric GBM as well as DIPG^21,22,23,24. Further, this receptor has been successfully targeted by drug delivery systems using surface-functionalized nanocarriers^25,26. Several IL13rα2 targeting peptides have been investigated for both diagnostic and therapeutic purposes. Peptides have been gaining momentum as cell-targeting moieties owing to their small size, ease of synthesis, and attractive pharmacokinetic profiles. Compared to proteins or antibodies, peptides are less immunogenic and are better able to penetrate and diffuse throughout the tumor tissue^27,28. A short linear 9 amino acid peptide (CGEMGWVRC) has been used to identify IL13Rα2 expressing cells and effectively deliver anti-cancer agents by receptor-mediated endocytosis^29,30,31,32. When injected intravenously into mice, this peptide homed to IL-13Rα2-expressing tumors in subcutaneous as well as intracranial models of GBM, suggesting the ability to cross the blood-brain tumor barrier (BBTB)³¹. Importantly, paclitaxel-loaded copolymer nanoparticles significantly reduced tumor volume and increased survival in mice bearing intracranial GBM tumors compared to non-targeted nanoparticles or free drug³². Similarly, cell-penetrating peptides have been widely shown to deliver anti-cancer agents across cell membranes; however, a major drawback is the lack of cell specificity³³. Despite this, cell-penetrating peptides can be easily coupled to nanomaterials to increase cell uptake³⁴. A 21-amino acid-long peptide (KETWWETWWTEWSQPKKKRKV), which was designed with a nuclear localization sequence, can facilitate not only cell membrane penetration but also nuclear delivery³⁵.

The overarching goal of our research is to develop more effective drug delivery systems using C-dots as the nano-carrier. Therefore, we employed a dual peptide system for enhanced tumor targeting and cancer cell drug delivery. We constucted a quadruple nanomodel using (1) a short peptide (shPep-1) capable of traversing the BBTB and specifically targeting high-grade gliomas, (2) a longer peptide (lnPep-1) to assist in cell uptake and nuclear cargo delivery, (3) a highly effective anti-cancer drug, epirubicin as well as (4) the small molecule 5-aminoimidazole-4-carboxamide (AIC), which we previously observed to increase the efficacy of epirubicin in a triple conjugated C-dot model¹⁴. In our previous work, we constructed a triple conjugated C-dot model using the protein transferrin as the targeting ligand, epirubicin, and the small molecule AIC. We found that combined C-dot delivery of epirubicin and AIC induced significantly more brain tumor death than either C-dot delivery of epirubicin or AIC alone¹⁴. Temozolomide is the most commonly used chemotherapy to treat GBM, however, at physiological pH, it metabolizes to 5(-3methyltriazen-1-yl) imidazole-4-carboxamide, which is then degraded to AIC and the methyldiazonium cation, which transfers the methyl group to the DNA, causing DNA damage.

The efficacy of the quadruple nanomodel was explored through in vitro cell viability studies in both cancer and non-cancer cell lines. Furthermore, the efficacy of the quadruple nanomodel was compared to single peptide models in the cancer cell lines, and the contribution of each peptide on nanoparticle cell uptake and nuclear localization was determined with bioimaging studies. Our results demonstrate that the quadruple nanomodel is superior to single peptide conjugate models in promoting high-grade glioma cell death, which was primarily due to enhanced nanoparticle tumor cell uptake and nuclear localization. A schematic for the synthesis of the quadruple and single peptide (triple) nanomodels is shown in Fig. 1.

**Fig. 1: Schematic representation of the synthesis of the triple C-dot conjugates with lnPep-1 and shPep-1 and the quadruple C-dot conjugate.**

Results

The peptides and small molecules were covalently conjugated onto C-dots via EDC/NHS coupling. Synthesis of the quadruple nanomodel and the reference single peptide conjugates was conducted as one-pot reactions. Carboxylic acid functionalized carbon dots were linked to each small molecule or peptide through the amide bond. The successful conjugation of each molecule on the C-dots was determined by the analytical techniques described below.

Physiochemical characterization of C-dots-shPep-1-lnPep-1-epi-AIC (Quadruple nanomodel)

Ultraviolet–visible spectroscopy (UV-vis) spectra in Fig. 2A, the red dotted lines at 496 and 230 nm exhibited the successful conjugation of epirubicin and the peptides on C-dots, respectively. The corresponding 230 nm peak denotes the tryptophan amino acid present in both peptides. Whereas the prominent peak at 330 nm in the temozolomide spectrum (purple) was absent in the quadruple conjugate nanomodel spectrum (black). Due to the structural rearrangement of temozolomide into aminoimidazole-4-carboxamide (AIC)³⁶. During the rearrangement of temozolomide to AIC, the N=N on temozolomide disappears, which hinders the π-π* transition represented by the 330 nm peak. Thus, it indicates that temozolomide dissociates into the AIC form during the conjugation process at pH 7.4, as has been reported³⁷.

**Fig. 2: Spectroscopic and mass spectrometric characterization of the quadruple nanomodel (C-dots-shpep-1-lnpep-1-epi-AIC).**

In addition to the UV-vis spectroscopy, the fluorescence spectroscopy data also revealed the successful conjugation of the peptides and epirubicin on C-dots. The excitation-independent emission bands at 350 and 556–591 nm on the quadruple nanomodel fluorescence emission spectrum exhibit the tryptophan emission of the peptides and epirubicin, respectively (Fig. 2B). The verification of each peptide binding on the C-dots was challenging to reveal by UV-vis or fluorescence spectroscopy.

Fourier-transform infrared spectroscopy (FTIR) spectral analysis, however, suggested the presence of all four ligands on the quadruple nanomodel (Fig. 2C). The four ligands were covalently conjugated to C-dots via -COOH groups of C-dots and -NH2 groups of ligands, resulting in the formation of the peptide (C-N) bond. The AIC structure possesses a prominent C=N imide stretching at 1662 cm⁻¹, whereas, at 1662 cm⁻¹, a C=N imide stretching appeared barely on the quadruple nanomodel, confirming the presence of AIC molecules. The appearance of a prominent N-H bending peak at 1608 cm⁻¹ supports the presence of two peptides on the C-dots. The appearance of a significant C-N stretching peak at 1320 cm⁻¹ supports the fact that the successful conjugation of two peptides on C-dots. Furthermore, alkene C=C bending peaks from the epirubicin appear at the 976 cm⁻¹ region of the quadruple conjugated C-dots’ spectrum. Finally, AIC’s C=C bending alkene peak is present at the 936 cm⁻¹ region. 1438.

Lastly, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis was conducted for the recognition of each peptide on the quadruple nanomodel. Because the molecules over 1000 Da are amenable to detection by MALDI-TOF, the binding of each peptide on C-dots can be distinguished by MALDI-TOF. The two peptides were displayed in two different m/z paradigms (Fig. 2D). The shPep-1, consisting of only 9 amino acids, indicated the molecular ion peak at 1040, whereas the lnPep-1, which contains 21 amino acids, displayed the molecular ion peak at 2848. The same two peaks were observed in the quadruple nanomodel MALDI-TOF spectrum, confirming the successful conjugation of each peptide on C-dots (Fig. 2E). The peak at 2077 m/z may represent a dimer formed during the ionization process, which is commonly observed in MALDI-TOF analyses of peptide mixtures. Specifically, peptides with cysteine residues like shPep-1 can form disulfide bonds, which result in a larger mass, less the mass of the hydrogens lost in bond formation. Since the quadruple nanomodel includes additional ligands, it’s possible that the 3136 m/z peak could represent a peptide-ligand adduct or matrix-peptide cluster. Despite this, MALDI-mass spectroscopic analysis remains a promising technique to distinguish the two peptides of the quadruple nanomodel.

Morphological and functional analysis

The morphology of the naked C-dots and the quadruple nanomodel was determined using Atomic force microscopy (AFM). The AFM profiles of over 300 bare C-dots and quadruple nanomodel C-dots were analyzed. Fig. 3A (bare c-dots) and 3B (quadruple nanomodel) show the individual C-dots with the height profiles. The histogram and the fitted Gaussian curve for the height distribution obtained by AFM imaging are shown in Fig. S1. The peak of the Gaussian fit and standard deviation is 1.41 ± 0.18 nm for bare carbon dots and 2.42 ± 1.25 nm for quadrupole carbon dots. The increased size of the quadruple confirms conjugation of the peptides and chemotherapeutic drugs on the C-dots.

**Fig. 3: Size, morphology, and DNA-binding properties of C-dot conjugates.**

To assess the physical interactions of C-dots on DNA, plasmid pUC19 DNA was incubated with the C-dots alone or with the non-peptide C-dot conjugates and was analyzed by agarose gel electrophoresis. As shown in Fig. 3C, neither the C-dots alone nor the C-dots-AIC conjugate changed the mobility of the DNA when compared to the DNA only lane; however, both the C-dots-epi and C-dots-epi-AIC slowed the migration of the DNA, similar to the epirubicin intercalation control. These results demonstrate that epirubicin DNA-binding functionality is preserved following nanoparticle conjugation, supporting the feasibility of the C-dot platform for nucleic acid–targeted delivery.

Drug loading content

Prior to determining the effects of the C-dots on cell viability, the loadability of epirubicin was calculated in each conjugate. As previously mentioned, temozolomide undergoes a structural rearrangement at pH 7.4, producing AIC³⁷. The loaded epirubicin concentration in each C-dot conjugate was analyzed by UV-vis spectroscopy. As shown in Fig. 2A, the prominent excitation wavelength of epirubicin is at 487 nm. Thus, the epirubicin calibration curve was plotted to analyze the epirubicin concentration of each conjugate in 60 mg L⁻¹. The percent drug loading content (DLC%) was calculated according to the equation below.

$${{{\boldsymbol{DLC}}}}{{{\boldsymbol{ \% }}}}=\frac{{Amount}\,{of}\,{epirubicine}\,{conjugate}d\,{onto}\,C-{dots}}{{Total}\,{amount}\,{of}\,{the}\,{conjugate}}\times 100$$

Interestingly, the lowest epirubicin content of 23.3% was found in the quadruple nanomodel (C-dots-lnPep-1-shPep-1-epi-AIC). The single peptide conjugates C-dots-shPep-1-epi-AIC and C-dots-lnPep-epi-AIC exhibited 61.6% and 66.6% DLC, respectively (Table 1). These results demonstrate that epirubicin drug loading onto the quadruple conjugated C-dots is less efficient than the reference conjugate samples with only one peptide.

Table 1 Epirubicin drug loading content (DLC) in conjugates

Full size table

In vitro results

IL13Rα2 expression

Although IL13Rα2 expression has been demonstrated in high-grade gliomas²⁴, we confirmed IL13Rα2 expression in our cancer cell lines by Western blot assay. IL13Rα2 protein expression levels were observed in all three cancer cell lines, whereas minimal IL13Rα2 expression was observed in the non-cancer cell line, H35 (Supplementary Fig. 2).

Biological activity

Next, we tested the cytotoxicity of the quadruple conjugate C-dots-shPep-1-lnPep-1-epi-AIC and the reference triple conjugates (C-dots-lnPep-1-epi-AIC; C-dots-shPep-1-epi-AIC) against the high-grade glioma cell lines SJ-GBM2, U87, and NP53. At concentrations as low as 50 nM, the quadruple conjugate drastically reduced cell viability in the adult GBM cell line, U87, pediatric GBM cell line, SJ-GBM2, and the DIPG cell line, NP53, to 58%, 54%, and 51%, respectively, of non-treated controls (Fig. 4A–C). Furthermore, the quadruple nanomodel induced significantly more cell death when compared to the single peptide C-dot conjugates, particularly at the lower concentrations. At the higher concentrations, robust cell death was observed for all conjugates tested. Lastly, as shown in Fig. 4D, the quadruple nanomodel was much less toxic to the non-cancer cell line, vascular smooth muscle cells (VSMC) with an IC₅₀ value of 2465 nM compared to 87.3, 64.2 and 63.1 nM for U87, SJ-GBM2 and NP53 cells, respectively (Fig. 4E). In addition, for both U87 and SJ-GBM2 cells, the IC₅₀ values were significantly lower for the quadruple compared to the single peptide reference conjugates. For the NP53 cells, the Ic₅₀ of the quadruple did not quite reach significance when compared to the lnPep-1 conjugate (p = 0.059). Interestingly when comparing the IC₅₀ values of the shPep-1 conjugates across the cell lines (one-way ANOVA revealed a significant effect of treatment [F(2, 16) = 9.76, p = 0.0017, η² = 0.55), the IC₅₀ of the shPep-1 conjugate was significantly lower for NP53 cells than that for U87 cells (p < 0.002) and SJ-GBM2 cells (P < 0.014) potentially suggesting high IL13Rα2 activity (cycling) for the DIPG cell line.

**Fig. 4: Cytotoxicity and selectivity of the quadruple-conjugated C-dot nanomodel in high-grade glioma cell lines.**

As detailed above, epirubicin drug loading was significantly reduced in quadruple nanomodel than for the single peptide conjugates. Therefore, the prominent anticancer effect of the quadruple nanomodel versus the single peptide conjugates is most likely due to enhanced C-dot cell uptake and nuclear drug delivery.

Cell uptake and nuclear localization analysis

To explore the cellular uptake and intracellular trafficking of the peptide conjugates, C-dots were conjugated to the fluorescent dye 5-(aminomethyl) fluorescein hydrochloride (5-Amfl), followed by conjugation to either shPep1, lnPep1, or both peptides. The cellular internalization and nuclear localization of each conjugate were investigated using fluorescent microscopy. SJ-GBM2 cells were treated for 1 h with the dual peptide conjugate, C-dots-5-Amfl -shPep-1-lnPep-1, and the reference conjugates of C-dots-5-Amfl -shPep-1, C-dots-5-Amfl -lnPep-1, or C-dots-5-Amfl. The dual peptide conjugate C-dots-5-Amfl -shPep-1-lnPep-1 (shown in green) displayed robust fluorescent intensity throughout the cells, including the nucleus. 5-Amfl and DAPI overlay also demonstrate strong nuclear localization of the dual peptide conjugate compared to the single peptide conjugates (Fig. 5E and F4). The C-dots-5-Amfl -lnPep-1 displayed the second-highest fluorescent intensity both in the cytoplasm and the nucleus (Fig. 5D and F3). The lnPep-1 is the cell-penetrating peptide that rapidly enters cells and delivers cargo to the nucleus, consistent with the observation of a higher localization of the C-dots-5-Amfl -lnPep-1 conjugate in the nucleus compared to the non-targeted C-dots-5-Amfl conjugate³⁸. Moreover, the C-dots-5-Amfl -lnPep-1 conjugate demonstrated overall cellular fluorescence at higher levels compared to C-dots-5-Amfl -shPep-1 and the non-targeted C-dots-5-Amfl conjugates. The lower level of cellular fluorescence observed in the C-dots-5-Amfl -shPep-1 cells (Fig. 5C and F2) may be due to the short incubation time. It has been demonstrated that when shPep-1 was incubated with GBM cells, the highest peptide internalization took place at 4 hours post exposure³¹. Further, line-scanning profile overlap of C-dots-5-Amfl -shPep-1 conjugate with Hoechst indicated less nucleus colocalization and more cytoplasm localization, confirming that the shPep-1 facilitates mainly cellular internalization (Fig. 5C, F2). Of note, the non-treated control group demonstrated no autofluorescence (Fig. 5A, F1).

**Fig. 5: Analysis of cell uptake and nuclear location of C-dot conjugates.**

Lastly, 100 cells from each treatment group were analyzed to determine Pearson’s correlation coefficient (PCC), which provides information about the degree of colocalization of C-dots and the nucleus. If the results of PCC indicate as +1 it represents the perfect correlation, 0 for no correlation, and −1 for perfect anti-correlation. The strongest nuclear correlation was observed for the quadruple conjugate, significantly higher than C-dots-5-Amfl, C-dots-5-Amfl-shPep-1, C-dots-5-Amfl-lnPep-1 (Fig. 5, F5), which agrees with the line-scanning profile analysis. Additionally, a strong nucleus colocalization correlation for the quadruple can also be seen from the 2D intensity histogram graphs (Supplementary Fig. 3). Therefore, it is likely that the combination of the two peptides increases both cellular uptake and nuclear localization, facilitating enhanced epirubicin cell killing.

Discussion

Nanomedicines offer hope for more effective cancer treatment by selectively targeting tumor cells, delivering effective anti-cancer agents at therapeutic levels, and minimizing toxic side effects. However, for aggressive tumors, it is unlikely that there will be a “one size fits all” nanomedicine. In the age of precision or personalized medicine, nanomedicines need to be cost-effective and easily and quickly synthesized to fulfill the demand for individualized therapy. C-dots can be synthesized from inexpensive, non-toxic reagents, and the one-pot synthesis process used in this study for C-dot nanomedicines is attractive as it eliminates the need for lengthy separation and purification of intermediate chemical compounds. As a result, one-pot synthesis method saves time and resources by enhancing the efficiency of C-dot conjugations, leading to greater yields.

Our quadruple nanomodel recognizes high-grade glioma brain tumors, including GBM and DIPG, by specifically targeting IL-13Rα2 present on the cell membrane. The shPep-1 peptide recognizes and binds to the IL-13Rα2 to induce receptor-mediated endocytosis, whereas the lnPep-1 peptide facilitates nucleus membrane permeability while enhancing cellular internalization. The significant anti-cancer efficacy of the quadruple nanomodel over the single peptide C-dot conjugates was demonstrated in vitro. Further, this effect was due to the presence of both peptides on the C-dot conjugate, rather than increased drug loading emphasizing the importance of cell and organelle targeting.

The IL13Rα2 targeting peptide (CGEMGWVRC) used for this study was previously shown not only to home to high-grade glioma tumors in vivo but also to cross the BBTB; however, the mechanism for this is unclear. Nevertheless, multiple studies demonstrate that peptide-targeted IL13eRα2 nanoparticles can decrease tumor size and prolong survival in orthotopic mouse high-grade glioma models^26,32,39. In addition, high expression of IL13Rα2 on breast cancer brain metastasis (BCBM) has been shown to correlate with worse prognosis, promoting cell proliferation and metastasis⁴⁰. Therefore, in addition to high-grade gliomas, IL13Rα2 is a promising target for drug delivery to BCBM as well as other cancers demonstrated to express increased levels of the receptor.

The use of C-dots as the nanocarrier for drug delivery has several advantages over other drug delivery systems. Non-toxic C-dots are inexpensive to synthesize, very easy to modify with multiple ligands, and importantly, the synthesis reactions can be scaled up to produce the necessary quantities for patient treatment. The high surface-to-volume ratio and the abundance of surface functional groups allow for high density of targeting peptides, thereby increasing the efficiency of the targeting process, enabling more precise and effective interactions with specific biological targets⁴¹. The enhanced surface area provided by C-dots allows for more customizable functionalization, improving their potential as targeted delivery systems in biomedical applications. These highly versatile nanoparticles can be easily and quickly modified to target a variety of cellular targets, pass through biological barriers, and deliver multiple drugs. GBM is a highly heterogeneous tumor, and it is unlikely that a single nanoparticle formulation will prove beneficial to all patients. Therefore, the ability to easily and quickly synthesize more personalized nanoformulations at a low cost should ultimately prove beneficial to the patients and the healthcare system in general.

Methods

Materials

lnPep-1 (KETWWETWWTEWSQPKKKRKV) was purchased from ABIOTIC (Escondido, CA). shPep-1 (CGEMGWVRC) was purchased from BiomatiK USA, LLC (Wilmington, DE). Epirubicin hydrochloride, temozolomide, N-hydroxy succinimide (NHS), 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), and 5-(aminomethyl) fluorescein hydrochloride (5-Amfl) were purchased from Sigma Aldrich (St Louis, MO). SYBR Green Nucleic Acid Gel Stain and agarose were obtained from ThermoFisher Scientific (Waltham, MA). The plasmid pUC19 was obtained from New England BioLabs (Ipswich, MA). Dialysis tubing with molecular weight cut-off (MWCO) 3500 Da was purchased from ThermoFisher Scientific (Waltham, MA), and Sephacryl S-300 was purchased from GE Healthcare (Uppsala, Sweden) for size exclusion chromatography (SEC). Deionized (DI) water Modulab 2020 water purification system (San Antonio, TX) has a resistivity of 18 MΩ cm and surface tension of 72.6 mN m⁻¹ at 20.0 ± 0.5 °C.

Synthesis of black C-dots

Carboxylic acid-functionalized C-dots were synthesized by acidic oxidation¹⁶. Briefly, 1 g carbon nanopowder was refluxed with sulfuric acid (36 mL) and nitric acid (12 mL) for 15 h at 110 °C in an oil bath. After the mixture was cooled to room temperature, the unreacted acids were neutralized with a saturated (1 M) sodium hydroxide solution (pH 14). The neutralized mixture was vacuum-filtered to remove the unreacted carbon powder. The supernatant was then kept in an ice bath to precipitate the salt that was produced during the acid neutralization step. A piece of sodium sulfate was added to avoid super-saturation. The filtration was repeated two additional times. Organic waste was removed by chloroform (60 mL). The purified solution was centrifuged at 3000 rpm (RCF = 935.76) for 30 min and subsequently transferred into a 3500 Da molecular weight cut-off (MWCO) dialysis membrane. The mixture was dialyzed against 4 L of Milli-Q-DI water for 5 days while replacing the water for every 4–10 h. Lastly, the purified C-dots solution was dried using a rotary evaporator.

Synthesis of C-dots-shPep-1-lnPep-1-epi-AIC, C-dots-shPep-1-epi-AIC and C-dots-lnPep-1-epi-AIC conjugates

To synthesize the quadruple nanomodel, the C-dots-shPep-1-lnPep-1-epi-AIC conjugate, black C-dots (8 mg) were dissolved in 3 mL of 25 mmol L⁻¹ phosphate buffer solution (PBS) (pH 7.4). EDC, 8 mg, was dissolved in 0.5–1.0 mL of PBS and added to the C-dot solution. The mixture was stirred at room temperature for 30 min. NHS, 10.7 mg, was dissolved in 0.5–1.0 mL of PBS) and added to the C-dot solution mixture. After 30 min of stirring, 1 mL of shPep-1 (3 mg mL⁻¹) and 1 mL of lnPep-1 (1.5 mg mL⁻¹) solutions were added into the C-dot reaction mixture. After stirring for an additional hour, 4.0 mg of epirubicin and 13.5 mg of temozolomide, which were pre-dissolved in 0.5–1.0 mL dimethyl sulfoxide (DMSO), were added to the C-dot reaction. The mixture was stirred at room temperature overnight. The following day the mixture was purified by membrane dialysis (3500 MWCO) for 4 days. The Milli-Q deionized (DI) water was replaced every 4–10 h. The purified solution of the quadruple nanomodel was frozen at −80 °C and lyophilized for 4 days to obtain the powdered product¹⁶. C-dots-shPep-1-epi-AIC synthesis occurred as detailed above, excluding the addition of lnPep-1. C-dots-lnPep-1-epi-AIC synthesis occurred as detailed above, excluding the addition of shPep-1. Temozolomide, a prodrug, was used to produce the AIC conjugates because, while stable at acidic pH, it hydrolyzes at neutral pH, producing the reactive methyl diazonium cation and its major metabolite AIC, which contains 2 primary amine groups to facilitate attachment to the C-dots³⁷.

Synthesis of C-dots-lnPep-1-shPep-1-5-Amfl, C-dots-lnPep-1-5-Amfl and C-dots-shPep-1-5-Amfl conjugates

To ensure equal loading of the Amfl, C-dots (32 mgs) were dissolved in 8 mL of 25 mmol L⁻¹ phosphate buffer solution (PBS) (pH 7.4). EDC (71.12 mg) was dissolved in 4.0 mL PBS and added into the C-dots solution. The mixture was stirred at room temperature for 30 min, and 42.8 mg NHS dissolved in 4.0 mL of PBS was added to the C-dot mixture. After 30 min of stirring, 4 mg 5-(aminomethyl) fluorescein hydrochloride (5-Amfl) in 2.0 mL of DMSO was added to the C-dot reaction. The mixture was stirred at room temperature overnight. The following day the mixture was purified using a dialysis membrane of 3500 MWCO for 3 days. Once the dialysis was completed, the C-dots-5-AMfl solution was divided into 4 aliquots with equal volumes.

For the synthesis of the C-dots-lnPep-1-shPep-1-5-Amfl conjugate, shPep-1 and lnPep-1 were added one of the 4 aliquots as follows. 17.78 mg of EDC, which was pre-dissolved in 0.5 mL of PBS, was incorporated into the C-dots-5-Amfl solution. This mixture was stirred at room temperature for 30 min. Then, 10.7 mg of NHS dissolved in PBS (0.5 mL) was added into the mixture. After 30 min of stirring, 1 mL of shPep-1 (3 mg mL⁻¹) and 1 mL (1.5 mg mL⁻¹) of lnPep-1 solutions were introduced into the reaction mixture. The mixture was stirred at room temperature overnight. The following day, the mixture was purified by using a dialysis membrane of 3500 MWCO for 4 days. The Milli-Q-DI water was replaced every 4–10 h and the solid powder was obtained by lyophilization. The second aliquot of C-dots-5-Amfl conjugate was used to synthesize the C-dots-5-Amfl -LnPep-1 conjugate via conjugation to the lnPep-1 peptide. The synthesis of this reference sample, C-dots-lnPep-1-5-Amfl, is exactly the same, except for the addition of shPep-1. The third aliquot of C-dots-5-Amfl conjugate was used to synthesize the C-dots-5-Amfl -shPep-1 conjugate via conjugation to the shPep-1 peptide. Similarly, the synthesis of this reference sample C-dots-shPep-1-5-Amfl is exactly the same, except for the addition of lnPep-1. The fourth aliquot of C-Dots-5-Amfl conjugate was frozen at -80 ⁰C and lyophilized to obtain the dried C-dots-5-Amfl powder. No peptides were conjugated.

Synthesis of C-dots-Epi-AIC, C-dots-Epi and synthesis of C-dots-AIC

C-dots (8 mg) were dissolved in 3 mL PBS (pH 7.4). EDC, 8 mg, was dissolved in 0.5–1.0 mL of PBS and added to the C-dot solution. The mixture was stirred at room temperature for 30 min. NHS, 10.7 mg, was dissolved in 0.5–1.0 mL of PBS and added to the C-dot solution mixture. After 30 min of stirring, 4.0 mg of epirubicin and 13.5 mg of temozolomide, which were pre-dissolved in 0.5–1.0 mL dimethyl sulfoxide (DMSO), were added to the C-dot reaction. The mixture was stirred at room temperature overnight. The following day the mixture was purified by membrane dialysis (3500 MWCO) for 4 days. The Milli-Q deionized (DI) water was replaced every 4–10 h. The purified solution of C-dots-epi-AIC nanomodel was frozen at −80 °C and lyophilized for 4 days to obtain the powdered product. The Synthesis of C-dots-Epi was conducted according to the method above, excluding the addition of temozolomide. Similarly, the synthesis of C-dots-AIC was conducted according to the method above, excluding the addition of epirubicin.

Characterization

To evaluate the physicochemical properties of the synthesized C-dot conjugates (20 μg/mL), multiple complementary characterization techniques were employed. To ensure reproducibility and batch-to-batch consistency, all characterization experiments were repeated using independently synthesized batches of C-dot conjugates. This verification step confirmed both the stability and reliability of the conjugation process.

Spectroscopic techniques

UV-visible spectroscopy measurements were carried out using a Shimadzu UV-2600 spectrophotometer with a 1 cm path length quartz cuvette. For fluorescence analysis, emission spectra were recorded on a Horiba Jobin Yvon Fluorolog-3 fluorometer, utilizing a consistent slit width of 5 nm for both excitation and emission to ensure reliable comparisons across samples. Fourier-transform infrared (FTIR) spectroscopy was conducted using a PerkinElmer Frontier spectrometer equipped with an attenuated total reflection (ATR) accessory. The ATR prism was used as both the sample interface and background reference, allowing for straightforward and reproducible spectral acquisition. To confirm molecular composition, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was performed using a Bruker Autoflex Speed instrument.

Atomic force microscopy (AFM)

The bare C-dots and the quadruple conjugated C-dots were dissolved in Milli-Q-DI at a concentration of 0.25 mg/ml, sonicated for a minimum of 2 h before applied to the substrate, consisting of freshly cleaved highly ordered pyrolytic graphite (HOPG) or MICA. Samples were incubated at RT for 15 min and dried using nitrogen gas before scanning. AFM was performed by Cypher S (Oxford Instruments, Asylum Research, Santa Barbara, CA). Topographic AFM images were acquired in tapping mode in air using the AC160TSA-R3 AFM probe. Multiple images were obtained for both the bare C-dot and quadruple conjugated C-dots. Approximately 300 of each C-dot was analyzed. The data were fitted to a Gaussian distribution using MATLAB to find the most frequent height and standard deviation.

Functional characterization and biological evaluation

Agarose gel electrophoresis

Agarose (1%, w/v) gel electrophoresis was performed in 20 mM Tris–acetate, 2 mM EDTA (TAE). DNA (250 ng PUC19 plasmid DNA) and incubated for 1 h with 50 ng epirubicin (positive control), black C-dots, or the C-dot conjugates; C-dot-AIC, C-dot-epi, and C-dot-AIC-epi (1 μg each) in a 10 mM TRIS, 100 mM NaCl buffer (pH 7.4) before loading the gels. Following electrophoresis, gels were stained with SYBR Green Nucleic Acid Gel Stain for 15 min and visualized using an Azure C400 Imager (Azure Biosystems).

Cell Culture

SJ-GBM2 is a post chemotherapy derived pediatric GBM cell line obtained from the Children’s Oncology Group (COG, Texas Tech University, Health Science Center, TX, USA). The DIPG cell line, NP53 was derived from a tumor arising in a DIPG genetically engineered mouse model induced by PDGF-B signaling, TP53 loss, and ectopic H3.3K27M expression⁴². The NP53 cell line was kindly provided by Dr. Oren Becher, Northwestern Medicine, Feinberg School of Medicine, Ann and Robert H. Lurie Children’s Hospital of Chicago. NP53 cells were cultured in Complete NeuroCult™ Proliferation Medium (Stemcell Technologies, Vancouver, Canada) supplemented with 1% penicillin/streptomycin (P/S) (Thermofisher Scientific, Waltham, MA). U87 (U-87 MG) cells were obtained from American Type Culture Collection (ATCC HTB-14). SJ-GBM2 and U87 were cultured and maintained in RPMI media supplemented with 10% heat-inactivated FBS (hi-FBS) (Thermofisher Scientific, Waltham, MA) and 1% P/S. Adult human neural progenitor cell line derived from the hippocampus, H35, was generously provided by Dr. Dennis A. Steindler (Tufts University, Boston, MA, USA)⁴³. H35 cells were cultured in DMEM media supplemented with N₂, 5% FBS, 20 μg/ml bovine pituitary extract 40 ng/ml of epidermal growth factor, and 20 ng/ml basic fibroblast growth factor (Stemcell Technologies, Vancouver, Canada). Human brain derived vascular smooth muscle cells (VSMC) were obtained from Sciencell (Carlsbad, CA, USA) and were cultured in DMEM/F12 supplemented with 10% hi-FBS and 1% P/S. All experiments using VSMCs were performed on minimally passaged cells (passages <8). All cell lines were routinely tested for mycoplasma using LookOut mycoplasma PCR detection kit (SigmaAldrich, St. Louis, MO, USA) according to the manufacturer’s instructions and were maintained at 37 °C in a humidified 5% CO₂ incubator. Authentication of U87 and SJ-GBM2 via STR profiling was performed by the providers. In general, the primary cell lines were characterized with functional and phenotypic assays by the provider. NP53 cell line was verified genetically through sequencing or PCR for defining mutations (PDGF-B overexpression, p53 loss, H3.3K27M) in the RCAS transgenic mouse model, with phenotypic consistency confirmed in publications. H35 was characterized as human neural progenitors via functional and phenotypic validation. VSMC is a primary cell line authenticated by immunofluorescence showing >90–95% purity with positive staining for α-smooth muscle actin, calponin, and smooth muscle myosin heavy chain, and negative for endothelial and fibroblast markers. No additional authentication was performed. All primary cell lines were expanded and frozen to maintain stock of relatively low passage numbers. None of the cell lines used are mis-identified as per the International Cell Line Authentication Committee (ICLAC)⁴⁴.

Western blot analysis

Western blotting studies were performed to determine the expression of IL13Rα2 in high-grade glioma cell lines using our previously published protocol⁴⁵. Briefly, H35, SJ-GBM2, U87, and NP53 cells were lysed in RIPA buffer, and protein concentrations were calculated by using BCA protein assay (BioRad Hercules, CA, USA). Equal amounts of protein (20 µg) were loaded onto 12% polyacrylamide gel (BioRad Hercules, CA, USA) for electrophoresis and subsequently transferred onto nitrocellulose membranes. The membranes were blocked for 1 h in 5% non-fat milk (Biorad, Hercules, CA, USA) at room temperature (RT) and incubated with the primary antibody diluted in 2.5% BSA overnight at 4 °C. IL13Rα2 polyclonal antibody (PA5-106798) was purchased from Thermo Scientific (Waltham, MA, USA), and GAPDH ((14C10) Rabbit Monoclonal Antibody, #3683) was purchased from Cell Signaling Technology (Danvers, MA, USA). These antibodies were previously validated by the manufacturer; no additional validation was performed. Finally, all membranes were incubated at room temperature with anti-mouse or anti-rabbit secondary antibodies for 1 h. Blots were processed by using SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).

Cell viability assay

The anti-cancer efficacy of the single peptide conjugates, C-dots-shPep-1-epi-AIC, and C-dots-ln-Pep-1-epi-AIC, and the quadruple conjugate, C-dots-shPep-1-lnPep-1-epi-AIC, was determined using 3 high-grade glioma cell lines: SJ-GBM2, U87, and NP53. The cytotoxicity of the quadruple conjugate was also investigated in the non-tumor VSMCs. Cell viability was determined by MTS assay as previously described⁴⁶. Briefly, 24 h prior to the drug treatment, the cells were cultured in 96-well plates with a cell density of 0.5–1.5 × 105 per well. Subsequently, the cells were treated with 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, or 10.0 μM of each C-dot conjugate. After 72 h incubation, viability was examined using CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) based on the manufacturer’s instructions. Absorbance at 490 nm was measured by using a BioTek Synergy HT Plate reader. Numerical source data for viability and IC₅₀ value graphs can be found in Supplementary data 1 file.

Fluorescent Microscopy

SJ-GBM2 cells were plated in 24-well plates at a density of 1 × 10⁵ cells/well. After the incubation for 24 h at 37 °C, cells were treated with C-dots-5-Amfl, C-dots-5-Amfl-shPep-1, C-dots-5-amfl-lnPep1, or C-dots-5-Amfl-shPep-1-lnPep-1 at 1 mM concentration for an hour. After this treatment, cells were washed with PBS and treated with 1 µg/mL Hoechst for 15 min. Then, cells were washed with PBS for two more times and fixed with 4% formaldehyde for 20 min. Once the fixation was completed, 100 µL of citifluor antifade agent was added on top of the cells for 2 days. Finally, cells were washed with PBS 3 times to wash off the antifading agent and examined by confocal microscopy.

Image analysis

Image analyses of 5-Amfl conjugated dual and single peptide nanomodels were performed using ImageJ v.1.53i. Quantification analyses of 5-Amfl fluorescence intensity were performed using GraphPad Prism 9.3.1 software. The pixel counts per fluorescence intensity (grey scale) quantification of fluorescence intensities of cells were determined using ImageJ v.1.53i. The relative pixel count is the number of pixels with a given fluorescence intensity within a selected region of interest (SJ-GBM2 cells), subtracted by the background pixels in that region. Fluorescent intensities and line scanning distance are represented as an XY line graph. Data were analyzed using GraphPad Prism Software (version 6; GraphPad Software Inc., La Jolla, CA, USA). Pearson’s correlation test was applied to determine the correlation between fluorescent intensities under scanning lines of Hoechst nucleus staining dye and 5-Amfl C-dots conjugates. PCC values used to generate graph can be found in Supplementary data 1 file.

Statistical analysis

In vitro anti-cancer efficiency data were represented as the average of a minimum of 3 separate biological experiments, each with four technical replicates. The viability was calculated as the percent of non-treated cells. Data is presented as the average viability ± standard error of the mean (SEM). Significance was determined using students’ T-test (two-tailed) or by one-way ANOVA followed by post hoc test, with post hoc Tukey’s HSD test (Online Web Statistical Calculators website; astatsa.com). Parametric tests were used assuming normality and homogeneity of variances based on the robustness of ANOVA with equal sample sizes; no formal tests of normality were performed. The consistency of the data was confirmed by analyzing different batches of C-dots-conjugates.

Inclusion and ethics statement

This manuscript does not contain studies with human participants or animals performed by any of the authors. Research involving human embryos or embryonic stem cells was not part of this study. All research involving cell lines was conducted in compliance with relevant institutional policies.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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Acknowledgements

This work was supported by grants from the National Science Foundation; grant numbers: 1809060, 2041413 (R.M.L), and 2102563 (R.P.), the Florida Department of Health Live Like Bella research grant #21L08 (R.M.G), Mystic Force Foundation (R.M.G), and HCA Florida University Hospital (R.M.G). We thank Dr. Oren Becher (Northwestern Medicine) for kindly providing the NP53 cell line, and Dr. Dennis A. Steindler (Tufts University) for generously providing the H35 human neural progenitor cell line.

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These authors contributed equally: Emel Kirbas Cilingir, Sajini D. Hettiarachchi.

Authors and Affiliations

Department of Chemistry, University of Miami, Coral Gables, FL, USA
Emel Kirbas Cilingir, Sajini D. Hettiarachchi, Parth Rathee, Yiqun Zhou, Braulio CLB Ferreira, Lukun Wang, Annu Joji, Kun Wang, Rajeev Prabhakar & Roger M. Leblanc
Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, USA
Emel Kirbas Cilingir
Dr. Kiran C. Patel College of Allopathic Medicine, Nova Southeastern University, Ft. Lauderdale, FL, USA
Sajini D. Hettiarachchi, Maria J. Moreno Hollweg, Steven Vanni & Regina M. Graham
Department of Chemistry and Physics, Halmos College of Natural Sciences and Oceanography, Nova Southeastern University, Fort Lauderdale, FL, USA
Carlos M. Gonzalez
Department of Physics, University of Miami, Coral Gables, FL, USA
Mehrdad Shiri & Kun Wang
Department of Neurological Surgery, University of Miami, Miller School of Medicine, Miami, FL, USA
Steven Vanni
HCA Florida University Hospital, Ft. Lauderdale, FL, USA
Steven Vanni

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Contributions

E.K.C. and S.D.H. equally contributed to the study by performing experiments, analyzing data, and writing the manuscript. Y.Z. performed confocal imaging. B.F. prepared the FTIR Figure. M.M.H. drew the graphical abstract and helped to edit the manuscript. A.J. synthesized the black C-dots, R.P. supervised P.R. and L.W., who performed experiments, K.W. supervised M.S., who performed the A.F.M. studies, C.M.G. and S.V. helped to revise the results and edit the manuscript. R.M.L. and R.M.G. advised E.K.C. and S.D.H. throughout the project and critically revised the manuscript. RMG also performed experiments.

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Correspondence to Roger M. Leblanc or Regina M. Graham.

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Cilingir, E.K., Hettiarachchi, S.D., Rathee, P. et al. Development of a quadruple-conjugated carbon dot nanomodel for targeted glioma therapy. Commun Chem 9, 96 (2026). https://doi.org/10.1038/s42004-026-01900-3

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Received: 03 December 2024
Accepted: 09 January 2026
Published: 30 January 2026
Version of record: 19 February 2026
DOI: https://doi.org/10.1038/s42004-026-01900-3