Development of AS1411 aptamer-conjugated chitosan-carbon dot nanocarriers for targeted drug delivery and fluorescent imaging in breast cancer therapy

Abstract

Theranostic nanomedicine, combining therapy with diagnostic imaging, offers a powerful strategy for real-time monitoring and targeted treatment of cancer. In the current study, we developed a pH-sensitive delivery system based on chitosan (CS) and poly caprolactone-poly ethylene glycol- poly caprolactone (PCL-PEG-PCL) copolymer, which was conjugated to the AS1411 Aptamer and tagged with carbon dots (CDs), abbreviated as DOX/P/CS-CD-Apt. Then, the theranostic potential of DOX/P/CS-CD-Apt in carrying DOX to MCF-7 breast cancer cells was evaluated both in vitro and in vivo. CDs were synthesized via a one-step hydrothermal method and chemically conjugated to the CS backbone along with AS1411 using EDC/NHS chemistry. On the other hand, DOX is encapsulated into the final carrier through the double emulsion-solvent evaporation method. The nanocarrier was characterized using FT-IR, FESEM, XPS, XRD, TEM, DLS, and zeta potential. Our results represented that DOX/P/CS-CD-Apt were uniform spherical morphology, high drug encapsulation, and controlled release under acidic conditions. Fluorescence microscopy revealed cytoplasmic entrance of DOX/P/CS-CD-Apt in MCF-7 cells, indicating effective nucleolin-mediated uptake. MTT assays and apoptosis evaluation demonstrated the higher cytotoxicity and apoptotic effects compared to free DOX or non-targeted formulations. Moreover, in vivo PET imaging confirmed the selective accumulation of nanoparticles at the tumor site in a 4T1 breast cancer model, along with a notable reduction in tumor size. These findings highlight DOX/P/CS-CD-Apt as a promising theranostic platform for targeted breast cancer therapy with integrated imaging capability.

Graphical Abstract

Introduction

Breast cancer is the most prevalent kind of cancer among women, with over two million cases diagnosed globally in 2018. Although chemotherapy is the primary treatment for cancer, it has significant drawbacks. One major issue is the non-specific distribution of chemotherapeutic agents, which affects both healthy and cancerous cells, thereby reducing the amount that reaches tumor cells and causing undesirable side effects. Furthermore, conventional chemotherapy suffers from limitations such as unpredictable drug delivery and premature drug clearance through systemic circulation and renal excretion [1]. To overcome these challenges, combination therapy has emerged as a promising approach by employing multiple therapeutic agents. This strategy can reduce adverse effects and enhance efficacy through synergistic mechanisms. However, its clinical application is hindered by issues such as systemic toxicity, non-specific drug accumulation at the tumor site, and limited cellular uptake [2]. In recent years, smart nanocarriers have been developed to address these limitations [3]. Functionalizing nanocarriers with stimuli-responsive moieties, which are sensitive to physiological cues such as pH [4], temperature [5], redox [5], and enzymatic activity [6], can enable precise, controlled drug release at the target site [7–10]. Among these stimuli, pH-responsiveness is particularly attractive for cancer therapy. This is due to the acidic microenvironments found in tumors (pH 6.5–7.0), endosomes (pH 5.0–6.0), and lysosomes (pH 4.5–5.0), in contrast to the neutral pH of normal tissues (pH 7.4). Exploiting these differences has become a widely adopted strategy for enhancing the specificity and efficacy of anticancer drug delivery systems. Until now, several nanocarrier systems have been developed to take advantage of the enhanced permeability and retention (EPR) effect observed in tumor tissues due to the permeability of tumor vasculature and defects in the lymphatic system, which facilitate passive targeting of tumor sites [11–14]. These nanocarriers provide several benefits, including customizable physicochemical characteristics, the ability to deliver drugs in a controlled and sustained manner, extended circulation time in the bloodstream, and the potential for both passive and active targeting of tumor sites [15].

Chitosan, a natural copolymer derived from the deacetylation of chitin, has gained significant attention as a drug carrier. It exhibits excellent physicochemical properties, including biocompatibility, biodegradability, non-toxicity, and ease of chemical modification [16]. The presence of reactive amino and hydroxyl functional groups on the backbone of chitosan enables facile conjugation with targeting ligands, chemotherapeutic agents, and responsive moieties, allowing for the development of multifunctional delivery systems [17]. Interestingly, chitosan exhibits pH-sensitive behavior due to the protonation of its amino groups under acidic conditions, which facilitates drug release in the acidic tumor microenvironment and in intracellular compartments, such as endosomes and lysosomes [18]. For example, a stepwise pH-responsive NPs based on dimethylmaleic acid-chitosan-urocanic acid was developed to promote the therapeutic effect of doxorubicin (DOX). The prepared nanoplatform exhibits a negative surface charge under physiological conditions and demonstrates improved stability during blood circulation, as well as enhanced accumulation in tumor sites via the EPR effect, followed by tumor cellular uptake. On the other hand, a switch to the positive surface charge allowed for the on-demand release of the drug from the NPs [19]. Additionally, functionalization of CS through ligand conjugation makes it a suitable platform for active targeted cancer therapy. Chauhan et al. reported the development of dual-functionalized theranostic chitosan–PLGA nanoparticles (NPs), modified with AS1411 aptamer and RGD peptide, for the targeted co-delivery of docetaxel (DTX) and upconversion nanoparticles (UCNPs) as model anticancer and imaging agents, respectively, in brain cancer therapy. The prepared NPs exhibited significantly enhanced cellular uptake, 89-fold improved cytotoxicity, improved cancer cell cycle arrest even at lower drug concentration, and superior bioavailability with extended mean residence time of DTX in systemic circulation and brain tissues [20]. Carbon dots (CDs), a new class of carbon-based nanomaterials, have gained considerable attention due to their excellent water solubility, anti-photobleaching, chemical stability, low cytotoxicity, and good biocompatibility. Furthermore, they offer a simple, cost-effective synthesis process and facile surface functionalization, making them advantageous over conventional fluorescent dyes [21–24]. To enable the selective targeting of cancer cells, CDs can be functionalized with cancer-specific ligands or biomarkers. In this context, Kim et al. developed folic acid functionalized carbon dot/polypyrrole NPs (FA-CD/PPy NPs) for the photothermal therapy (PTT) of folate receptor (FR) positive HeLa cancer cells. Upon near-infrared (NIR) laser irradiation, the viability of HeLa cells incubated with 200 µg/mL FA-CD/PPy-NPs was dramatically decreased to 25.02 ± 1.85%, attributed to the high photothermal conversion efficiency (η = 40.80 ± 1.54%) [25].

The tumor-selective properties of aptamers have increasingly attracted attention for their potential use in drug delivery systems, particularly in cancer therapeutics and imaging probes [26]. Among various tumor-targeting aptamers, AS1411 has emerged as a promising candidate for receptor-targeted delivery of anti-cancer drugs. AS1411 is a non-immunogenic, biocompatible 26-mer guanine-rich DNA/RNA oligonucleotide aptamer that has undergone clinical evaluation due to its potent antiproliferative and targeting capabilities. It forms a stable G-quadruplex structure that shows resistance to nuclease degradation and exerts anticancer effects by disrupting downstream signaling pathways. AS1411 specifically binds to nucleolin, a multifunctional 76 kDa protein overexpressed on the surface of specific tumor cells. Nucleolin is involved in diverse cellular processes such as transcription, ribosomal RNA packaging and transport, DNA replication, and nucleocytoplasmic shuttling [27]. Recently, Iman et al. reported the preparation of PEGylated liposomal DOX functionalized with the AS1411 aptamer (AS-PLD) for nucleolin-targeted delivery to C26 tumor cells. The biodistribution and pharmacokinetic studies demonstrated significant tumor accumulation of AS-PLD, with peak localization observed 72 h post-injection [28]. The objective of our current research is to develop an innovative, targeted nanoparticle system with a controlled and sustained drug release profile, aiming to minimize side effects while enhancing the bioavailability and half-life of the drug. To achieve this, we engineered a delivery platform composed of chitosan conjugated with fluorescent CDs and AS1411 aptamer, combined with PCL-PEG-PLC (PCEC) copolymer, to facilitate both active and passive delivery of DOX to the tumor site through the nucleolin-targeting ability of AS1411 aptamer and permeability of tumor vasculature system, respectively, while also enabling cellular imaging through the fluorescent properties of the CDs. To the best of our knowledge, this is the first report on the synthesis of DOX-loaded chitosan-CD NPs functionalized with the AS1411 aptamer, presenting a promising strategy for simultaneous cancer therapy and bioimaging.

Materials and methods

Materials

Chitosan (medium molecular weight), Epichlorohydrine (≥ 98% [w/v] aqueous solution), glacial acetic acid (99.5%), Sodium hydroxide (NaOH), Dichloromethane (DCM), diammonium phosphate, citric acid, 1-ethyl-3(3-dimethylaminopropyl) Carbodiimide (EDC), N-hydroxy-succinimide (NHS), polyvinyl alcohol (PVA, average Mw = 89,000–98,000), penicillin G, streptomycin, Dialysis membrane (MWCO12 kDa), MTT powder, dimethyl sulfoxide (DMSO), were purchased from Sigma-Aldrich (Steinem, Germany) and Amicon^® Ultra Centrifugal Filter Devices (Amicon Ultra-15 Membrane MWCO 100 KDa) were purchased from Merck company (Germany). The PCEC copolymer with the PEG molecular weight 6000Da was kindly donated to us by Dr. Vaghefi [29]. DMEM/high glucose, EDTA trypsin and fetal bovine serum (FBS), were purchased from Gibco. Annexin V, Propidium iodide, and other biological reagents were purchased from Invitrogen, USA. The breast cancer (MCF-7) cell lines were purchased from the National Cell Bank Pasteur Institute of Iran (Tehran, Iran). AS1411, with sequence 5′carboxy C10-d(GGTGGTGGTGGTTGTGGTGGTGGTGG)-3′, was purchased from GenFanavaran (Iran). Analytical-grade chemicals and a source of double-distilled water were used for all testing.

Physicochemical characterization

Particle size and morphology of the prepared nanoparticles were determined by Transmission Electron Microscopy (TEM) (LEO 906, Germany), Field Emission Scanning Electron Microscopy (FE-SEM) (MIRA3 FEG-SEM, Tescan, Czech), and dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Instruments, Malvern, UK) at 25 °C, using distilled water as the dispersant. The SEM samples were obtained by pouring diluted nanomaterials onto carbon-coated copper grids and drying them overnight at RT. Fourier Transform Infrared (FTIR) spectroscopy (Bruker Tensor 27 spectrometer, Germany) was used to confirm the creation of nanoparticles by detecting their major functional groups. The spectra of samples were recorded from 400 to 4000 cm^− 1. X-ray diffraction (XRD) was used to investigate the crystallinity of CDs. XRD patterns were obtained with a Tongda TD-3700 diffractometer with Cu Kα irradiation (λ = 1.5406 Å) at room temperature, operating at a voltage of 30 kV. These experiments were performed at a scan rate of 1° min^− 1 within the scan range of 2θ = 10 ° to 80 °. XPS was performed on an ESCALAB MK II X-ray photoelectron spectrometer using Mg as the excitation source. Fluorescence emission spectra were collected on a NOVA fiber-coupled spectrometer.

Synthesis and preparation of CDs

The synthesis was performed using a one-step hydrothermal method. A solution of 0.3 g citric acid and 0.754 g diammonium phosphate (in a 1:4 molar ratio) was prepared in 20 mL of deionized water. The mixture was transferred into an autoclave and heated at 180 °C for 4 h. After cooling to room temperature, the product was purified using a dialysis membrane with a molecular weight cutoff (MWCO) of 2000 Da to obtain uniformly sized particles. The resulting dark brown, transparent CDs were then freeze-dried and stored at 4 °C for further use [27, 30].

Quantum yield measurement

The quantum yield (QY) of the CDs was measured according to the previously described method [31]. In this method, the QY of the CDs was determined relative to quinine sulfate (QS) as a reference (Q = 0.54 in 0.1 M H₂SO₄). The solutions of CDs and QS were prepared in distilled water and 0.1 M H₂SO₄, respectively. The absorbance at the excitation wavelength was ≤ 0.05 to minimize inner-filter effects. Emission spectra were acquired at an excitation wavelength of 350 nm (the excitation wavelength that produced the maximum emission for the CDs) and integrated to obtain total emission intensity. The absorbance of sample and reference at 350 nm were measured and matched when possible. Quantum yield was calculated using the comparative equation:

where is quantum yield, I is the measured integrated emission intensity, OD is the absorbance at the excitation wavelength, and n is the refractive index of the solvent (both solutions had n ≈ 1.33). The subscript r refers to the reference fluorophore of known Q, for example, QS in the present work.

Conjugation and characterization of AS1411 aptamer and CDs with CS

To ensure the AS1411 aptamer achieves its functional secondary structure, a thermal annealing process was performed. Lyophilized aptamer was dissolved in an appropriate volume of water, heated to 85 °C for 2 min, and then allowed to cool gradually to room temperature (25 °C) over 10 min [32]. This procedure promotes proper folding and stabilization of the aptamer’s G-quadruplex structure, enhancing its target recognition efficiency [33].

For the preparation of the CS-CD-Apt conjugate, 4 µL of aptamer (100 µM) was dispersed in 2 mL of diethyl pyrocarbonate (DEPC) water and stirred at room temperature. Subsequently, 2 mg of CDs were added to the solution and thoroughly mixed. To facilitate covalent bonding, 7 mg of EDC and 7 mg of NHS were added, and the mixture was stirred in the dark for 3 h at room temperature. Following this activation step, 10 mg of CS powder was added to the reaction, and stirring was continued under the same conditions for an additional 12 h. The resulting product was purified by washing with DEPC water using an Amicon^®Ultra centrifugal filter (100 kDa) to remove unbound aptamer and CDs. Successful conjugation of the aptamer was evaluated via agarose gel electrophoresis. The CS-CD-Apt conjugate was loaded onto a 3% agarose gel alongside a molecular weight ladder and subjected to electrophoresis at 110 V for 30 min. The gel was subsequently stained with a nucleic acid-safe stain for visualization.

Preparation of DOX-loaded chitosan nanoparticles (DOX/P/CS, DOX/P/CS-CD, and DOX/P/CS-CD-Apt)

In this study, the double emulsion solvent evaporation method was employed. The inner aqueous phase (W1) consisted of 15 mg of CS, or Apt-and CD-conjugated CS, dissolved in 1 mL of 1% (w/v) acetic acid, along with 1.25 mL of DOX solution (2 mg/mL). In the first step, W1 was emulsified into the oil phase (O), which contained 10 mg of PCEC dissolved in 2 mL of DCM, using homogenization at 3000 rpm for 5 min. The resulting primary W1/O emulsion was then gradually added to 10 mL of an aqueous PVA solution (0.5% w/v), followed by homogenization at 17,000 rpm for 10 min to form the W1/O/W2 double emulsion. The pH of the emulsion was adjusted to 7 using 2 N NaOH. Subsequently, 50 µL of ECH, diluted in 5 mL of 0.5% (w/v) PVA solution, was added dropwise under continued homogenization (17,000 rpm, 10 min). The nanoparticles formed upon overnight stirring at room temperature, allowing for complete evaporation of the organic solvent. The nanoparticles were then collected by centrifugation at 13,000 rpm for 20 min. The supernatant was used to determine the amount of unloaded drug via UV-Vis spectrophotometry at a wavelength of 480 nm (λ_max). Finally, the nanoparticles were freeze-dried and stored at 4 °C for further use. Entrapment efficiency (EE) and loading content (LC) were calculated using the following equations:

In vitro drug release study

The in vitro release profile of DOX from DOX/P/CS-CD-Apt NPs was evaluated using a dialysis method. A dialysis bag (molecular weight cut-off: 12 kDa) was employed in phosphate-buffered saline (PBS) at two different pH values: 7.4 (physiological) and 5.4 (lysosomal). Briefly, 2 mL of drug-loaded nanoparticles (1 mg/mL) were placed into separate dialysis bags, which were then immersed in 25 mL of release medium at the respective pH values. The samples were incubated at 37 °C with gentle shaking at 50 rpm. At predetermined time intervals (0.5, 1.5, 2.5, 3.5, 4.5, 6, 24, 48, and 72 h), 1 mL of the release medium was withdrawn and replaced with an equal volume of fresh medium to maintain sink conditions. The amount of DOX released was quantified by UV-Vis spectrophotometry at 480 nm, and the cumulative release (CR) percentage was calculated using the following equations [34]:

: volume of sample withdrawn (ml), : the bath volume (ml), : drug concentration at time t (µg/ml), : the total amount of drug (µg), and : drug release (%) at t and t-1.

Drug release kinetics analysis

The release profile of DOX from the DOX/P/CS-CD-Apt nanoparticles was further evaluated by applying several established kinetic models to understand the underlying release mechanism. Four commonly used models were analyzed: (i) the zero-order model, which assumes a release rate independent of drug concentration [35]; (ii) the first-order model, where the release rate is proportional to the remaining drug concentration [36]; (iii) the Korsmeyer-Peppas model, often used for polymeric matrices to distinguish between Fickian and non-Fickian diffusion [37]; and (iv) the Higuchi model, which describes drug release from porous or insoluble matrices as a diffusion-controlled process [38].

i

where C is the amount of drug released at time t, C₀ is the initial concentration, and k₀ is the zero-order release constant.

ii

where k₁ is the first-order release constant.

iii

where Mt/M∞​ is the fraction of drug released at time t, K_KP​ is the release-rate constant, and n is the diffusional exponent indicative of the release mechanism.

iv

where K_H​ represents the Higuchi dissolution constant, reflecting matrix characteristics.

The cytotoxicity assay and cell culture

The MCF-7 cell line was supplied by the Pasteur Institute of Iran. It was subsequently cultivated in a DMEM-F12 medium, containing 10% v/v FBS and 1% of a penicillin-streptomycin combination solution. 5% carbon dioxide, 37 °C, and 90% humidity were all present in the incubator (Memmert, Germany) [39]. The MTT test was used to assess the cytotoxicity of the DOX/P/CS, DOX/P/CS-CD-Apt, and free DOX on MCF-7 cells. In order to achieve adequate confluency, cells were seeded onto 96-well plates at a density of 5 × 10³ per well and incubated for 24 h. The medium was then swapped out for a replacement medium that included various treatment concentrations (0.2, 0.4, 0.8, 1.6, and 3.2 µM). The control group consisted of untreated cells cultured in a standard medium. Following incubation for 24, 48, and 72 h, the media were taken out and replaced with MTT solution (5 mg/ml) for four hours. Following the removal of the medium, 100 µl of DMSO was added and placed in a shaker to melt the formazan crystals that had been produced. Lastly, the absorbance of each well at 570 nm was recorded using the ELISA plate reader (Multiskan MK3, Thermo Electron Corporation, USA). The viability (%) was calculated using the following equation:

Cellular uptake using fluorescence microscopy

Fluorescence microscopy was employed to examine the cellular uptake of DOX/P/CS-CD-Apt in MCF-7 cells. Cells were cultured in dishes at 37 °C using DMEM growth medium supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. The cells were subsequently incubated at approximately 70% confluence until normal morphology was attained. Afterward each dish was filled with 0.59 µM of the DOX/P/CS-CD-Apt prepared in DMEM medium and incubated for the specified duration 0.5, 1, and 1.5 h post treatment. The cells underwent three washes with 0.1 M PBS (pH 7.4) to eliminate unattached compounds. The cells were visualized via fluorescence microscopy (Olympus BX50) and an attached camera (Olympus DP72). The captured photos were analyzed using Image J software.

Cell apoptosis analyses

The Annexin V-FITC labeling method was employed to evaluate cell apoptosis. MCF-7 cells were plated in 6-well plates at a density of 5 × 10⁵ cells per well and allowed to attach and divide for 24 h. Subsequently, cells were exposed to free DOX, CS-Dox, and CS-CD-DOX-Apt at their respective IC₅₀ concentrations and incubated for 72 h. The cells subjected to PBS treatment were designated as the control group. Subsequently, the cells were trypsinized, collected, rinsed with PBS, and transferred to separate falcon tubes. Ultimately, they were centrifuged, dispersed in 1 ml of PBS, and suspended in 200 µl of binding buffer. The cells were stained with Propidium Iodide and Annexin V-fluorescein isothiocyanate (FITC). The proportion of early and late apoptosis was analyzed using FACS Calibur flow cytometry (Becton Dickinson).

In vivo protocols

To evaluate the effectiveness of the synthesized nanoparticles for diagnostic purposes, a tumor model was established, and a biodistribution study was performed in experimental animals. BALB/c albino mice, aged 2–3 months and weighing between 25 and 30 g, were procured from the Tehran University of Medical Science Laboratory Animal Ltd. (Iran) and housed under standard circumstances. All animal experiments were conducted in accordance with protocols assessed and authorized by the Ethics Committee of Tabriz University of Medical Sciences.

Subcutaneous tumors were induced by administering 50 µL of concentrated 4T1 mouse breast cancer cells in saline solution, with a cell concentration of approximately 6 × 10⁶ cells/mL (Pulaski and Ostrand-Rosenberg 2000). Following cell inoculation, the animals were monitored daily until approximately one week post-inoculation, at which point subcutaneous tumors became palpable, measuring between 10 and 15 mm in diameter. The body weight of the tumor-bearing mice was regularly assessed to monitor their health during the tumor’s progression. The studies commenced on the fourteenth day following tumor implantation.

Small-animal optical imaging

The mice were housed at a standard temperature of 22 °C ± 2 °C and a relative humidity of 55% ± 10% under a 12:12 h light: dark cycle. Four mice participated in the investigation. The control mouse was administered 200 µL of the control solution, while the other animals received 200 µL of CS-Dox-CD-Apt nanoparticles at a dosage of 0.59 µM. CS-DOX-CD-Apt nanoparticles were administered to mice (n = 3) via the tail vein, and tumor progression was assessed in both prone and supine postures using fluorescence imaging at intervals post-injection (blank), 30 min, 1 h, 4 h, 24 h, 48 h, and 72 h across all treatment groups. In vivo imaging was conducted with the KODAK imaging system (FX Pro) at the Preclinical Core Facility (TPCF) at Tehran University of Medical Sciences, employing fluorescent mode with 5-minute exposure duration. The excitation and emission filters were calibrated at 510 nm and 535 nm, respectively. The light emitted by the mice was captured by the KODAK camera system, processed, digitized, and shown.

Small-animal PET imaging and image analysis

Micro-PET scans of small animals were conducted using an Xtrim PET scanner at the Preclinical Core Facility (TPCF) located at Tehran University of Medical Sciences on several days. Mice were administered approximately 0.3 mCi of 18FDG via the tail vein while under general anesthesia.

Statistical analysis

Data were subjected to statistical analysis using analysis of variance (ANOVA), with a p-value of less than 0.05 considered indicative of a statistically significant difference. All experimental procedures were conducted in triplicate. Data were presented as mean ± SEM, and graphics were generated and quantified using GraphPad Prism and ImageJ software.

Results

Preparation of DOX/P/CS-CD-Apt nanocarriers

In this study, CDs were synthesized via a one-step hydrothermal method. Subsequently, a double emulsion solvent evaporation technique, followed by crosslinking with ECH, was employed to encapsulate DOX within the CS-PCEC matrix. The PCEC copolymer used in this study was the same material previously synthesized via ring-opening polymerization and fully characterized by our group [29]. The crosslinking mechanism is illustrated in Scheme 1A. The resulting DOX/PCEC/CS nanoparticles were further functionalized with CDs and the AS1411 aptamer to enhance cellular imaging capabilities and improve tumor-specific adhesion and permeability. The complete fabrication process of the CD- and aptamer-modified DOX/PCEC/CS nanoparticles (DOX/P/CS-CD-Apt NPs) is depicted in Scheme 1B. For targeted delivery, the 5′-carboxyl-modified AS1411 aptamer, a 25-mer oligonucleotide with high binding affinity and specificity for nucleolin, a glycoprotein overexpressed on the membrane of MCF-7 breast cancer cells, was selected to enhance nanoparticle targeting and cellular uptake [3, 40].

Scheme 1.

A) The crosslinking mechanism of CS with ECH. B) The reaction path for the fabrication of DOX/P/CS-CD-Apt NPs

Characterization

FTIR analysis

FTIR spectroscopy was used to verify the functional groups in DOX, CS, PCEC, DOX/P/CS, DOX/P/CS-CD, and DOX/P/CS-CD-Apt (Fig. 1). The FTIR spectrum of DOX represents a characteristic band at 1705 cm^{− 1}, corresponding to the C = O stretching vibration of the carbonyl group. The peak at 1640 cm^{− 1} corresponds to the C = C ring stretching vibration. The band at 1078 cm^{− 1} confirms C-O-C asymmetric vibrations. In CS’s spectrum, the broad peak at 3100–3600 cm^{− 1} is due to overlapping N-H and O-H stretching vibrations. The double peaks at 2870 and 2925 cm^{− 1} correspond to aliphatic C-H groups. CS’s characteristic peaks at 1643 and 1617 cm^{− 1} signify amide I (C = O) stretching and amide II (N-H) bending vibrations. The absorption bands at 1456 and 1379 cm^{− 1} indicate CH₂ bending and CH₃ symmetrical deformation modes. The peak at 1421 cm^{− 1} shows NH₂ bending vibrations of primary amine groups. The band at 1159 cm^{− 1} confirms C-O-C asymmetric vibrations. Peaks at 1084 and 1028 cm^{− 1} are linked to C-O stretching vibrations [41]. The characteristic peak of PCEC is assigned at 1723 cm^{− 1}, corresponding to the C = O stretching vibration of the carbonyl ester group in the PCL block. Also, the double peaks representing C-H aliphatic groups are assigned at 2861 and 2946 cm^{− 1}. Other absorption bands at 1174 and 1240 cm^{− 1} are attributed to the C-O-C and -COO- stretching vibration, respectively [29]. As can be seen in Fig. 1, after the loading of DOX, the characteristic peaks of CS and PCEC are observed in the spectrum; however, the presence of DOX broadens these peaks. Additionally, the absorption band at 1650 cm^{− 1} corresponds to the C = C bonds in DOX, confirming its successful loading into the nanocarrier [42]. The conjugation of the CDs with the amine groups of CS leads to a decrease in the intensity of the peak at 3320 cm^{− 1}, indicating the formation of amide bonds between the amine groups of CS and the carboxylic acid groups of the CDs. Furthermore, in the spectrum of the DOX/P/CS-CD complex, the intensity of absorption bands associated with carbonyl ester groups in PCL and C-H aliphatic groups is reduced compared to those in previous spectra. After the conjugation of the aptamer, the DOX/P/CS-CD-Apt complex exhibits a distinctive absorption band at 1076 cm^{− 1}, which can be assigned to the O-P stretching vibration of phosphate groups [43]. The conjugation of the aptamer also causes shifts in the absorption bands of the characteristic peaks, leading to overlaps with peaks at lower wave numbers.

Fig. 1.

A-B) FTIR spectra of DOX, CS, PCEC, DOX/P/CS, DOX/P/CS-CD, and DOX/P/CS-CD-Apt

Size distribution and morphological analysis

To evaluate the morphology and size distribution of the DOX/P/CS-CD-Apt NPs, we utilized FESEM (Fig. 2A-C). The FESEM images revealed that the nanoparticles were well-distributed, without any aggregations or adhesions. They exhibited a similar shape and smooth surfaces, with an average diameter of 81.12 ± 9.86 nm, as calculated using ImageJ software. This size range indicates that the nanoparticles are less likely to be phagocytized after intravenous injection compared to larger nanoparticles [44]. TEM, shown in Fig. 2D, was also employed to assess the size distribution and morphology of the CDs. The average diameter of the CDs was approximately 4.03 ± 1.10 nm. As depicted in the figure, the CDs appear as dark dots against a lighter background due to their high electron density. The hydrodynamic size distribution and surface charge of the prepared nanocarriers were analyzed using DLS and zeta potential measurements (Fig. 2E). According to the DLS data, the average size of the DOX/P/CS-CD-Apt NPs was 235.8 nm. The discrepancy between the particle sizes obtained from FESEM and DLS is expected for polymeric nanocarriers. FESEM provides the dry-state diameter, reflecting only the compact solid core of the particles (~ 81 nm). In contrast, DLS measures the hydrodynamic diameter of nanoparticles dispersed in aqueous media, which includes the surrounding solvation layer and the hydration shell associated with the hydrophilic components of the system. The presence of chitosan and PEG-containing PCL-PEG-PCL segments promotes water absorption and polymer-chain swelling, leading to an expanded corona around the particles and consequently a larger measured size (235.8 nm). Furthermore, electrostatic repulsion among protonated amine groups of chitosan at physiological pH may induce chain relaxation and an extended conformation in solution, contributing additional enlargement of the hydrodynamic radius. Such behavior is characteristic of responsive polymeric nanoparticles and is consistent with the pH-dependent swelling and release behavior observed in the release kinetics study. Importantly, despite this hydration-associated increase in size, nanoparticles within the 200–250 nm hydrodynamic range are widely reported to remain suitable for tumor accumulation through the enhanced permeability and retention (EPR) effect, and the presence of the AS1411 aptamer further ensures active, nucleolin-mediated uptake. Consistent with this, our in vivo PET imaging results confirmed efficient tumor localization of the DOX/P/CS-CD-Apt nanocarrier, indicating that the hydrated particle size did not hinder biodistribution or targeting performance.

Fig. 2.

A-C) FESEM micrographs, and size distribution histogram of DOX/P/CS-CD-Apt NPs. D) TEM image of CDs along with a corresponding particle size distribution histogram. E) (Upper panel) zeta potential values for CD, DOX/P/CS, and DOX/P/CS-CD-Apt. Data are represented as mean ± standard deviation, n = 3. (Lower panel) hydrodynamic size distribution of DOX/P/CS-CD-Apt NPs

Zeta potential is a crucial parameter for evaluating nano drug delivery systems. The electrostatic interactions between nanoparticles and their payloads can influence zeta potential values. The zeta potentials for CD, DOX/P/CS, and DOX/P/CS-CD-Apt were found to be -9.70 mV, + 12.9 mV, and + 10 mV, respectively. The observed changes in zeta potential indicate these electrostatic interactions. Notably, the slight reduction in zeta potential for DOX/P/CS-CD-Apt upon binding with CD and Apt suggests successful attachment to the carrier. Overall, the presence of a positive surface charge on the nanoparticles enhances cellular uptake [45].

Photoluminescence properties of CDs

The optical properties of the prepared CDs dispersed in deionized water were investigated using UV-visible absorption and fluorescence emission spectroscopy. As shown in Fig. 3A, the CDs exhibited two absorption peaks at 258 nm and 370 nm. These peaks correspond to the π→π* transition of the carbon-carbon (C = C) aromatic bond and the n→π* transition of the carbon-oxygen (C = O) bond, respectively [46]. The unheated precursors, which consisted of citric acid and diammonium phosphate, showed maximum absorption at 303 nm. Importantly, the peak at 258 nm resulted in negligible photoluminescence (PL) emission. The n→π* transition, on the other hand, produced significant emission at 445 nm, indicating the capture of excited state energy by surface states (as depicted in Fig. 3B) [47, 48]. The inset in Fig. 3B shows photograph of the aqueous CD solution under normal daylight (right) and ultraviolet (UV) light (left) illumination. The fluorescence spectra of the CD solution exhibited excitation-dependent behavior, with the strongest emission peak occurring at an excitation wavelength of 350 nm. As the excitation wavelength varied from 310 nm to 390 nm, a redshift in the emission peak was observed, along with a reduction in peak intensity, as demonstrated in Fig. 3C. This excitation-dependent photoluminescence of the CDs is believed to be related to the size distribution of the CDs and/or the variation of different emissive sites within them [49]. The quantum yield of the CDs was determined to be 38% when quinine sulfate in an aqueous solution was used as a standard.

Fig. 3.

A) The UV-vis absorption spectrum of the CDs. B) The PL spectrum of the CDs at an excitation wavelength of 350 nm. (Inset: a photograph of the aqueous CD solution under normal daylight on the right and under UV light (λ = 365 nm) illumination on the left. C) The PL spectra of the CDs at various excitation wavelengths

X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) analysis

X-ray photoelectron spectroscopy (XPS) was utilized to investigate the surface elemental composition and chemical functionalities of the synthesized CDs. The survey spectrum (Fig. 4A) displays three prominent peaks at binding energies of 285 eV (C 1s), 400 eV (N 1s), and 532 eV (O 1s), indicating that the CDs are primarily composed of carbon, nitrogen, and oxygen. Elemental analysis revealed that the CDs consist predominantly of carbon (53.56%) and oxygen (41.26%), with a minor contribution from nitrogen (5.18%). The high-resolution C 1s spectrum (Fig. 4B) was deconvoluted into four components located at 283.62 eV, 285.05 eV, 286.30 eV, and 287.77 eV, corresponding to C = C/C–C, C–N, C–O, and C = O functional groups, respectively. These components confirm the presence of various sp² and sp³ hybridized carbon atoms along with oxygen- and nitrogen-containing functionalities. The O 1s spectrum (Fig. 4C) exhibited three peaks at 530.26 eV, 531.00 eV, and 532.16 eV, attributed to carbonyl (O = C), O–C = N/C–O–C, and hydroxyl (C–OH) groups. These peaks indicate significant surface oxidation, suggesting abundant carboxyl, hydroxyl, and ether/imino groups. The N 1s high-resolution spectrum (Fig. 4D) was resolved into three peaks centered at 398.44 eV, 399.71 eV, and 400.75 eV, which are assigned to N–H, N-C-C, and graphitic nitrogen ((C)₃–N), respectively. This distribution confirms successful nitrogen doping and incorporation into both aromatic and aliphatic environments [50]. Together, these results validate the formation of oxygen- and nitrogen-rich surface functionalities, such as –COOH and –NH₂ on the CDs. These functional groups are expected to enhance water dispersibility, biocompatibility, and offer potential sites for further conjugation or surface engineering. The atomic percentages corresponding to each functional group are summarized in Table 1.

Fig. 4.

Surface chemical composition and structural characterization of the synthesized CDs. (A) Survey XPS spectrum showing the presence of C 1s, O 1s, and N 1s elements. High-resolution XPS spectra of (B) C 1s, (C) O 1s, and (D) N 1s, revealing the chemical bonding environments of carbon, oxygen, and nitrogen, respectively. (E) XRD pattern of CDs displaying a broad peak at 2θ = 26°

Table 1.

Atomic percentage values ​​of each bond extracted from the XPS test

Sample	Peak	Bond	Peak location (eV)	Peak area (CPS.eV)	Atomic percentage (%)
CDs	C1s	C = C/C-C	283.62	18542.02	48.60
		C-N	285.05	2529.75	6.63
		C-O	286.30	6893.73	18.05
		C = O	287.77	10211.07	26.72
	O1s	O = C	530.26	27602.21	27.12
		O-C = N/C-O-C	531.00	40144.56	39.43
		C-OH	532.16	34070.82	33.45
	N1s	N-H	398.44	3120.16	41.57
		N-C-C	399.71	2263.09	30.13
		(C)3-N	400.75	2125.87	28.29

The XRD pattern (Fig. 4E) shows a broad diffraction peak centered around 2θ = 26°, which is characteristic of amorphous or poorly crystalline carbon structures. This peak corresponds to the (002) plane of graphitic carbon, suggesting the presence of disordered graphitic-like layers typically observed in CDs.

In vitro loading and release study of DOX from the DOX/P/CS-CD-Apt NPs

In this study, a double-emulsion solvent-evaporation method followed by crosslinking was employed to encapsulate DOX within a polymeric matrix. The encapsulation of DOX was facilitated by hydrophobic interactions, electrostatic attractions, and hydrogen bond formation between the drug molecules and the polymeric network. The encapsulation efficiency (EE) and drug loading content (LC) were found to be 92% and 9.2%, respectively, using a drug-to-nanocarrier feeding ratio of 1:10. The pH values selected for the release study (pH 7.4 and 5.4) were chosen to represent the physiological blood environment and the acidic intracellular compartments, respectively. Although the extracellular tumor microenvironment is mildly acidic (pH 6.5–7.0), numerous studies have demonstrated that nanoparticle-mediated DOX delivery relies primarily on endocytic uptake, followed by drug release in endosomal/lysosomal vesicles, where the pH typically falls to 5.0–5.5 [51, 52]. After internalization, DOX-loaded nanoparticles traffic into these acidic compartments, leading to accelerated drug release through pH-responsive mechanisms and subsequent diffusion of DOX into the cytosol and nucleus [51, 53]. Therefore, pH 5.4 was selected to model the intracellular release environment, which represents the dominant stage of drug activation in polymeric nanosystems.

The cumulative release profile of DOX from the DOX/P/CS-CD-Apt nanoparticles at pH 5.4 and 7.4 at 37 °C is presented in Fig. 5A. At both pH conditions, DOX exhibited a sustained release behavior, attributed to its interactions with the nanocarrier matrix. After 120 h of incubation, the cumulative release of DOX reached approximately 52.32% at physiological pH (7.4) and 84.41% at acidic pH (5.4), mimicking lysosomal conditions. The pH-responsive release behavior is primarily due to the protonation of chitosan’s free amine groups (pKa ≈ 6.5) at acidic pH, which leads to increased electrostatic repulsion among positively charged amines. This repulsion disrupts the interwoven polymeric chains, enhancing DOX diffusion into the surrounding medium. Moreover, the increased protonation at lower pH reduces the stability of hydrogen bonds between DOX and chitosan, further contributing to the destabilization of the drug-carrier complex and promoting drug release. The proposed mechanism for DOX release from the nanocarrier is illustrated in Fig. 5B. This controlled and pH-sensitive release behavior is advantageous for cancer therapy as it reduces systemic side effects while enhancing drug accumulation and therapeutic efficacy at the tumor site.

Fig. 5.

A) In vitro release profile of DOX from the DOX/P/CS-CD-Apt NPs at pH 5.4, 7.4, and 37 °C. Data represent mean ± standard deviation, n = 3. B) A schematic representation demonstrating the interactions involved in the loading of DOX onto nanocarriers

Drug release kinetics and mechanistic modelling

To understand the mechanism underlying DOX release from the nanocarrier, experimental release data at pH 7.4 and pH 5.4 were fitted to several commonly used kinetic models, including zero-order, first-order, Korsmeyer-Peppas, and Higuchi models. The fit quality for each model was assessed by comparing their correlation coefficients (R² values), calculated using KinetDS software. As shown in Table 2, the Korsmeyer-Peppas model had the highest R² value for DOX release at pH 7.4 (R² = 0.9661), indicating this model best describes the release behavior under physiological conditions. In the Korsmeyer-Peppas framework, the release mechanism is analyzed based on the diffusional exponent n, where n ≤ 0.45 indicates Fickian diffusion (case I), 0.45 ≤ n < 0.89 denotes anomalous (non-Fickian) diffusion, n = 0.89 signifies zero-order release (case II), and n > 0.89 suggests super case II transport. The DOX release exponent at pH 7.4 (n = 0.1013) is well below 0.45, confirming that DOX release from the nanocarrier follows a Fickian diffusion-controlled mechanism. At pH 5.4, the Korsmeyer-Peppas model again showed the highest R² value (R² = 0.9726), with a corresponding release exponent of n = 0.1030, indicating diffusion-driven drug release, similar to what was observed at physiological pH. Although the Higuchi model also provided an acceptable fit at pH 5.4 (R² = 0.9726), the higher R² of the Korsmeyer-Peppas model and the low n value both support a mainly Fickian diffusion mechanism. According to the Higuchi theory, drug release is controlled by diffusion through a hydrated porous matrix, and initial drug loading influences the development of a concentration gradient responsible for sustained diffusion into the surrounding medium [54]. The relatively high Higuchi R² value at acidic pH suggests that increased polymer swelling and protonation of chitosan may enhance DOX mobility; however, the overall release behavior remains primarily diffusion-controlled. These analyses together confirm that DOX release from the DOX/P/CS-CD-Apt nanosystem is governed by Fickian diffusion, with faster drug release under acidic conditions consistent with the endosomal and lysosomal microenvironment.

Table 2.

Kinetic modelling parameters for DOX release from DOX/P/CS-CD-Apt nanoparticles at pH 7.4 and pH 5.4, calculated using KinetDS software

Release Model	Zero-Order	Korsmeyer-Peppas		First-Order	Higuchi
	R 2	R 2	n*	R 2	R 2
DOX (7.4)	0.5041	0.9661	0.1013	0.0605	-1.34
DOX (5.4)	0.5322	0.9726	0.1030	0.0670	0.9726

* Diffusion or release exponent

Confirmation of the AS1411 aptamer conjugation onto the surface of nanocarriers

To confirm the successful conjugation of the AS1411 aptamer to the surface of the nanocarriers via amide bond formation between the carboxylic acid groups of the aptamer and the amine groups of CS, agarose gel electrophoresis was performed using a 3% gel. As shown in Fig. 6A, different components were loaded onto the gel: DNA aptamer at concentrations of 5, 10, and 20 pmol (Lanes 2, 3, and 4, respectively), DNA ladder (Lane 1), DOX/P/CS-CD-Apt nanocarriers (Lanes 5 and 6), the filtrate (Lane 7), and bare CDs (Lane 8). The gel was electrophoresed in Tris-borate-EDTA (TBE) buffer at 110 V for 30 min, and DNA bands were visualized using a standard transilluminator. The results demonstrated successful aptamer conjugation. Notably, the DOX/P/CS-CD-Apt samples (Lanes 5 and 6) did not migrate toward the positive electrode, indicating covalent bonding between the aptamer and the nanocarrier, which limited their electrophoretic mobility. In contrast, free aptamer bands (Lanes 2, 3, and 4) showed expected migration patterns. The observed band shift and immobilization in Lanes 5 and 6, compared to the free aptamer, confirmed that conjugation altered the surface charge and mobility. Additionally, no significant band was observed in the filtrate (Lane 7), suggesting that most aptamers were successfully bound to the nanocarrier surface, further confirming the efficiency of the conjugation process. In this study, the AS1411 aptamer was employed as a dual-function agent, serving both as a targeting ligand and a carrier for the specific delivery of DOX to MCF-7 breast cancer cells.

Fig. 6.

A) Gel electrophoresis mobility shift characterization of the formation of DOX/P/CS-CD-Apt nanocarriers. Lane 1: DNA Marker; Lane 2, 3, and 4: Apt with concentrations of 5, 10, and 20 pmol, respectively. Lane 5, 6: DOX/P/CS-CD-Apt nanocarrier. Lane 7: the mixture under the filter. Lane8: CDs. The rectangle indicated AS1411 conjugated successfully. B-D) In vitro cytotoxicity of various DOX formulations against MCF-7 cells after being exposed to different concentrations of DOX, DOX/P/CS-CD, DOX/P/CS-CD-Apt, and control after (B) 24 h, (C) 48 h, and (D) 72 h. All tests were repeated three times, n = 3, and the data are shown based on (mean ± SD). *p < 0.05, **p < 0.01, and ***p < 0.001

In vitro cytotoxicity as say

To evaluate the therapeutic potential and safety profile of the developed DOX-loaded nanocarriers, an MTT assay was conducted to assess the cytotoxicity of blank nanoparticles (control), free DOX, DOX/P/CS-CD-Apt, and DOX/P/CS-CD against MCF-7 cells over 24, 48, and 72 h. Untreated MCF-7 cells served as the negative control and ANOVA test was used to analyze the data. As shown in Fig. 6B-D, cell viability decreased in a dose- and time-dependent manner for all DOX-containing treatments. At 24 hours (Fig. 6B), a reduction in cell viability was observed at higher doses in all treatments (but not significant among groups). After 48 hours (Fig. 6C), differences were seen: DOX/P/CS-CD-Apt exhibited significantly enhanced cytotoxicity compared to free DOX at concentrations of 0.2, 0.4, and 3.2 μM (*p < 0.05). By 72 hours (Fig. 6D), all DOX-loaded formulations led to a marked decrease in cell viability, with DOX/P/CS-CD-Apt showing the strongest effect. At the highest concentration (3.2 μM), cell viability dropped to approximately 26.3% for the targeted formulation, significantly lower than for free DOX or DOX/P/CS-CD alone (*p < 0.05). The IC₅₀ values after 72 h further support these findings, calculated as 0.70 µM for free DOX, 0.66 µM for DOX/P/CS-CD, and 0.59 µM for DOX/P/CS-CD-Apt. This trend demonstrates that DOX encapsulation into the P/CS-CD matrix slightly enhances cytotoxicity, while aptamer functionalization further boosts cellular uptake and therapeutic efficacy due to nucleolin-mediated targeting. Notably, the blank nanoparticles (without DOX) showed negligible cytotoxicity across all time points, confirming their biocompatibility. Collectively, these results suggest that the DOX/P/CS-CD-Apt formulation provides improved therapeutic performance compared to free DOX, particularly over longer incubation periods, owing to its enhanced internalization and sustained release properties.

Cell apoptosis analysis

To find out the levels of cell necrosis and apoptosis after treatments, flow cytometry was used. Quadrants Q1, Q2, Q3, and Q4 correspond to necrotic (FITC-/PI+), late apoptotic (FITC+/PI+), early apoptotic (FITC+/PI-), and viable cells (FITC-/PI-), respectively. The flow cytometry diagram illustrates the quadrant regions. MCF-7 cells stained with Annexin V-FITC underwent an apoptosis assessment, indicating that the cells exhibited greater sensitivity to DOX/P/CS-CD compared to free DOX. Furthermore, functionalizing the complexes with the Apt enhanced this sensitivity, reduced the rate of cell division, and raised the amount of apoptosis. Our apoptosis percent frequencies were analyzed by ANOVA method. According to the findings, within 72 h, there were roughly 26.90 ± 0.537 apoptotic cells in the cells treated with free DOX (9.80 ± 0.735% early and 17.10 ± 1.2% late apoptosis), which was associated with reduced drug uptake (Fig. 7). In the group treated with DOX/P/CS-CD NPs, the percentage of apoptotic cells reached 35.582 ± 2.676%, with the majority (17.90 ± 1.10%) undergoing late-stage apoptosis (Fig. 7). After receiving DOX/P/CS-CD-Apt treatment, the percentage of apoptotic cells rose to 67.75 ± 3.50%, with the majority of those cells (26.30 ± 1.27%) at a late stage and (41.45 ± 2.23%) at an early stage of apoptosis (Fig. 7). On MCF-7 cells, a nanocarrier containing DOX/P/CS-CD-Apt caused almost 1.55 ± 0.74% necrosis.

Fig. 7.

Annexin V and PI staining were used to identify viable cells (annexin V−, PI−), early apoptotic cells (annexin V+, PI−), late apoptotic cells (annexin V+, PI+), and necrotic cells (annexin V−, PI+). The apoptotic effects of cells, determined by flow cytometry after 72 h in MCF-7 cells for control (A), free DOX (0.70µM (B), DOX/P/CS-CD (0.66µM) (C), and DOX/P/CS-CD-Apt (0.59 µM) (D). Quantitative results of apoptotic effects evaluated by Annexin V/FITC assay (E). All tests were repeated three times, n=3, and the data are shown based on (mean±SD). *p < 0.05, **p < 0.01, and ***p < 0.001

Figure 7 shows the apoptotic effects of cells, determined by flow cytometry after 72 h in MCF-7 cells. According to Fig. 7A, the highest number of live cells was observed in the control group, where the average live cells (%) in this group were 93.5 ± 0.73, and the lowest number was observed in the DOX/P/CS-CD-Apt group, where the average live cells (%) in this group were 30.8 ± 4.11. The DOX, DOX/P/CS-CD, and DOX/P/CS-CD-Apt groups showed a significant decrease compared to the Control group) p < 0.05(. The DOX/P/CS-CD-Apt group had the highest rate of early apoptosis (Fig. 7), with a mean of 41.45 ± 2.23, while the control group had the lowest rate, with a mean of 1.85 ± 0.74. Comparing the DOX, DOX/P/CS-CD, and DOX/P/CS-CD-Apt groups to the control group, there was a substantial increase (p < 0.05). The DOX/P/CS-CD-Apt group had the highest rate of late apoptosis (Fig. 7), with a mean of 26.3 ± 1.27; the control group had the lowest rate, with a mean of 2.76 ± 0.50. Comparing the DOX, DOX/P/CS-CD, and DOX/P/CS-CD-Apt groups to the control group, there was a substantial increase (p < 0.05). With an average of 2.4 ± 0.594 necrotic cells (%) (Fig. 7), the DOX group had the most, whereas the DOX/P/CS-CD group had the fewest, with an average of 1.018 ± 0.25. Comparing the DOX, DOX/P/CS-CD, and DOX/P/CS-CD-Apt groups to the control group, no discernible changes were seen (p > 0.05). With an average of 67.75 ± 3.50, the DOX/P/CS-CD-Apt group had the highest rate of total apoptosis (%) (Fig. 7), whereas the Control group had the lowest rate, averaging 4.61 ± 0.24. When compared to the Control group, the DOX, DOX/P/CS-CD, and DOX/P/CS-CD-Apt groups displayed a substantial increase (p < 0.05). This rise in apoptosis may be linked to increased drug penetration by NPs into the cells. Cells could therefore adjust and make up for their circumstances in response to drug concentration. The groups that received free medication and nanocarrier treatment differed significantly, as seen in Fig. 7.

Cell uptake study

The intracellular uptake behavior of the DOX/P/CS-CD-Apt NPs is investigated by fluorescent imaging of the MCF-7 cells at different time intervals, as shown in Fig. 8A. To facilitate visualization of nanoparticle internalization, CDs were covalently attached to the nanocarriers as a blue fluorescent probe, while DOX itself served as the red fluorescent marker to identify the cells. The cells were treated with about 0.59 µM DOX/P/CS-CD-Apt NPs for 0.5, 1, and 1.5 h to determine the time-dependent uptake of the CD-conjugated nanoparticles. At 0.5 h, free DOX displayed strong red fluorescence due to its rapid diffusion across the cell membrane, whereas the blue fluorescence from CD-labeled nanocarriers was relatively weak, indicating the early stage of receptor-mediated internalization. At 1 h, an increase in blue fluorescence was observed, reflecting progressive uptake of the aptamer-functionalized nanocarriers. By 1.5 h, the CD signal became more intense and widely distributed within the cells, showing efficient internalization of the NPs. Overall, the increasing fluorescence intensity over time demonstrates a clear time-dependent internalization process. By increasing the incubation time, the fluorescence intensity increased, indicating a time-dependent internalization process (Fig. 8B). These findings indicate that DOX/P/CS-CD-Apt NPs primarily enter MCF-7 cells through receptor-mediated endocytosis, enabling sustained and targeted intracellular accumulation of DOX compared to the free drug.

Fig. 8.

(A) Fluorescence images of MCF-7 cells incubated with DOX/P/CS-CD-Apt (0.59 µM) at 37 °C for 0.5, 1, 1.5 h. (B) The mean fluorescent intensity of uptake images were analyzed by Image J and reported as histogram graphs. The uptake was time depandant and in 1.5 h become densely fluorescent. *** p < 0.001

In vivo small-animal optical imaging

Fluorescence imaging was conducted to assess the uptake of conjugates, and qualitative analysis was performed at different times. Figure 9 illustrates the accumulation of DOX/P/CS-CD-Apt NPs at 30 min to 72 h post-injection at the tumor site, in comparison to the control group. The figure shows that, compared to the control group, the nanocarrier accumulated in the tumor areas five minutes after the mice were injected with the nanocarrier DOX/P/CS-CD-Apt. The quantity of accumulation and luminous intensity also grew at 30 min, 1 h, 2 h, and 4 h after injection, but at 24, 48, and 72 h, the fluorescent amount reduced. One of the reasons for this decrease can be attributed to the effectiveness of the drug on cancer cells. This effectiveness is confirmed by the data related to cell viability and DOX release, which was at its highest after 50 to 96 h. Additionally, the results of the PET scan, which are shown in Fig. 9, as well as the reduction in tumor size in comparison to the control group, also confirm this effectiveness.

Fig. 9.

(A) Whole body fluorescence imaging acquired from mice. Accumulation of DOX/P/CS-CD-Apt in mice after 5 min injection (blank), 30 min, 1 h, 2 h, 4 h, 24 h, 48 h and 72 h post injection. (B) Accumulation of DOX/P/CS/CD (non-targeted CS) in mice

Small-animal PET imaging and image analysis

Representative small-animal PET images for the treated and control groups are shown in Fig. 10. The figure presents 18 F-FDG PET scans of 4T1 tumor-bearing mice in the control group (untreated) and the treatment group (injected with DOX/P/CS-CD-Apt NPs). The control group consists of tumor-bearing mice (the tumor is marked in red on their flanks). The control group received 0.3 Mci of the 18 F-FDG and was imaged for 30 min after injection. As expected, we witnessed the accumulation of 18 F-FDG in the tumor area. In the treated samples, DOX/P/CS-CD-Apt NPs were injected into the same mice, and PET imaging was performed again after 72 h, which reduced tumor size and decreased the reduction in 18 F-FDG accumulation compared to the control, indicating the positive effect of tumor size reduction.

Fig. 10.

The small animal PET images of mice treated with DOX/P/CS-CD-Apt NPs and control group

Discussion

Nanoparticle-based targeted therapies have been widely utilized in cancer treatment to reduce the systemic toxicity associated with conventional chemotherapeutic agents. However, repeated administration may still offer serious risks, including nanoparticle-related toxicity and off-target effects, which can be life-threatening. Therefore, it is vital to develop safer and more effective strategies that improve tumor-specific accumulation and cytotoxicity while reducing adverse effects on healthy tissues [55]. In this study, we synthesized CDs via a one-step hydrothermal method. Subsequently, we encapsulate DOX within the CS-PCEC matrix using a double emulsion solvent evaporation technique, followed by crosslinking with ECH. For targeting and diagnostic purposes, the AS1411 aptamer and CDs were conjugated to chitosan using EDC/NHS chemistry. Successful conjugation was confirmed through DLS, FT-IR, electrophoresis, SEM, TEM, XPS, and XRD analyses. A notable decrease in zeta potential and the absence of band formation in gel electrophoresis confirmed the successful attachment of CDs and aptamer to the chitosan backbone. The prepared NPs indicate the LC of 9.2% and EE of 92%. In vitro release studies demonstrated that DOX release was significantly higher at acidic pH 5.4 compared to physiological pH 7.4. DOX is a potent and widely used first-line chemotherapeutic agent with well-established anticancer activity [35]. This study is the first to chemically conjugate the AS1411 aptamer for targeted delivery to cancer cells while simultaneously employing fluorescent CDs for diagnostic imaging, thereby integrating therapy and diagnosis into a single nanoplatform. Although previous studies have examined chitosan or CD based nanocarriers functionalized with different aptamers, our system distinctively assimilates covalently attached CDs inside a pH-responsive chitosan/PCL-PEG-PCL hybrid, reaching a dually controllable release, and confirmed by preclinical (in vivo) theranostic evaluation.

The results demonstrated that DOX exerted a cytotoxic effect on MCF-7 cells, and this effect was significantly enhanced when delivered via DOX/P/CS-CD and DOX/P/CS-CD-Apt nanocarriers. Notably, the incorporation of the AS1411 aptamer in DOX/P/CS-CD-Apt resulted in approximately 10–15% higher cytotoxicity compared to the non-targeted DOX/P/CS-CD formulation. Supporting this observation, Pina et al. reported that aptamer-mediated active targeting using the 5TR1 aptamer led to a 20% increase in cancer cell death compared to non-targeted nanocarriers [56]. The enhanced cytotoxicity observed in our study can be attributed to several factors, including the reduced particle size of the nanoparticles, which facilitates greater cellular uptake, improves colloidal stability, and promotes deeper penetration into tumor cells [57]. Additionally, the modified surface morphology of the DOX/P/CS-CD-Apt nanocarrier may contribute to overcoming multidrug resistance mechanisms, an advantage not observed with free DOX [58]. AS1411 facilitates cellular uptake via nucleolin-mediated macropinocytosis, a rapid internalization pathway compared to passive endocytosis, which typically leads to lysosomal entrapment [59]. This mechanism allows for faster intracellular trafficking and more efficient cytoplasmic release of the pH-responsive drug payloads, thereby minimizing lysosomal degradation and reducing exposure of healthy cells to high drug concentrations. Additional potential mechanisms for cell death that have been previously documented include cell cycle arrest, blocking specific signaling pathways such as NF-κB, and direct antiproliferative activity of the aptamer itself [60]. These multifaceted mechanisms contribute to the superior anticancer efficacy of the targeted nanocarrier system.

Several studies have explored the use of CS, CDs, and the AS1411 Apt for targeted drug delivery to cancer cells. For instance, Mohammadzadeh et al. developed a novel cancer diagnostic agent by conjugating AS1411 Apt with a generation 2 anionic linear globular dendrimer (G2), loaded with the Iohexol contrast agent. The targeting capability, biocompatibility, and imaging performance of the formulation were evaluated through a series of in vitro and in vivo studies. Their results demonstrated that this Apt-functionalized system could selectively recognize and accumulate in cancer cells while exhibiting minimal toxicity to healthy tissues. The targeted nature of this diagnostic platform makes it a promising candidate for tumor-specific computed tomography (CT) imaging [61]. A similar approach utilizing the AS1411 aptamer was reported by Behrooz et al., who developed a PAMAM dendrimer-PEG nanocarrier conjugated with 5-FU for the targeted treatment of gastric cancer. The aptamer-functionalized dendrimer, referred to as a “smart bomb,” was designed to enhance selective drug delivery to cancer cells with subsequent enhanced tumoricidal effects [62].

In a related study, Kong et al. developed a fluorescent imaging nanosystem by modifying CDs with the AS1411 aptamer for selective targeting of nucleolin-overexpressing cancer cells. The high-luminescence CDs were synthesized via a hydrothermal method using citric acid and subsequently coated with polyethylenimine (PEI) to facilitate electrostatic assembly. The resulting CDs–PEI complex was functionalized with AS1411 aptamer through electrostatic interactions, forming the CDs–PEI–AS1411 nanosystem. Its targeting efficiency was evaluated using MCF-7 cells (high nucleolin expression) and L929 cells (low nucleolin expression) through fluorescence imaging and flow cytometry. The results demonstrated significantly enhanced cellular uptake of the nanosystem in MCF-7 cells compared to L929 cells, confirming its potential as a targeted imaging probe for cancer diagnostics [43].

In the present study, the enhanced internalization of the DOX/P/CS-CD-Apt nanosystem into MCF-7 cells can be attributed to the high binding affinity between the AS1411 aptamer and nucleolin receptors. To further investigate this, we assessed the cellular uptake of the DOX/P/CS-CD-Apt nanosystem using fluorescence microscopy. MCF-7 cells treated with the targeted nanoparticles exhibited significantly higher fluorescence intensity compared to control cells, indicating superior internalization. This difference is likely due to the elevated expression of nucleolin on the surface of MCF-7 cells, which enhances the efficiency of AS1411-mediated uptake. These findings were further validated by flow cytometry analysis, which confirmed the microscopy results and demonstrated a high cellular uptake efficiency of the DOX/P/CS-CD-Apt formulation.

Also, CDs were utilized in this investigation due to their well-established advantages, including high quantum yield, excellent resistance to photobleaching, strong tissue penetrability, and minimal phototoxicity [63]. In addition, the large specific surface area of CDs facilitates higher drug-loading capacity, thereby enhancing the overall anticancer performance of the nanocarrier. The ultrasmall size of CDs (< 10 nm) further contributes to the reduced dimensions of the final construct, enabling efficient movement through biological tissues, improved endocytic uptake, and effective intra- and intercellular trafficking [64].

Along with their biocompatibility and inherent photostability, the synthesized CDs exhibited a quantum yield of ~ 38%, which is comparable to high-performing hydrothermally prepared CDs such as CATris (QY ≈ 37%) [65] and falls within the typical 10–60% range reported for bottom-up carbon dots [66]. When compared to standard fluorophores, the brightness of our CDs remains well within the operational range for biological imaging. For example, although fluorescein can achieve QY ≈ 0.93 under strongly basic conditions, its monoanionic form, dominant near physiological pH, exhibits a much lower QY (~ 0.37) [67]. Similarly, Cy5 commonly displays QYs of 0.20–0.30 in aqueous or buffered environments due to aggregation and solvent quenching [68]. Carbon dots also exhibit superior resistance to photobleaching, as demonstrated for citric acid-derived CDs that retain fluorescence under prolonged UV exposure [69], with broader reviews confirming their enhanced long-term photostability compared with many organic dyes [70]. Collectively, these comparisons confirm that the optical properties of our CDs, brightness, environmental stability, and photostability, are well-suited for reliable imaging within theranostic cancer nanomedicine.

Together, these findings support the conclusion that AS1411-functionalized nanoparticles incorporating CDs not only improve selective accumulation in cancer cells but also enhance therapeutic efficacy and imaging capability. The AS1411 aptamer facilitates receptor-mediated endocytosis via nucleolin binding, while CDs contribute to sensitive fluorescence tracking, thereby enabling simultaneous targeted delivery and real-time detection within the tumor microenvironment. Our study has some limitations. First, in vitro targeting efficacy was assessed in a single nucleolin-overexpressing breast cancer cell line; inclusion of nucleolin-low expression or negative controls would strengthen targeting specificity claims. Next, in vivo evidence is constructed mainly on imaging data within the evaluated time window; future work should comprise quantitative biodistribution analysis such as ex vivo organ fluorescence or radioactivity, and pharmacokinetics, expanded animal numbers, and longer monitoring. Finally, full safety evaluations histopathology, biochemistry, and immunogenicity are needed to describe the therapeutic index and translational significance.

Future effort will focus on confirming this uptake pathway by quantitative investigation of nucleolin expression, competitive binding analyses with free AS1411, and co-localization examining with lysosomal and endosomal markers to clarify intracellular transferring directions. Also, our in vivo results represent qualitative biodistribution patterns rather than quantitative organ-level uptake which could be focused in future investigations.

Conclusion

In conclusion, we developed a multifunctional theranostic nanocarrier composed of CS, fluorescent CDs, and the nucleolin-targeting aptamer (AS1411) for DOX delivery and fluorescence imaging. The physicochemical analyses confirmed effective nanocarrier fabrication and aptamer conjugation. The DOX/CS-CD-Apt showed a controlled, pH-responsive release profile in vitro. In MCF-7 breast cancer cells (nucleolin-overexpressing), the aptamer-functionalized nanocarrier demonstrated higher cellular uptake, elevated cytotoxicity, and apoptosis relative to non-targeted nanocarrier under the tested conditions. In vivo fluorescence and small-animal PET imaging indicated obvious accumulation of the DOX/CS-CD-Apt in the tumor region in the employed model, supporting its potential for combined imaging and drug delivery. Together, this work is limited to the experimental models and endpoints evaluated. More validation across additional breast cancer models (including nucleolin-low expressing controls), larger cohorts, quantitative organ biodistribution, longer follow-up, and comprehensive safety and efficacy comparisons will be necessary before translational utility. Overall, our results support the feasibility of the DOX/CS-CD-Apt platform as a candidate theranostic system and provide a basis for further optimization and prolonged preclinical assessment.

Author contributions

MJM: Writing- original draft; Investigation; Methodology. EA: Conceptualization; Methodology; Supervision; Validation, review & editing; Project administration. SVM.: Conceptualization; Formal analysis; Investigation; Methodology; Validation; Writing- review & editing; Project administration. NSA: Methodology, Validation; AN: Investigation.

Funding

This work was supported by the Deputy of Research and Technology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran, grant number: 66652.

Data availability

The data and analyses generated during this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable. This study did not involve human participants, data, or biological materials that required ethical approval.

Consent for publication

Consent for publication was not applicable, as this manuscript does not include any individual person’s data in any form. All authors have reviewed and approved the final draft and consented to its submission for publication.

Competing interests

The authors declare no competing interests.