Folic Acid-Decorated Chitosan-PLGA Nanobiopolymers for Targeted Drug Delivery to Acute Lymphoblastic Leukemia Cells: In Vitro Studies

Abstract

Objectives

This study developed a drug delivery system (DDS) using folic acid (FA)-functionalized chitosan (CS) and poly (lactic-co-glycolic acid) (PLGA) nanocarriers for targeted sodium butyrate (NB) delivery to leukemia cells (NALM6). The goal was to enhance NB's therapeutic efficacy while reducing its cytotoxicity to non-malignant cells.

Methods

FA-CS-PLGA nanocarriers were synthesized and characterized using Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), zeta potential analysis, transmission electron microscopy (TEM), and thermogravimetric analysis (TGA). Encapsulation efficiency, release kinetics, cytotoxicity, and apoptosis induction were assessed using MTT assays and flow cytometry in NALM6 cells.

Results

The FA-CS-PLGA nanocarriers had a surface charge of 34.2 ± 0.12 mV and a size range of 40–60 nm. Encapsulation efficiency was 16%, with 16% of NB released within the first 4 h. MTT assays showed a reduction in leukemia cell viability to 26% after 24 h with 400 nM FA-CS-PLGA-NB, compared to over 50% viability with pure NB. The IC50 was around 300 nM. Flow cytometry revealed that FA-CS-PLGA-NB induced apoptosis in over 20% of leukemia cells, far exceeding the 5% induced by unmodified NB.

Conclusion

FA-CS-PLGA nanocarriers show significant promise as a targeted DDS for leukemia therapy, enhancing NB delivery to leukemia cells and improving therapeutic efficacy while minimizing off-target toxicity. These results support further in vivo studies and potential clinical applications.

Keywords

chitosan PLGA folic acid targeted drug delivery acute lymphoblastic leukemia

Introduction

Acute lymphoblastic leukemia (ALL) is a hematological malignancy characterized by the clonal proliferation of immature lymphoid cells within the bone marrow, peripheral blood, and other tissues.¹ It remains the most prevalent form of leukemia in children, although it also significantly affects adults, particularly those over 65 years of age.² Globally, the incidence of ALL is on the rise, contributing to approximately 25–30% of all pediatric cancer cases.³ Despite notable advancements in chemotherapy, immunotherapy, and bone marrow transplantation, the treatment of ALL continues to present major challenges due to high relapse rates and the emergence of drug resistance.⁴ In addition, conventional therapies are frequently associated with severe adverse effects, including immunosuppression, organ toxicity, and an elevated risk of secondary malignancies.⁵ The heterogeneity of ALL, driven by diverse genetic and molecular abnormalities, complicates treatment strategies, highlighting the urgent need for more targeted and less toxic therapeutic approaches.⁶ Addressing these challenges necessitates ongoing research into novel therapeutic modalities, including the application of nanotechnology and biopolymer-based drug delivery systems (DDSs), which have demonstrated promise in enhancing therapeutic outcomes while minimizing systemic toxicity.⁷

Nanoparticles as DDSs offer a significant advantage by improving the efficacy of cancer treatments.⁸ Among the various nanocarrier systems, biopolymers stand out due to their ability to evade immune responses, protect bioactive compounds from enzymatic degradation, extend half-life by reducing renal clearance, prevent liver filtration, and prolong therapeutic circulation time.^9,10 Of the available biopolymers, chitosan (CS) and polylactic-co-glycolic acid (PLGA) have been approved by the U.S. FDA for their applications in biomedical fields, thanks to their excellent biocompatibility, biodegradability, and low immunogenicity.^11,12 Chitosan, a natural polysaccharide composed of poly[β-(1-4)-linked-2-amino-2-deoxy-D-glucose], has been extensively investigated for its suitability in drug delivery systems due to its stability, hydrophilicity, solubility in biological fluids, and favorable pharmacokinetic and pharmacodynamic properties.^13–15

However, despite these advantages, the use of CS and PLGA-based DDSs for cancer treatment faces several challenges, including limited drug loading capacity, inadequate control over drug release, and suboptimal targeting specificity, all of which reduce therapeutic efficacy.^16–19 To address these limitations, recent advancements have focused on surface functionalization strategies, such as incorporating targeting ligands like folic acid (FA).²⁰ FA-functionalized DDSs enhance selective targeting of cancer cells by binding to folate receptors, which are often overexpressed on the surface of many cancer cells, including leukemia cells.²¹ These systems not only improve targeted drug delivery but also benefit from the enhanced permeability and retention (EPR) effect, allowing for more efficient accumulation of the DDS in tumor tissues.^21,22 Furthermore, FA-functionalization enhances serum stability, solubility, and overall pharmacokinetics by preventing premature degradation of nanoparticles in the bloodstream and eNBling controlled drug release at the tumor site.^23,24

Incorporating naturally derived bioactive compounds, such as sodium butyrate (Na-OOCCH2CH2CH3, NB), into cancer therapy holds considerable potential for the development of innovative, nature-based DDSs targeting a variety of cancers, including lung, breast, kidney, liver, and leukemia.²⁵ Sodium butyrate is known for its potent anti-cancer effects, as it inhibits vascular endothelial growth factor (VEGF) by suppressing histone deacetylases (HDACs), thereby reducing VEGFR-mediated angiogenesis and blocking protein kinase B (Akt) signaling, which ultimately leads to cell cycle arrest at the G2/M phase.²⁶ dditionally, NB decreases the levels of proteins and cytokines associated with angiogenesis and apoptosis—such as Akt, Bcl2, IL6, and IL17—further reducing cancer cell viability, making it an effective therapeutic candidate for integration into targeted drug delivery systems.^26,27

This study aims to address the current shortcomings of existing drug delivery systems by developing a novel FA-functionalized CS-PLGA nanocarrier system designed to enhance the targeted delivery of sodium butyrate to leukemia cells. This approach seeks to improve therapeutic efficacy while minimizing systemic toxicity, overcoming the limitations seen in conventional DDS strategies for cancer treatment.

Materials and Methods

Materials and Apparatus

Chitosan with a molecular weight of 50 000 to 190 000 Da and viscosity ranging from 20 to 300 cp, polylactic-co-glycolic acid (PLGA) (50:50, Mw: 30 000-60 000 Da), folic acid (FA) (Mw: 441.4 g/mol), sodium butyrate (NB, ≥ 98.5% GC, Mw: 110.09 g/mol), tripolyphosphate (TPP), 1-Ethyl-3-(3-dimethylaminopropyl)-carbodi-imide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) were all obtained from Sigma-Aldrich. Additionally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), penicillin, streptomycin, fetal bovine serum (FBS), and cellulose dialysis membranes (Mw = 12 000 Da) were procured from Sigma-Aldrich. The NALM6 leukemia and HDF normal cell lines were sourced from the Pasteur Institute of Iran.

Characterization of the synthesized products was performed using various analytical tools, including a NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific), Fourier-transform infrared (FT-IR) spectrometer (Perkin-Elmer 843), thermogravimetric analysis (TGA) (STA 1500; Rheometric Scientific), transmission electron microscopy (TEM) (Philips EM 208S; Netherlands), and dynamic light scattering (DLS) spectroscopy (NanoBrook 90 Plus; Brookhaven, USA).

Synthesis of CS-PLGA (CS-P) Nanoparticles

PLGA was conjugated to CS using an activation process. Initially, 40 mg of PLGA was dissolved in dichloromethane and stirred at 70 rpm. A mixture of EDC and NHS (1:1 molar ratio) was added to the PLGA solution under a nitrogen atmosphere and dark conditions, with stirring at 70 rpm for 3 h to complete the activation. Separately, 50 mg of CS was dissolved in 6 mL of 1% acetic acid and stirred at 70 rpm for 2 h. The activated PLGA solution was then combined with the CS solution, and the conjugation reaction was allowed to proceed overnight under light-protected conditions in a nitrogen atmosphere, with continuous stirring at 70 rpm. The final solution was washed three times with a 3:1 water-ethanol mixture to remove unreacted PLGA molecules. The solution was then split into two portions: one half was used to prepare spherical CS-P nanoparticles, and the other half was saved for subsequent synthesis. For nanoparticle fabrication, 1 mL of TPP was slowly added to the CS-P solution under ultrasonication, and the sample was stored at 4 °C for further analytical and biomedical assessments.

Synthesis of FA-CS-PLGA (FA-CS-P) Nanoparticles

The synthesis of FA-CS-P was initiated by activating FA with NHS and EDC. Briefly, 10 mg of FA was dissolved in DMSO and stirred at 70 rpm, followed by the addition of an EDC/NHS mixture (1:1 molar ratio). This reaction was conducted for 3 h under a nitrogen atmosphere, dark conditions, and stirring at 70 rpm. The activated FA solution was then mixed with the prepared CS-P solution and allowed to react overnight with vigorous stirring at room temperature in dark conditions. The resulting FA-CS-P solution was centrifuged at 4000 rpm for 4 min to separate any unreacted FA molecules. The solution was then divided into two parts: one portion was used to prepare spherical FA-CS-P nanoparticles by adding 1 mL of TPP under ultrasonication, and the other portion was reserved for drug entrapment. The spherical FA-CS-P nanoparticles were stored at 4 °C for future analysis.

Preparation of NB-Entrapped FA-CS-P Nanoparticles

Sodium butyrate (NB) was loaded into the FA-CS-P nanoparticles through the following procedure. First, 5 mg of NB was dissolved in 8 mL of deionized (DI) water and stirred at 80 rpm for 4 h to obtain a homogenous solution. This NB-containing solution was added to 6 mL of the previously prepared FA-CS-P solution, and the mixture was stirred at 50 rpm for 5 h at 37 °C. To form the spherical drug delivery system (DDS), 1 mL of TPP was gradually added to the NB-loaded FA-CS-P solution under ultrasonication at 60% amplitude for 90 s while keeping the sample on an ice bath. The NB-loaded FA-CS-P nanoparticles were centrifuged at 4000 rpm for 8 min to ensure the efficient separation of unloaded nanocarriers, facilitating the removal of unencapsulated NB.

Characterization of Synthesized Nanoparticles

The characterization of the fabricated FA-CS-P nanoparticles was conducted using several analytical techniques. FT-IR spectroscopy was employed to assess the formation and deformation of chemical bonds and to verify successful drug entrapment. The KBr pellet method was used, and spectra were obtained between 400 cm⁻¹ and 4000 cm⁻¹. DLS was used to determine the surface charge and hydrodynamic diameter of the nanoparticles. Freshly prepared samples were ultrasonicated for 15 min at 37 °C before measurement. The size, dispersity, and morphology of the NB-loaded FA-CS-P nanoparticles were evaluated using TEM. For TEM analysis, the samples were dispersed onto a copper grid, air-dried at 25 °C, and imaged at an accelerating voltage of 120 kV. Thermogravimetric analysis (TGA) was performed to evaluate both the amount of sodium butyrate (NB) encapsulated within the FA-functionalized chitosan-PLGA (FA-CS-P) nanoparticles and to assess the thermal stability of the synthesized samples. In this study, approximately 4 mg of each sample was accurately weighed and placed into the TGA instrument. The samples were then heated from 50 °C to 800 °C at a constant heating rate of 10 °C per minute under a continuous nitrogen flow of 40 mL/min to prevent oxidation during heating. The mass loss observed at different temperature ranges corresponds to the degradation of various components, including the polymer matrix and the encapsulated NB.²⁸

Drug Release Study

The release behavior of NB from FA-CS-P nanoparticles was investigated in phosphate-buffered saline (PBS) to simulate the in vitro tumor microenvironment. The FA-CS-P-NB nanoparticles (3 mL) were placed in a dialysis membrane (12 000 Da molecular weight cutoff), sealed, and incubated in 100 mL of PBS at 37 °C with shaking at 80 rpm. The release medium was prepared at either acidic pH (5.0 and 6.0) or neutral pH (7.4). At specific time intervals (0.5, 1, 2, 3, 4, 8, 16, 32, and 64 h), 10 µL of the release medium was withdrawn and replaced with an equal volume of fresh PBS. The absorbance of the collected samples was measured at 480 nm using a NanoDrop UV-Vis spectrophotometer, and the percentage of NB released was calculated.

Cell Culture

The NALM6 leukemia cell line and HDF normal cell line were cultured in RPMI medium (Gibco, UK), supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 μg/mL streptomycin, and 100 IU/mL penicillin. Cells were maintained in a humidified incubator at 37 °C with 5% CO2.

Cell Viability Assay

The anti-cancer activity of the synthesized nanocarriers, including CS, CS-P, FA-CS-P, FA-CS-P-NB, and NB, was evaluated using the MTT assay. The assay is based on the reduction of MTT to formazan by mitochondrial dehydrogenase in viable cells.²⁹ For this, serial dilutions of the synthesized samples were prepared at concentrations of 100, 200, 300, and 400 nM in a serum-supplemented tissue culture medium and sterilized by filtration (0.2 µm). A total of 5000 cells/100 μL were seeded in 96-well plates and incubated at 37 °C with 5% CO2 for 24 h. After cell attachment, the culture medium was replaced with 150 μL of the prepared samples, and plates were incubated for 24 or 48 h. The medium was then replaced with 200 μL of serum-free RPMI, and 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, followed by incubation for 4 h at 37 °C. Finally, the medium was replaced with 200 μL of DMSO, and absorbance was measured at 570 nm using an ELISA reader.

Apoptosis Assay

Apoptosis induction by the nanocarriers was evaluated using flow cytometry based on Annexin V-FITC/propidium iodide (PI) staining.³⁰ Approximately 2 × 10⁵ NALM6 cells were seeded in six-well plates and incubated overnight at 37 °C with 5% CO2. Cells were treated with 200 nM concentrations of the nanocarriers for 5 h, washed with PBS, and centrifuged at 4000 rpm for 2 min. The cell pellet was resuspended in 100 µL of binding buffer, and cells were stained with Annexin V-FITC and PI for 15 min in the dark. Apoptosis was quantified using a flow cytometer.

Statistical Analysis

All experiments were performed in triplicate, and data were reported as mean ± standard deviation. Statistical differences between groups were evaluated using one-way analysis of variance (ANOVA), with p < .05 considered statistically significant.

Results and Discussion

In this study, we developed FA-conjugated chitosan-PLGA (FA-CS-P) nanocarriers for the targeted delivery of sodium butyrate (NB) to acute lymphoblastic leukemia cells, demonstrating their potential for enhanced therapeutic efficacy. The experimental results highlight key aspects of nanocarrier behavior, including biocompatibility, drug release, and targeted cytotoxicity, which are discussed in the context of existing literature.

FT-IR Analysis

In this study, Fourier-transform infrared (FT-IR) spectroscopy was utilized to confirm the surface modification, element conjugation, and therapeutic presence in the synthesized samples, including CS, CS-P, FA-CS-P, and FA-CS-P-NB. The FT-IR spectra, illustrated in Figure 1, demonstrate key absorption bands that reflect the structural features of the different nanocarrier formulations. For CS nanoparticles, characteristic absorption peaks were observed at approximately 3417 cm⁻¹, corresponding to the stretching vibrations of –NH₂ and –OH groups, and at 3336 cm⁻¹, indicating O−H and N−H stretching vibrations. Further evidence of the CS structure was provided by peaks at 1634 cm⁻¹, associated with C = O stretching, and at 1073 cm⁻¹, which correlated with C−N bond vibrations.³¹ Stretching vibrations in the range of 2800 cm⁻¹ to 3000 cm⁻¹ were attributed to CH, CH₂, and CH₃ groups, while the absorptions between 1470 cm⁻¹ and 1490 cm⁻¹ corresponded to the C−H stretching in methyl groups.³²

Figure 1.

FT-IR Spectra of synthesized nanocarriers. FT-IR results of syntheses, including CS, CS-P, FA-CS-P, and FA-CS-P-NAB. A. The absorption peaks of CS were attributes at around 3417 cm⁻¹ (–NH₂ and –OH groups stretching vibration), 3336 cm⁻¹ (O−H and N−H stretching vibrations), 1634 cm⁻¹ (C = O), 1470 cm⁻¹ to 1490 cm⁻¹ (C–H), and 1073 cm⁻¹ (C−N bond); B. The signals at around 1643 cm⁻¹ and 1706 cm⁻¹ (stretching vibration of the C = O groups in P), 1021 cm⁻¹ (C−O stretching of ester groups), and 1241 cm⁻¹ (C−H groups) were recorded for P in the compartment of CS-P; C. For the FA-CS-P nanocarrier, the characteristic peaks at around 3465 cm⁻¹ (–NH₂ and –OH groups stretching vibration and primary amine) 2924 cm⁻¹ (alkyl C−H and C = C stretch), 1653 cm⁻¹ (aromatic C = C bending), and 1415 cm⁻¹ (CH−NH−C = O amides bond formation) were assigned due to the presence of FA; D. Regarding the FA-CS-P-NAB sample, the shifted peaks at around 1646 cm⁻¹ (C = O stretching) and 2924 cm⁻¹ to 2921 cm⁻¹ (C–H stretching vibration) were indicator of NAB.

For the CS-P sample, the distinctive peaks at 1643 cm⁻¹ and 1706 cm⁻¹ were attributed to the C = O stretching vibrations of PLGA, confirming the successful conjugation of PLGA to CS. Additional peaks at 1021 cm⁻¹ and 1241 cm⁻¹ were associated with C−O stretching of ester groups and C−H groups in PLGA, respectively.³³ These findings were further supported by the presence of C−H stretching vibrations between 2700 cm⁻¹ and 3000 cm⁻¹, indicating PLGA's presence within the nanocarrier.³⁴

In the FA-CS-P sample, a sharp absorption peak at 3465 cm⁻¹ indicated the stretching vibrations of –NH₂ and –OH groups, confirming FA conjugation to CS. Additional peaks at 2924 cm⁻¹, 1728 cm⁻¹, and 1653 cm⁻¹ confirmed the presence of FA within the nanocarrier through alkyl C−H, C = O, and aromatic C = C stretching vibrations, respectively.^35,36 This suggests successful FA incorporation into the nanocarrier.

The FT-IR spectrum of FA-CS-P-NB revealed a shift in the C = O stretching peak from 1646 cm⁻¹, indicating the presence of NB within the nanocarrier. A minor shift in the C−H stretching vibration from 2924 cm⁻¹ to 2921 cm⁻¹ was also observed, further supporting NB entrapment within the nanocarrier.

Size and Morphology Analyses

In this study, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were employed to evaluate the hydrodynamic diameter, size distribution, surface charge, and shape of the synthesized CS-P, FA-CS-P, and FA-CS-P-NB nanocarriers. Table 1 and Figure 2 present the results from both analyses, providing critical insights into the physicochemical properties of these nanocarriers, which play a key role in their drug delivery performance. These properties directly influence the solubility, stability in serum, ability to penetrate cancer tissues, drug entrapment efficiency, and drug release kinetics of the nanocarriers.³⁷ These techniques provide complementary information about the nanoparticles, and the observed discrepancy between the DLS and TEM measurements reflects differences in the way these techniques assess particle size.

Figure 2.

TEM micrograph of FA-CS-P-NAB nanocarriers. Transmission electron microscopy (TEM) image showing the morphology of the FA-CS-P-NAB nanocarriers. The spherical nanoparticles with a size range of 40–60 nm confirm their suitability for drug delivery.

Table 1.

the Average Hydrodynamic Diameter Fabricated Samples Using DLS Analyzer.

Fabricated samples	Dispersed in	Diameter/nm
CS-P	H₂O	130.5 ± 6.3
FA-CS-P	H₂O	263 ± 8.9
FA-CS-P-NB	H₂O	338.5 ± 15.2

DLS measurements showed that the average hydrodynamic diameter of CS-P was approximately 130 nm, which increased to 263 nm after the conjugation of FA. This increase in size indicates the successful surface modification of the nanocarrier. Following the entrapment of NB, a further increase in diameter was observed, confirming successful drug loading. The increase in size is attributed to both the entrapment of NB within the nanocarrier matrix and possible surface interactions, such as hydrogen bonding and electrostatic interactions, that occur between NB and the nanocarrier surface.³⁸ DLS captures the dynamic, hydrated state of the nanoparticles in suspension, providing a measurement of the overall particle size in solution, including the solvent shell and any loosely associated molecules.

The surface charge of FA-CS-P-NB was measured at approximately 34.2 ± 0.12 mV, reflecting the positive charge of the amino groups from chitosan and FA on the nanocarrier surface. This positive charge plays an important role in enhancing the stability and cellular uptake of the nanocarriers.

In contrast, TEM analysis provided a core size range of 40–60 nm for FA-CS-P-NB nanocarriers, which is notably smaller than the DLS results. This size discrepancy is common in nanomaterial studies and can be attributed to the differences in measurement techniques. DLS measures the hydrodynamic size, which includes the nanoparticle core along with any surrounding solvent layers, while TEM provides the dry core size of the particles,³⁹ TEM also revealed a spherical shape with a homogenous distribution, which is advantageous for drug delivery, as spherical and nanoscale DDSs are better suited for efficient penetration into cancer tissues.^40,41 The larger size reported by DLS can also be attributed to potential nanoparticle aggregation in suspension. DLS is sensitive to the presence of aggregates or loosely associated clusters, which can skew the average size measurement upwards. TEM, however, images individual particles, reducing the likelihood of observing these aggregates. Additionally, the polydispersity index (PDI) obtained from DLS suggests a broad size distribution within the sample. Larger aggregates or particles in the sample may have contributed to the larger average size measured by DLS, whereas TEM provides a more direct measurement of the core particles themselves.

The nano-sized FA-CS-P-NB nanocarriers are particularly well-suited for cancer therapy, as nanocarriers smaller than 200 nm are ideal for exploiting the enhanced permeability and retention (EPR) effect, which allows for their preferential accumulation in tumor tissues.⁴² The gelatinous layer observed around the core of the particles may be due to the presence of conjugated FA, further supporting their potential for targeted delivery in cancer treatment.

TGA Analysis and Drug Release Profile

Thermogravimetric analysis (TGA) was employed to assess the physical state, thermal stability, and drug content of the FA-CS-P-NB nanocarriers by analyzing weight loss at different temperatures.⁴³ The TGA was conducted across a temperature range from 50 °C to 800 °C, where shifts in the weight loss curve were indicative of both thermal stability and the amount of NB entrapped within the nanocarrier. As shown in Figure 3, two primary weight loss events were observed. The first, at around 90–100 °C, resulted in approximately 5% weight loss, which was attributed to the evaporation of residual water from the nanocarrier samples. The second, more significant, weight loss occurred between 180–500 °C for FA-CS-P and 200–400 °C for FA-CS-P-NB, with the former showing 60% weight loss and the latter 35%. These losses are likely due to the degradation of the nanocarrier components. The difference in total weight loss between the two samples (16.08%) corresponds to the amount of NB successfully entrapped in the FA-CS-P-NB nanocarrier.

Figure 3.

Thermogravimetric analysis and drug release profile. A and B. Drug content, thermal stability, and drug release profile of the synthesized FA-CS-P and FA-CS-P-NAB nanocarriers. There are two main weight losses for both samples. According to the TGA curves of FA-CS-P and FA-CS-P-NAB samples, a weight loss of 16.08% was measured by comparing the TGA curves of two samples; C. On the other hand, the drug release manner of FA-CS-P-NAB nanocarrier indicated controlled NAB release compared to acidic medium and free NAB.

The high drug entrapment efficiency demonstrated in this study is crucial for the effectiveness of drug delivery systems (DDS), particularly in cancer therapy.⁴⁴ This result is consistent with prior studies that have used TGA to quantify drug loading and assess the thermal properties of polymer-based nanocarriers. For instance, Gholami et al (2019) reported similar thermal degradation profiles for drug-loaded nanoparticles, further validating the use of TGA as an effective method for such analyses.^45,46

The drug release behavior of the FA-CS-P-NB nanocarrier was also investigated, focusing on the rate of NB release over time. Controlled and sustained drug release is a key characteristic of advanced DDS, offering a significant advantage over conventional therapies like chemotherapy.⁴⁷ As depicted in Figure 3, NB exhibited an initial burst release, with approximately 16% of the drug released within the first 4 h. After this, a more controlled and sustained release profile was observed. In contrast, pure NB showed a significantly faster release rate, with 82% of the drug released in the first 8 h compared to only 36% from the nanocarrier

A key feature of this nanocarrier system is its pH-sensitive release behavior. When tested in different pH environments, the release rate of NB from FA-CS-P-NB was found to be more than twice as fast in acidic conditions compared to neutral pH. This is particularly important for cancer treatment, as tumor tissues typically have an acidic microenvironment due to their high metabolic activity.⁴⁸ The faster drug release in acidic environments enhances the efficacy of the DDS in targeting cancer cells while minimizing side effects on normal tissues. Previous studies, such as those by Birlik Demirel et al (2020), have also demonstrated the pH-responsive drug release capabilities of FA-conjugated nanocarriers, supporting the potential of these systems for cancer therapyv.^49,50

In summary, the TGA results confirm the efficient NB loading and thermal stability of FA-CS-P-NB nanocarriers, while the controlled, pH-sensitive drug release profile highlights their potential for targeted cancer therapy. These findings are consistent with other studies that emphasize the advantages of PLGA-based nanocarriers for sustained and controlled drug release in cancer treatment.^51,52

Cell Viability Assay

The MTT assay was conducted to evaluate the cytotoxicity and cancer cell growth suppression capabilities of the synthesized samples, including CS, CS-P, CS-P-FA, CS-P-FA-NB, and NB. The study was performed on NALM6 leukemia cells and HDF normal cells using varying concentrations (100, 200, 300, and 400 nM) over 24 and 48-h periods. The results, as shown in Figure 4, reveal significant differences in biocompatibility and cytotoxicity among the samples.

Figure 4.

Cytotoxicity assay of synthesized nanocarriers. Cytotoxic effects of synthesized samples, including CS, CS-P, CS-P-FA, CS-P-FA-NAB, and NAB. This test was performed on the NALM6 (A and B) and HDF (C and D) cell lines after 24 h and 48 h of treatment with 100, 200, 300, and 400 nM concentrations. As can be seen, The IC₅₀ values in cancer cells were observed at 300 nM and 200 nM for CS-P-FA-NAB nanocarrier after 24 h and 48 h of treatment in NALM6 cells, respectively.

The data indicated that CS, CS-P, and CS-P-FA exhibited high biocompatibility across both cell lines. Specifically, NALM6 cells treated with 400 nM concentrations of these samples showed 91%, 98%, and 94% cell viability, respectively, after 24 h of treatment. A similar trend was observed in HDF cells, where the cell viabilities were 98%, 98%, and 96%, respectively, after 24 h of treatment with the same concentrations. These results demonstrate that CS, P, and FA are not only biocompatible but also potentially biodegradable, making them suitable candidates for drug delivery, particularly in non-cancerous cells.^12,15,53

Moreover, a linear relationship between the concentrations of CS, CS-P, and CS-P-FA and the corresponding cell viability values was observed, further confirming the materials’ safety. This linearity suggests that even at increasing concentrations, these drug-free samples do not induce significant cytotoxic effects, validating their potential as safe carriers for therapeutic agents.⁵⁴

In contrast, the CS-P-FA-NB sample displayed notable cytotoxicity, particularly in cancer cells. The data indicated a reverse relationship between the increasing concentration of CS-P-FA-NB and cell viability in NALM6 cells. After 24 h, cell viability ranged from 61% at 100 nM to 26% at 400 nM. The cytotoxic effect was further amplified after 48 h, with viability dropping to 15% at 400 nM, while HDF cells-maintained viability above 70% under the same conditions. This selective cytotoxicity can be attributed to the higher expression of folate receptors in cancer cells, leading to greater internalization of the FA-conjugated nanocarriers compared to normal cells.^55,56

Interestingly, pure NB showed considerable toxicity to normal cells, with cell viability dropping to 48% after 48 h at 400 nM, compared to the more favorable biocompatibility of the CS-P-FA-NB sample. These results suggest that the FA-conjugated nanocarrier system enhances the selective targeting of cancer cells, while minimizing adverse effects on normal cells. This is further supported by the IC50 values, which were around 300 nM and 200 nM for the CS-P-FA-NB nanocarriers after 24 and 48 h, respectively, compared to 400 nM for pure NB after 48 h.

The findings from this assay align with previous studies, such as the work by Pina et al, who developed an aptamer-modified chitosan system for targeted drug delivery in breast cancer cells, reporting over 75% cytotoxicity in MCF-7 cells following treatment.⁵⁷ Overall, the results support the potential of FA-conjugated CS-PLGA nanocarriers as an effective DDS for targeting leukemia cells, while offering a promising avenue for further cancer treatment applications.

Apoptosis Assay

The apoptosis-inducing ability of the fabricated nanocarrier systems, including CS-P, CS-P-FA, CS-P-FA-NB, and free NB, was assessed in NALM6 leukemia cells using flow cytometry. This experiment was conducted after 5 h of treatment with a 300 nM concentration, which corresponds to the IC50 value. Flow cytometry was employed to quantify live cells, early apoptosis, late apoptosis, and necrosis. Figure 5 illustrates the results of the test, with the data divided into four quadrants: Q1 for necrosis, Q2 for late apoptosis, Q3 for early apoptosis, and Q4 for live cells.

Figure 5.

Apoptosis induction in leukemia cells. Apoptosis results of NALM6 cells after treatment with CS-P (A), CS-P-FA (B), NAB (C), and CS-P-FA-NAB (D) samples using flow cytometry technique. As can be seen, more than 22% of apoptosis was attained for cancer cells treated with the CS-P-FA-NAB sample while pure NAB did lead to only 5% apoptosis.

The results demonstrate minimal apoptosis in cells treated with CS-P (1%) and CS-P-FA (2%), confirming the biocompatibility of these samples. However, when cells were treated with the CS-P-FA-NB nanocarrier, the apoptosis rate significantly increased to over 22%, indicating that the FA-CS-P-NB system is highly effective in inducing apoptosis in cancer cells. This suggests that the targeted and efficient performance of CS-P-FA-NB as a drug delivery system (DDS) is superior to the other tested formulations.^11,13 Additionally, free NB caused higher necrosis (13%) compared to apoptosis (5%) in NALM6 cells, emphasizing that the nanocarrier formulation not only improves targeted delivery but also enhances apoptosis over necrosis, a desirable outcome in cancer treatment.

When comparing the CS-P-FA-NB nanocarrier to free NB, the nanocarrier demonstrated an apoptosis-inducing capability nearly four times higher than that of free NB. This significant enhancement can be attributed to the increased internalization of the nanocarrier by cancer cells due to FA-targeting. The results of this test are consistent with those from the MTT assay, further verifying the effectiveness of the CS-P-FA-NB nanocarrier system. Moreover, our findings align with previous studies, such as the research by Lee et al (2020), which demonstrated that triphenylphosphonium (TPP)-modified glycol chitosan microspheres loaded with doxorubicin induced similar levels of apoptosis and necrosis in HeLa and HepG2 cancer cells.⁵⁸

The FA-CS-P-NB nanocarrier showed enhanced apoptosis induction and cytotoxicity, particularly in cancer cells, which underscores its potential as an effective therapeutic platform for leukemia treatment. This is supported by previous studies, such as Hassan et al (2023), which demonstrated that FA-conjugated nanocarriers significantly improve drug internalization and induce greater levels of apoptosis in cancer cells compared to non-targeted systems.⁵⁹ Our results further confirm the potential of FA-CS-P-NB nanocarriers as effective drug delivery systems for leukemia treatment.

Conclusion

In this study, a novel biocompatible CS-P-FA-NB nanocarrier was successfully developed for targeted delivery of sodium butyrate (NB) to NALM6 leukemia cells. A suite of analytical techniques, including Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), thermogravimetric analysis (TGA), and transmission electron microscopy (TEM), were employed to thoroughly characterize the physicochemical properties of the synthesized nanocarrier. The analysis confirmed efficient surface functionalization, nanometric size, globular morphology, and significant drug loading capacity in the CS-P-FA-NB system. Moreover, the release profile demonstrated controlled NB release at physiological pH and a more rapid, pH-sensitive drug release in acidic conditions, typical of tumor microenvironments.

To evaluate the therapeutic efficacy, cell viability assays and flow cytometry were performed, demonstrating the nanocarrier's potential to inhibit cancer cell proliferation and induce apoptosis. These findings strongly indicate that the CS-P-FA-NB nanocarrier holds significant promise as a targeted drug delivery system (DDS) for cancer therapy. The results of this research underscore the potential for further in vivo studies and clinical applications of the developed nanocomposite for cancer treatment.

Footnotes

Abbreviations

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Fatemeh Ramezani

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