Anticancer efficacy of albumin nanoparticles co-loaded with silver nanoparticles and 5FU in animal model of colon cancer

Abstract

Colorectal cancer (CRC) remains a major challenge to global health and chemotherapy, while effective, often suffers from non-specificity, limited efficacy and severe side effects. 5-Fluorouracil (5FU), a cornerstone of chemotherapy for colorectal cancer, has a short half-life and systemic toxicity. Targeted delivery systems are crucial to overcome these limitations. The aim of this study was to develop and evaluate albumin nanoparticles (ANPs) co-loaded with green-synthesized silver nanoparticles (AgNPs) and 5FU (Ag-5FU-ANPs) as a potential strategy to improve chemotherapeutic efficacy and reduce toxicity in the treatment of colorectal cancer. The AgNPs were synthesized from a green tea extract, characterized (UV-Vis, TEM/SEM, DLS) and showed a spherical morphology with an average size of 89.9 nm. Four nanoparticle formulations (ANP, Ag-ANP, 5FU-ANP, Ag-5FU-ANP) were prepared using a solvent displacement method. Characterization revealed successful encapsulation efficiency (EE) (%EE > 70–80% efficiency) and controlled release kinetics (according to the Higuchi model, > 90% release in 3 days). In vitro studies in normal human fibroblast cells (HFF) showed acceptable cytotoxicity for Ag-5FU-ANP compared to free agents, with minimal hemolysis. In a 21-day colon cancer model using Wistar rats with CT26-induced tumors, intravenous administration of Ag-5FU-ANP showed the most significant anticancer effect, reducing tumor size and tumor weight compared to other groups. Histopathological analysis confirmed increased apoptosis and decreased necrosis in the Ag-5FU-ANP group. However, while the combination therapies showed increased renal toxicity compared to ANP, Ag-5FU-ANP showed less severe hematological toxicity (anemia, leukocytosis) than 5FU monotherapy. The blood analysis confirmed these results. These results suggest that Ag-5FU-ANPs represent a promising dual drug delivery system for colorectal cancer, improving therapeutic outcomes through better localization of the drug and potential exploitation of the anti-cancer properties of AgNPs, while mitigating some systemic side effects associated with 5FU monotherapy through controlled release. Further optimization is required to balance efficacy and toxicity for potential clinical application.

Introduction

Cancer is a life-threatening disease that can develop in virtually any organ or tissue of the body when abnormal cells grow uncontrollably¹. These cells can invade the surrounding tissue and spread to other organs. Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide². Although the exact cause of colorectal cancer is unknown, several factors such as genetics, diet and non-cancerous conditions such as colorectal polyps, colorectal adenomas, ulcerative colitis and Crohn’s disease may play a role in its development³. There are several treatment options for CRC, including chemotherapy, radiotherapy, surgery and immunotherapy⁴. Chemotherapy is one of the most common treatment options and involves the systemic administration of one or more cytotoxic drugs. These drugs can spread non-specifically throughout the body and inhibit the growth of cancer cells by suppressing cell division or disrupting the structure and function of DNA. While chemotherapy prolongs patients’ lives, its non-specific nature and effects on healthy cells lead to severe side effects such as myelosuppression (bone marrow suppression), mucositis, diarrhea, malnutrition and neurotoxicity. To overcome these limitations, new strategies have been developed, such as targeted drug delivery systems^5–7. These systems aim to deliver cytotoxic drugs specifically to the tumor rather than to healthy tissue in order to increase treatment efficacy and reduce side effects^8,9. A targeted drug delivery system usually consists of three components: Ligands that target the tumor, anticancer drugs, and a drug carrier¹⁰. 5-Fluorouracil (5FU), a uracil derivative, is one of the most commonly used drugs in the chemotherapy of colorectal cancer. However, its short half-life and non-specific distribution in the body limit its clinical application¹¹. Nanostructured materials have proven to be promising drug carriers for targeted drug delivery. Metal and protein nanoparticles, with their unique physicochemical properties, have made significant contributions to medicine by increasing the therapeutic index of drugs, preventing multidrug resistance and effectively delivering therapeutic agents^12,13. Green synthesis of metallic nanoparticles offers an innovative and environmentally friendly approach that provides economic and environmental advantages compared to chemical and physical synthesis methods¹⁴. Among the various nanoparticles, protein nanoparticles such as albumin nanoparticles have attracted considerable attention as promising carriers for targeted drug delivery in cancer treatment due to their biocompatibility, biodegradability, non-antigenic and non-immunogenic properties, availability and cost-effectiveness¹⁵. Albumin nanoparticles loaded with anticancer drugs such as 5FU can specifically target tumor tissue and increase treatment efficacy by improving drug bioavailability and reducing side effects. In addition, the combination of albumin nanoparticles with silver nanoparticles synthesized using an environmentally friendly method can have a significant anticancer effect. Silver nanoparticles have great potential for cancer therapy due to their antimicrobial, anti-inflammatory and anticancer properties¹⁶. Green synthesis of metallic nanoparticles is an emerging field in nanotechnology that offers economic and environmental advantages as an alternative to chemical and physical methods. Therefore, the co-incorporation of silver nanoparticles and the drug 5FU into albumin nanoparticles could be a promising strategy to improve the efficacy of colorectal cancer treatment. This targeted drug delivery system could deliver the cancer drug specifically to the tumor tissue, reduce side effects and increase therapeutic efficacy. Chemotherapy combined with albumin and silver nanoparticles could be more effective in treating cancer than a standard chemotherapy formulation. Therefore, the aim of this study was to investigate the anticancer effect of albumin nanoparticles loaded with green-synthesized silver nanoparticles and the drug 5FU on colorectal cancer in an animal model. To overcome the challenges of conventional chemotherapy and harness the potential of nanotechnology, this study focuses specifically on the development and evaluation of a novel nanoformulation. Our plan involves the synthesis of green-synthesized silver nanoparticles (AgNPs) using a non-toxic green tea extract, followed by their co-loading with the chemotherapeutic agent 5-fluorouracil (5FU) into biocompatible albumin nanoparticles (ANPs) using a solvent displacement method. The primary target of the developed Ag-5FU-ANP nanostructure is the tumor microenvironment in colorectal cancer. It is well known that albumin nanoparticles act passively in tumors through the effect of enhanced permeability and retention (EPR) and preferentially accumulate in tumor tissue due to leaky vasculature and poor lymphatic drainage. By encapsulating both 5FU and AgNPs in the albumin matrix, we aim to achieve several important goals: (1) Enhance tumor-specific delivery of 5FU, thereby increasing local concentration and therapeutic efficacy while reducing systemic exposure and associated toxicity. (2) Utilize the inherent anti-cancer properties of AgNPs, which could lead to a synergistic therapeutic effect. (3) Improve the pharmacokinetics and stability of the two therapeutic agents by controlled release from the nanoparticle carrier. (4) Mitigate the side effects commonly associated with monotherapy with free 5FU. This approach is in line with recent advances in nanomedicine, where multifunctional nanocarriers are being developed to overcome the limitations of conventional treatments. This is demonstrated by studies on the development of complex systems such as polydatin-conjugated zinc MOFs encapsulated in liponiosomes to specifically induce apoptosis¹⁷, or zingerone-loaded zinc MOFs coated with niosomes to enhance their antimicrobial and anticancer properties¹⁸. Our study aims to contribute to this field by investigating the feasibility and efficacy of a simpler but potentially effective albumin-based dual drug delivery system for the treatment of colorectal cancer. The main research question of this study is: Can the co-encapsulation of green-synthesized silver nanoparticles and 5-fluorouracil in albumin nanoparticles provide superior anticancer efficacy against colorectal cancer while reducing systemic toxicity compared to conventional 5FU monotherapy?

Materials and methods

Silver nitrate (AgNO₃, ≥ 99.8%) and Bovine Serum Albumin (BSA, ≥ 96%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 5-Fluorouracil (5FU, ≥ 98%) was obtained from Merck KGaA (Darmstadt, Germany). Glutaraldehyde (25% solution in water) and Ethanol (≥ 99.5%) were supplied by Merck KGaA (Darmstadt, Germany). Dimethyl sulfoxide (DMSO, ≥ 99.5%) was acquired from Sigma-Aldrich (St. Louis, MO, USA). Phosphate Buffered Saline (PBS, pH 7.4) was prepared in the laboratory. Fetal Bovine Serum (FBS) and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Penicillin-Streptomycin solution and Trypsin-EDTA solution were also supplied by Gibco (Thermo Fisher Scientific, Waltham, MA, USA). MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Green tea extract was provided by Gia Kala Company (Iran). The normal human fibroblast cell line (HFF) was purchased from Pasteur Institute of Iran.

Synthesis of silver nanoparticles

The silver nanoparticles were produced using a green synthesis method. Green tea leaf extract, prepared in deionized water, was used as a reducing agent. Five milliliters of the 4% green tea extract (Gia Kala Company, Iran) was slowly added to 30 ml of the 1 mM silver nitrate solution and stirred at 50 °C for 5 h. Then the solution was protected from light for 20 h. The resulting solution was washed three times with ethanol and deionized water and centrifuged at 8000 rpm. The resulting precipitate was dried in an oven at 40 °C. The synthesized silver nanoparticles were characterized by UV-Vis spectrophotometry, DLS, TEM, SEM. DLS and TEM were used to determine the particle size and charge, while SEM was used to study the morphology of the nanoparticles. The presence of silver nanoparticles in the final solution was confirmed by UV-Vis spectrophotometry.

Synthesis of albumin nanoparticles and drug-loaded albumin nanoparticles

Albumin nanoparticles (ANPs) were synthesized using a desolvation method. BSA (200 mg) was dissolved in 2 mL of deionized water and heated at 40 °C for 5 min to initiate denaturation. The denatured BSA solution was then rapidly mixed with 8 mL of cold ethanol (pre-cooled to 4 °C) using a magnetic stirrer at 500 rpm for 10 min. Glutaraldehyde (250 µL of a 25% solution) was added dropwise to the mixture to crosslink the proteins, and the reaction was continued with stirring at room temperature (25 °C) for 20 h. The resulting nanoparticle suspension was centrifuged at 10,000 rpm for 30 min at 4 °C to pellet the nanoparticles. The supernatant was carefully removed and the pellet was washed three times with cold deionized water (4 °C) to remove unreacted glutaraldehyde and ethanol. After each wash, it was centrifuged under the same conditions (10,000 rpm for 30 min at 4 °C). The final pellet was resuspended in 1 mL of deionized water and, if necessary, sonicated for 5 min in an ice bath with a Sonicator (200 W, 50% amplitude) to break up aggregates and ensure uniform dispersion. For the 5FU-ANPs, the same protocol was followed, but 5 mL of 5-fluorouracil solution (50 mg/mL in deionized water) was added to the 8 mL of cold ethanol before mixing with the denatured BSA solution. For the Ag-ANPs, 2 mL of a 0.2% (w/v) suspension of green-synthesized silver nanoparticles (AgNPs) in deionized water was added to the 8 mL of cold ethanol after BSA denaturation before mixing with the BSA solution. The double-loaded Ag-5FU-ANPs combined both modifications: 5 mL of the 5FU solution and 2 mL of the AgNP suspension were added to the 8 mL of cold ethanol before mixing with the BSA solution. All variants underwent the same cross-linking, washing and (if necessary) sonication steps as described for the ANPs. Characterization included FTIR (functional group analysis), DLS (size and zeta potential) and SEM (morphology). The methods ensured consistent formation of nanoparticles, with integration of the drug achieved by successive additions during solvent displacement. The key variables were the timing of drug incorporation (AgNPs or 5FU before or after mixing BSA in ethanol) and the use of glutaraldehyde for structural stabilization.

Evaluation of encapsulation efficiency percentage (%EE) and release kinetics of silver nanoparticles and 5-fluorouracil from synthesized nanostructures

The %EE of silver nanoparticles and 5-FU in albumin nanoparticles was determined using a spectroscopic method. After synthesizing the respective nanoparticle formulations (Ag-ANP, 5FU-ANP, Ag-5FU-ANP), the unbound drug and AgNPs were separated from the nanoparticle suspension by centrifugation at a certain speed (e.g. 10,000 rpm) for a certain duration (e.g. 30 min) at a certain temperature (e.g. 4 °C). The supernatant was carefully collected and the concentration of the remaining free 5-FU was measured spectrophotometrically at 266 nm using a UV-Vis spectrophotometer. The concentration of AgNPs in the supernatant was measured at 460 nm. The concentration of 5-FU was determined using a previously prepared calibration curve.

%EE was calculated using the following equations¹⁹:

In vitro release studies were performed using dialysis. A known amount of the nanoparticle formulation (2 ml) was placed in a dialysis membrane bag and immersed in a release medium (phosphate buffered saline, pH 7.4) at 37 °C. A sample was taken from the release medium at predetermined intervals and the concentration of the drugs was measured spectrophotometrically. The removed volume was replaced with fresh medium. The percentage of cumulative release was calculated and the release kinetics were analyzed using suitable models. The choice of pH 7.4 as the release medium was based on the intended intravenous route of administration of the nanoformulations, where the primary release environment would be the systemic circulation and the tumor microenvironment, both of which maintain physiological pH conditions around 7.4.

Cytotoxicity evaluation

The cytotoxicity of the synthesized nanostructures was investigated in HFF cells using an MTT assay. The HFF cells were seeded in 96-well plates and incubated. They were then exposed to different concentrations (100–1000 µg/ml) of albumin nanoparticles loaded with silver nanoparticles and 5-fluorouracil for 48 h. After incubation with MTT solution, the resulting formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm using an Enzyme-Linked Immunosorbent Assays (ELISA) reader.

Cell viability was calculated using the following equation:

(Where Abs_570nm_Experimental is the absorbance of the wells treated with the nanostructures, and Abs_570nm_Control is the absorbance of the wells treated with the control (usually cells treated with the vehicle or untreated cells)^20,21.

Cell viability was calculated from the absorbance values, with experiments performed in triplicate and data expressed as mean ± standard deviation.

Evaluation of hemolysis

Hemolysis was measured using an absorbance method. Diluted erythrocyte samples were mixed with different concentrations (100, 250, 500, 800 and 1000 µg/ml) of the synthesized nanoparticles, free 5-fluorouracil and silver nanoparticles. Four albumin-based synthesized nanostructures, one negative control and one positive control were tested.

Evaluation of designed nanostructures in an animal model

The anti-cancer effect of the developed nanostructures was investigated in Wistar rats with surgically induced colon cancer. After anesthesia with ketamine (80 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally and a small abdominal incision²², CT26 cancer cells were injected into the colon, the wound was closed and postoperative care was performed. Once palpable tumors had developed, the rats were divided into six groups, which received the following: (1) albumin nanoparticles (10 mg/kg), (2) albumin nanoparticles loaded with silver nanoparticles (10 mg/kg ANPs + 2 mg/kg AgNPs), (3) albumin nanoparticles loaded with 5-fluorouracil (10 mg/kg ANPs + 15 mg/kg 5FU), (4) a combination of 5-fluorouracil and albumin nanoparticles loaded with silver nanoparticles (10 mg/kg ANPs + 2 mg/kg AgNPs + 15 mg/kg 5FU), (5) free 5-fluorouracil (15 mg/kg) or (6) normal saline as a control.

Treatments were administered intravenously every three days for 21 days, with all groups maintained under identical conditions. Tumor volume was measured every three days using calipers, and the longest diameter (length, L) and the shortest diameter (width, W) of the tumor were recorded. The tumor volume (V) was calculated using the following formula: V = (L × W²) / 2 (Where L is the length in mm and W is the width in mm, and the result is in mm³). Blood samples were taken for biochemical analysis at the beginning and end of the treatment period. After 21 days, the rats were euthanized by an intraperitoneal overdose of ketamine (150 mg/kg) and xylazine (15 mg/kg). Following the confirmation of death, the tumors were removed and measured, and the tumor and kidney tissue samples were processed for histological examination, including fixation, sectioning, staining with hematoxylin and eosin, and microscopic evaluation of tissue changes.

Results and discussion

Characterization of silver nanoparticles

In this study, silver nanoparticles were successfully synthesized by a green method using green tea leaf extract (Fig. 1). DLS analysis revealed an average nanoparticle size of 89.9 nm with a relatively uniform distribution (polydispersity index (PDI) of 0.6) and high stability due to a strongly negative zeta potential (-46.5 mV). These results are in agreement with other studies^23–29. Electron microscopy (TEM and SEM) confirmed the spherical morphology and crystalline nature of the nanoparticles, with most being less than 100 nm in size, which is consistent with the DLS results. UV-Vis spectroscopy showed peak absorption at 460 nm, further confirming the formation of silver nanoparticles. The green synthesis method using plant extracts is preferred over physical/chemical methods and microorganisms due to its cost efficiency, environmental friendliness and ease of scalability^30–36. Green tea extract proved to be successful in the production of stable silver nanoparticles.

Fig. 1.

Characterization of synthesized nanoparticles via green method using green tea extract. (A) DLS (Size distribution), (B) DLA (Zeta potential), (C) SEM, (D) TEM, (E) Spectroscopy.

Characterization of albumin nanoparticles, silver nanoparticles loaded albumin nanoparticles, 5-fluorouracil-loaded albumin nanoparticles, and silver nanoparticles with 5-fluorouracil loaded albumin nanoparticles

Albumin nanoparticles were synthesized by desolvation method and their properties were investigated by DLS and SEM. According to the DLS results (Fig. 2), the average size of albumin nanoparticles was 202.7 nm, the PDI was 0.4 and the zeta potential was − 7 mV. Albumin nanoparticles containing silver nanoparticles had an average size of 171.9 nm, a PDI of 0.39 and a zeta potential of -49.6 mV, indicating a reduction in particle size and increased stability. Albumin nanoparticles containing 5-fluorouracil had an average size of 224 nm, a PDI of 0.7 and a zeta potential of -91.3 mV, indicating an increase in size and a decrease in particle uniformity, but their stability was higher. Albumin nanoparticles containing silver nanoparticles and 5-fluorouracil had an average size of 109.55 nm, a PDI of 0.8, and a zeta potential of -58 mV, which brought the particle size to the ideal limit but increased their dispersion^37,38. Studies have shown that the size of nanoparticles is important for therapeutic and diagnostic applications and influences factors such as half-life in the bloodstream, targeting and cellular uptake³⁹. The nanoparticles synthesized in this study have a suitable size for cellular penetration and enable the release of drugs within the cell and the extracellular matrix. Langer et al.⁴⁰ also used the desolvation method to produce albumin nanoparticles with a controllable diameter (150–250 nm), which is consistent with the results of this study. The pH of the environment is also effective in controlling particle size, and a neutral and alkaline environment leads to the production of smaller nanoparticles^41,42. Size heterogeneity and colloidal instability are the main challenges in protein-based nanoparticles⁴³.

Fig. 2.

DLS images of albumin nanoparticles: size (A), zeta potential (B), albumin nanoparticles containing silver nanoparticles: size (C), zeta potential (D), albumin nanoparticles containing fluorouracil: size (E), zeta potential (F), albumin nanoparticles containing both silver nanoparticles and fluorouracil: size (G), zeta potential (H).

Studies have shown that albumin nanoparticles suspended in the aqueous phase remain stable for two months at 4 °C⁴³. Langer et al.⁴⁰ showed that Human Serum Albumin (HSA) nanoparticles are absorbed by cells.

In addition, the organic solvents and cross linkers used in desolvation can have toxic effects⁴⁰. Therefore, drying the albumin nanoparticle solution is not recommended as it leads to its aggregation. In this study, drying the synthesized albumin nanoparticles also led to the aggregation phenomenon. The SEM images of albumin-based nanoparticles shown in Fig. 3 indicate that most of the nanoparticles are in the nanoscale, which is consistent with the DLS results. All four types of synthesized nanoparticles-albumin nanoparticles, silver-containing albumin nanoparticles, nanoparticles containing 5-fluorouracil, and nanoparticles containing both silver and fluorouracil - exhibit spherical morphology and average size in the nanometer range, which is consistent with the results of Costa et al.⁴⁴. The spherical morphology improves the physical, chemical and biological properties of nanoparticles due to their higher surface-to-volume ratio, as demonstrated by various studies^45,46. This shape improves the interactions with the environment and makes them more effective as carriers of drugs or to improve their properties. In addition, it improves the rheological and dynamic properties in biological systems and ensures uniform distribution in drug delivery systems⁴⁷. This efficient distribution can lead to improved drug efficacy and reduced side effects. The SEM images in this study clearly show the spherical morphology of the nanoparticles. FTIR spectroscopy was used to evaluate the functional groups in the albumin nanoparticles and the results are summarized in Fig. 4. The spectral pattern for silver nanoparticles showed peaks at 3306 cm-¹ (O-H stretching), 2167 cm-¹ (C ≡ C or C ≡ N stretching), 1634 cm-¹ (C = O stretching) and 727 cm-¹ and 504.11 cm-¹ (Ag-O or Ag-N vibrations), which is consistent with previous studies^48–51. These peaks indicate interactions with oxygen- or nitrogen-containing reducing agents and the presence of organic residues. Albumin nanoparticles exhibited characteristic peaks at 3288.4 cm-¹ (O-H stretching), 2935.3 cm-¹ (C-H stretching), 1650.7 cm-¹ (C = O stretching, amide I), 1532.7 cm-¹ (N-H stretching, amide II) and others, consistent with published studies^44,52. These peaks confirm the presence of peptide bonds, hydroxyl groups and aromatic amino acids in albumin. The spectrum of 5-fluorouracil (5FU) showed peaks at 3338.3 cm-¹ (N-H stretching), 1627 cm-¹ (C = O stretching), 1524.1 cm-¹ (C = C stretching) and 786.05 cm-¹ (C-F stretching), which is consistent with its structure (53, 54). For silver-containing albumin nanoparticles, peaks at 3287.2 cm-¹ (N-H/O-H stretching), 1651.7 cm-¹ (C = O stretching, amide I) and 805.7 cm-¹ (possible cysteine interaction) were observed, indicating interactions between albumin and silver nanoparticles. Albumin nanoparticles loaded with 5FU showed peaks at 3275.7 cm-¹ (N-H/O-H stretching), 1645.5 cm-¹ (C = O stretching) and 607.9 cm-¹ (aromatic ring vibrations), indicating successful loading with 5FU and possible interactions with albumin. For albumin nanoparticles carrying both 5FU and silver, peaks were observed at 3284.3 cm-¹ (N-H/O-H stretching), 1638.4 cm-¹ (C = O stretching) and 1397.6 cm-¹ (CH3 bending), indicating successful loading of both components and possible interactions between albumin, 5FU and silver nanoparticles. The observed peak shifts and intensity changes suggest structural interactions within the nanoparticle system.

Fig. 3.

SEM images of (A) albumin nanoparticles, (B) albumin nanoparticles loaded with silver nanoparticles, (C) albumin nanoparticles loaded with fluorouracil drug, and (D) albumin nanoparticles loaded with both silver nanoparticles and fluorouracil drug.

Fig. 4.

FTIR spectra of 5FU drug (1), albumin nanoparticles (2), silver nanoparticles (3), albumin nanoparticles containing 5FU (4), albumin nanoparticles containing silver nanoparticles (5), and albumin nanoparticles containing both silver nanoparticles and 5FU drug (6).

Implications of particle size and zeta potential on therapeutic efficacy

The average size of 89.9 nm for green-synthesized silver nanoparticles is in the optimal range for cell uptake and tumor penetration. Nanoparticles in the size range of 50–200 nm can effectively exploit the EPR (Enhanced Permeability and Retention) effect, which allows preferential accumulation in tumor tissue through leaky vessels while preventing rapid excretion through the reticuloendothelial system⁵⁵. The strongly negative zeta potential of -46.5 mV for AgNPs indicates excellent colloidal stability, which prevents aggregation and ensures consistent biodistributive and therapeutic performance⁵⁶. For albumin-based formulations, the observed size variations (109.55–224 nm) are within the therapeutically relevant range for passive tumor targeting⁵⁷. The Ag-5FU-ANP formulation exhibited the smallest size (109.55 nm), which may contribute to improved tumor penetration and cell uptake. The changes in zeta potential upon drug loading provide insight into surface interactions and stability⁵⁸. The strongly negative zeta potential of the 5FU-loaded ANPs (-91.3 mV) indicates strong electrostatic stabilization, while the Ag-5FU-ANP formulation (-58 mV) exhibits sufficient stability for systemic circulation. These physicochemical properties correlate directly with the observed therapeutic results. The optimal size range facilitates effective tumor accumulation through the EPR effect, while the negative surface charge promotes prolonged circulation time by reducing protein adsorption and macrophage uptake. The controlled size distribution (PDI < 0.8) provides predictable pharmacokinetics and uniform drug release, contributing to the superior anticancer efficacy observed in the in vivo studies^59,60. In addition, the appropriate size and surface charge properties likely contributed to the lower systemic toxicity compared to free drugs, as evidenced by the improved hematologic parameters and reduced renal complications in the nanoformulated groups^61,62.

EE percentage and release kinetic evaluation

Table 1 show that over 80% of AgNPs and 5FU were encapsulated in single systems. When loaded in combination, AgNPs achieved over 70% encapsulation, while 5FU achieved over 60%. Similar studies to Liu et al.⁶³ and Motavalli et al. reported high encapsulation efficiencies, with Liu et al. achieving 96 ± 3% EE for BSA-AgNCs. The desolvation method used in this study is highlighted as ideal for the synthesis of albumin nanoparticles due to its reproducibility, simplicity and versatility for encapsulation of hydrophilic and hydrophobic drugs. Key parameters affecting nanoparticle size and drug encapsulation efficiency include the rate of ethanol addition, solution pH, and albumin concentration, which were optimized in this study. Release kinetics studies (Table 2; Fig. 5) showed that all nanoparticle formulations (AgNPs, 5FU-loaded and dual-loaded) demonstrated sustained release over three days, with over 90% of the drug being released. The results of the in vitro drug release kinetics are summarized in Table 2; Fig. 5. The silver nanoparticles showed a sustained release profile, with approximately 90% of the silver being released within three days. The release kinetics followed the Higuchi model, indicating that the release was diffusion controlled. The 5FU-loaded nanoparticles exhibited a sustained release profile, with approximately 95% of the 5FU released within three days. The release kinetics also followed the Higuchi model, suggesting that the release was mainly controlled by diffusion. The double-loaded nanoparticles showed a sustained release profile for both silver and 5FU, with approximately 85% of the silver and 90% of the 5FU released within three days. The release kinetics for both drugs followed the Higuchi model, suggesting diffusion-controlled release. Similar studies on albumin nanoparticles confirmed these results and emphasized the stabilizing effect of albumin on drug release^64,65.

Table 1.

Percentage of encapsulation efficiency of silver nanoparticles and 5-fluorouracil in synthesized albumin-based nanoparticles.

Type of nanoparticle	Percentage of silver nanoparticle encapsulation	Percentage of 5-fluorouracil encapsulation
Albumin nanoparticle containing silver nanoparticle	85%	–
Albumin nanoparticle containing 5-fluorouracil	–	82%
Albumin nanoparticle containing both materials	72%	69%

Table 2.

Release rates of silver nanoparticles and 5-fluorouracil from albumin-based synthesized nanoparticles over 3 days.

Time (days)	Silver nanoparticle release percentage (from nanoparticles containing only silver)	Fluorouracil release percentage (from nanoparticles containing only fluorouracil)	Silver nanoparticle release percentage (from nanoparticles containing both substances)	Fluorouracil release percentage (from nanoparticles containing both substances)
0.5	15%	20%	10%	15%
1	30%	40%	25%	35%
1.5	45%	55%	40%	50%
2	60%	70%	55%	65%
2.5	75%	85%	70%	80%
3	90%	95%	85%	90%

Fig. 5.

Release kinetic of silver nanoparticles and fluorouracil from albumin-based synthesized nanoparticles over 3 days. Albumin nanoparticles (ANP), albumin nanoparticles containing silver nanoparticles (ANP-Ag), albumin nanoparticles containing fluorouracil (ANP-5FU), and albumin nanoparticles containing silver nanoparticles and fluorouracil (ANP-Ag-5FU).

The Higuchi model describes the release of drugs from insoluble matrices as the square root of a time-dependent process based on Fick’s diffusion. The model is given by:

The Higuchi model provided the best fit for the release data, with (R2) values greater than 0.9, indicating that the release mechanism is primarily diffusion-driven.

The Korsmeyer-Peppas model is used to describe drug release from polymeric systems and is given by:

The Korsmeyer-Peppas model provided a good fit for the release data, with (R2) values above 0.9. The release exponent (n) was between 0.45 and 0.89, indicating non-Fickian (anomalous) diffusion.

The first-order kinetic model describes the release of drug, where the release rate is proportional to the amount of drug remaining in the system. The model is given by:

The first-order kinetic model also provided a good fit to the release data, with (R2) values above 0.9. This model suggests that the release rate decreases over time as the drug is released from the nanoparticles.

The sustained release profiles observed for all nanoparticle formulations indicate that the drugs are released in a controlled manner, which is beneficial for maintaining the therapeutic drug concentration over a prolonged period of time. The good fit of the Higuchi model suggests that the release mechanism is primarily diffusion controlled, which is desirable for sustained drug release. The good fit of the Korsmeyer-Peppas model and the release exponent (n) values indicate that the release mechanism involves both diffusion and erosion, which is consistent with the properties of albumin nanoparticles. The good fit of the first-order kinetics model suggests that the release rate decreases over time, which is consistent with the observed sustained release profiles. The R² values for all models were above 0.9, indicating a very good quality of fit. Gaurav et al.⁶⁶ supported these results and confirmed the suitability of the Higuchi model for the description of drug release kinetics. Overall, the study shows efficient encapsulation, sustained release and successful kinetic modeling of drug release from albumin nanoparticles.

In vitro investigations

The synthesized albumin-based nanoparticles were tested for cell toxicity and blood toxicity in the laboratory.

Hemolysis assay

Figure 6 shows the results, where 100% hemolysis is defined by a positive control (1% Triton) and 0% by a negative control (PBS). All albumin-based nanoparticles exhibited less than 1% hemolysis, with the percentage of hemolysis increasing in proportion to the concentration of nanoparticles. The order of highest to lowest hemolysis among the nanoparticles was: albumin nanoparticles containing silver nanoparticles and fluorouracil, albumin nanoparticles containing silver nanoparticles, albumin nanoparticles containing fluorouracil, and albumin nanoparticles. These values were all below 1%. The study concludes that the synthesized nanoparticles didn’t cause significant hemolysis at the concentrations tested. In particular, free silver nanoparticles and free 5-fluorouracil showed higher blood toxicity than their nano-encapsulated forms. Free 5-fluorouracil showed a hemolysis rate of 5.2%, while free silver nanoparticles showed less than 3% hemolysis. The text emphasizes that 5FU is known to cause hemolysis, which is supported by clinical studies. For example, the study by Sandvei et al. is cited, in which a patient developed acute intravascular hemolysis during treatment with 5FU⁶⁷. Fatal immune hemolysis due to antibodies against 5FU metabolites is also a known risk⁶⁸. Current study shows that nanoencapsulation reduces this hemolytic side effect of 5FU. Although the exact mechanism of AgNP-induced hemolysis is not yet fully understood, possible mechanisms include direct interaction with red blood cells, release of free silver ions, and disruption of cell function⁵⁶. The study by Chen et al. showed that smaller AgNPs (15 nm) induced more severe hemolysis and membrane damage, which they attributed to direct interaction, oxidative stress and membrane damage⁶⁹. Consistent with Chen et al., negligible hemolytic activity was observed for AgNPs close to 100 nm in size in the current study. Overall, the study showed that the toxicity of all nanostructures was below the internationally recognized standard of 5% for pharmaceutical compounds⁷⁰. This lower toxicity of the encapsulated forms is likely due to the controlled release of the drugs in the diluted blood environment, reducing the exposure of red blood cells to high concentrations of AgNPs and 5FU. The study concludes that the synthesized nanoparticles prepared using environmentally friendly methods for AgNP synthesis and desolvation for drug delivery do not cause significant hemolysis. Albumin is highlighted as beneficial due to its availability, low cost and biocompatibility as a major plasma protein.

Fig. 6.

Percentage of hemolysis in different concentrations of albumin nanoparticles (ANP), albumin nanoparticles containing silver nanoparticles (ANP-Ag), albumin nanoparticles containing fluorouracil (ANP-5FU), and albumin nanoparticles containing silver nanoparticles and fluorouracil (ANP-Ag-5FU).

Cytotoxicity evaluation (MTT assay)

The cytotoxicity of the synthesized nanostructures was thoroughly investigated in HFF cells using an MTT assay. The results are shown in Table 3. A clear concentration- and time-dependent toxicity was observed for most of the tested compounds, as evidenced by a progressive decrease in cell viability with increasing nanoparticle concentration and exposure duration. This pattern is statistically confirmed by the decreasing p-values (indicating increasing statistical significance of toxicity) with increasing concentration and duration, especially at higher concentrations. Significantly, only the albumin nanoparticles (ANP) showed excellent biocompatibility with normal human fibroblast cells. Percent cell viability was consistently above 100% at all concentrations and time points tested and showed no statistically significant toxicity (p > 0.05) compared to untreated control cells. This high biocompatibility, consistent with previous studies, underscores the advantages of protein nanocarriers^71–73. Among the active formulations, nanoparticles containing silver (ANP-Ag) and 5-fluorouracil (ANP-5FU) alone showed toxicity that generally increased with concentration and time. However, the most striking result was the pronounced synergistic effect observed with the double-loaded ANP-Ag-5FU nanoparticles. This formulation consistently yielded the lowest percentages of cell viability (e.g., 51.12% at 2 mg/mL at 48 h) and the most statistically significant toxicity (p < 0.001) across most active concentrations and time points, demonstrating superior cytotoxic potential compared to ANP-Ag or ANP-5FU when administered individually. This underscores the improved efficacy of the combination of AgNPs and 5FU in a single nanocarrier. In addition, the results clearly indicate that the free forms of 5-fluorouracil (Free 5FU) and silver nanoparticles (Free AgNPs) exhibited higher toxicity to normal HFF cells than their respective nano-encapsulated forms. For example, free 5FU at 2 mg/mL resulted in a viability of 65.1% at 48 h compared to 80.3% for ANP-5FU, and free AgNPs showed a viability of 64.7% compared to 80.9% for ANP-Ag. While both the free and encapsulated forms showed significant toxicity at higher concentrations (p < 0.001), the encapsulated versions consistently showed less severe effects, suggesting that the nanocarrier system effectively mitigates some of the immediate cellular toxicity associated with the free agents through controlled delivery. Concentration- and time-dependent toxicity was also demonstrated in similar studies with albumin nanoparticles loaded with different substances^74–76. In agreement with similar studies, our results also showed that albumin nanoparticles have no toxicity to normal cells^77–80. Protein nanocarriers, especially albumin-based nanoparticles, have numerous advantages compared to other nanomaterial. Biocompatibility, biodegradability, lower immunogenicity and lower cell toxicity are among the desirable properties of protein nanocarriers⁸¹. This high biocompatibility for albumin nanoparticles was also demonstrated in our study. This biocompatibility is related to the nature of the amino acids in the protein nanoparticles. The most important result in this section is the demonstration of the synergistic effect. Nanoparticles containing silver and 5-fluorouracil (ANP-Ag-FU) showed very high toxicity compared to other nanoparticles, including ANP-Ag and ANP-FU alone, at most concentrations and times. This indicates the synergistic effect of silver and 5-fluorouracil in the destruction of cancer cells. Overall, silver-containing nanoparticles show moderate toxicity, which increases with increasing concentration and time. Nanoparticles containing 5-fluorouracil show higher toxicity compared to ANP-Ag, especially at high concentrations. ANP-Ag-FU nanoparticles exhibit the highest toxicity, showing a significant decrease in cell viability (12.51) at a concentration of 2 mg/mL and a time of 48 h. The results of this table also show that the free 5-fluorouracil verdict has higher toxicity than the nanoparticle-containing drug 5FU at both time periods studied. As expected, the toxicity of the nanoparticles studied increased with increasing concentration and time. This pattern is consistent with the results of other studies on the toxicity of nanoparticles containing anticancer agents^82,83. An increase in the concentration of nanoparticles leads to an increase in their accumulation in the cell and consequently to an increase in the release capacity of active substances, resulting in increased oxidative stress, DNA damage and ultimately cell death^84–86. These in vitro cell toxicity studies thus highlight the potential of the desolvation method for the synthesis of nanoparticles that not only effectively encapsulate and deliver therapeutic agents, but also modulate their release and cellular interactions. This approach can potentially improve the therapeutic index by reducing off-target toxicity to normal cells while enhancing the combined cytotoxic effect on cancer cells (as suggested by the strong synergy observed even in normal HFF cells, suggesting even greater efficacy against cancer cells). This pattern is consistent with the results of other studies on the toxicity of nanoparticles containing anticancer drugs^87–89. Accordingly, an increase in nanoparticle concentration leads to increased accumulation and release of drugs, resulting in increased oxidative stress, DNA damage and ultimately cell death^90,91.

Table 3.

Percentage of viability of normal cells treated with different concentrations of albumin nanoparticles (ANP), albumin nanoparticles containing silver nanoparticles (ANP-Ag), albumin nanoparticles containing fluorouracil (ANP-FU), and albumin nanoparticles containing silver nanoparticles and fluorouracil (ANP-Ag-FU) on normal cell line (HFF) at 24 and 48 h.

Treatment time (h)	Concentration (mg/mL)	Free AgNPs	Free 5FU	ANP	ANP-Ag	ANP-5FU	ANP-Ag-5FU	p-value vs. untreated control
24	0.2	108	99.5	121	125.7	137.3	115.7	> 0.05
	0.5	97.5	87.9	118.2	109.9	122.8	86.7	< 0.05
	1	96.2	85.9	115.3	100.5	111.6	82.1	< 0.05 (ANP-Ag-5FU: < 0.01)
	1.5	95.02	75.9	105.8	100.1	96.2	74.7	< 0.01 (ANP-Ag-5FU: < 0.001)
	2	78.4	75.1	101.2	98.8	93.3	60.1	< 0.001
48	0.2	105.8	103.7	142.01	110.5	117.2	103.3	> 0.05
	0.5	102.1	94.1	128.1	99.4	109.6	98.7	< 0.05 (ANP-Ag-5FU: > 0.05)
	1	88.2	79.4	125.2	91.5	89.2	55.04	< 0.01 (ANP-Ag-5FU: < 0.001)
	1.5	81.9	74.7	124.7	87.1	85.1	54.6	< 0.01 (ANP-Ag-5FU: < 0.001)
	2	64.7	65.1	113.8	80.9	80.3	51.12	< 0.001

In vivo studies

Male rats with colon cancer were used to investigate the anti-cancer effect of synthesized albumin-based nanoparticles.

Tumor’s size and weight

The mean and standard deviation of tumor weight and tumor size in each group after 21 days of treatment are shown in Table 4. The results show that tumor weight and size were significantly reduced in groups 2, 3, 4 and 5 compared to the control group and group 1. The lowest values of tumor weight and size were observed in group 4, which showed a significant difference to all other groups. The difference between groups 2 and 3 is also not significant, but the difference between these two groups and group 5 is significant (Table 5). The results obtained show a significant reduction in tumor weight and tumor size in the silver-containing albumin nanoparticle sample compared to the control sample with 5-fluorouracil. The presence of silver nanoparticles and their interaction with the drug 5-fluorouracil caused this reduction in tumor size. Considering the appropriate synthesis method of the nanoparticles containing silver and 5-fluorouracil, good stability of the release rate in the body and high efficiency may be effective in these animal experiments. In the study by Zhao et al.⁹², the antitumor activity of H22 mouse models bearing a tumor was used to investigate whether the improvement of the pharmacokinetic behavior of 5FU through the formation of a drug-albumin complex leads to increased therapeutic efficacy. The results showed that the group treated with 5-fluorouracil showed a moderate anti-tumor effect in delaying tumor growth compared to the control group. On the other hand, the rats treated with 5-fluorouracil-containing albumin showed a significant reduction in tumor volume, and these results are consistent with our results.

Table 4.

Comparison of mean tumor weight and size among different groups treated with albumin nanoparticles, albumin nanoparticles containing silver nanoparticles, albumin nanoparticles containing 5-fluorouracil, and albumin nanoparticles containing silver nanoparticles and fluorouracil.

Group	Mean tumor weight (g) ± standard deviation	Mean tumor size (mm) ± standard deviation	p-value vs. control group
Group 1 (albumin)	1.23 ± 0.45	12.5 ± 2.1	0.123
Group 2 (albumin + silver)	0.89 ± 0.32	10.2 ± 1.8	0.034
Group 3 (albumin + 5FU)	0.65 ± 0.21	8.7 ± 1.5	0.001
Group 4 (albumin + 5FU + silver)	0.45 ± 0.15	6.5 ± 1.2	< 0.001
Group 5 (5FU)	0.98 ± 0.36	11.0 ± 2.0	0.042
Group 6 (control)	1.35 ± 0.50	13.8 ± 2.3	–

Table 5.

Comparison of mean tumor weight and size among different groups treated with albumin nanoparticles, albumin nanoparticles containing silver nanoparticles, albumin nanoparticles containing 5-fluorouracil, and albumin nanoparticles containing silver nanoparticles and 5-fluorouracil.

Group	Group 1 (Albumin)	Group 2 (Albumin + Silver)	Group 3 (Albumin + 5-FU)	Group 4 (Albumin + 5FU + Silver)	Group 5 (5-FU)	Group 6 (Normal Saline)
Group 1 (Albumin)	–	0.123	0.001	< 0.001	0.042	0.123
Group 2 (Albumin + Silver)	0.123	–	0.034	0.001	0.001	0.034
Group 3 (Albumin + 5-FU)	0.001	0.034	–	0.001	0.001	0.001
Group 4 (Albumin + 5FU + Silver)	< 0.001	0.001	0.001	–	0.001	< 0.001
Group 5 (5-FU)	0.042	0.001	0.001	0.001	–	0.042
Group 6 (Normal Saline)						–

Tumor histology

In this section, the results of tumor histology in the different groups are discussed and compared. The histological evaluations include the percentage of necrosis, microscopic changes, and other features of the tumor tissue (Table 6; Fig. 7).

Table 6.

Comparison of histopathology results of tumors in different groups treated with albumin nanoparticles, albumin nanoparticles containing silver nanoparticles, albumin nanoparticles containing 5-fluorouracil, and albumin nanoparticles containing silver nanoparticles and 5-fluorouracil.

Group	Percentage of Necrosis (%) ± Standard Deviation	Tumor Differentiation Grade (1 to 3) ± Standard Deviation	Number of Apoptotic Cells (per field of view) ± Standard Deviation	Other Histopathology Changes
Group 1: Albumin Nanoparticles Only	25.0 ± 5.0	2.5 ± 0.5	5.0 ± 1.0	Tumor masses with moderate differentiation
Group 2: Albumin Nanoparticles with Silver	20.0 ± 4.0	2.3 ± 0.4	7.0 ± 1.5	Decreased tissue differentiation and presence of inflammatory cells
Group 3: Albumin Nanoparticles + 5-FU	15.0 ± 3.0	1.8 ± 0.3	10.0 ± 2.0	High concentration of apoptotic and necrotic cells
Group 4: Albumin Nanoparticles + 5FU + Silver	10.0 ± 2.0	1.5 ± 0.2	12.0 ± 2.5	Reduced live cells due to extensive apoptosis
Group 5: 5FU	18.0 ± 4.0	2.0 ± 0.3	8.0 ± 1.0	Mild necrotic changes and inflammation
Group 6 (Control): Normal Saline	30.0 ± 6.0	3.0 ± 0.5	2.0 ± 0.5	Tumor masses with high differentiation

Fig. 7.

Comparison of histopathology results of tumors in different groups treated with albumin nanoparticles (1), albumin nanoparticles containing silver nanoparticles (2), albumin nanoparticles containing 5-fluorouracil (3), albumin nanoparticles containing silver nanoparticles and 5Fu (4), 5FU (5), and control (6).

From the table and the figure above, the following results can be analyzed and reproduced:

Percentage of necrosis: The percentage of necrotic area was measured in each tumor. Group 4 had the lowest percentage of necrosis, indicating the positive effect of albumin nanoparticles and the drug 5FU.

Degree of differentiation of the tumor: This scale indicates the degree of differentiation of the tumor cells. Group 4 had the lowest degree of differentiation with a value of 1.5, indicating low differentiation and less malignancy.

Number of apoptotic cells: The number of apoptotic cells in the microscopic field of view was recorded. Group 4 had the highest number of apoptotic cells with a value of 0.12, indicating the positive effect of the combination of nanoparticles and the drug.

Other tissue changes: Here you will find a general description of tumor-related tissue changes. Group 4 also showed widespread apoptosis and a decrease in viable cells, indicating the therapeutic effect of the combination of nanoparticles and the drug 5FU.

A comparison between the groups shows that:

Group 1 (albumin nanoparticles alone) has a higher percentage of necrosis compared to Group 4, and the degree of tumor differentiation is also significantly higher.

Group 2 (albumin nanoparticles with silver) showed a decrease in the percentage of necrosis and an increase in the number of apoptotic cells compared to group 1, indicating the positive effect of silver. Group 3 (albumin nanoparticles + 5FU) shows a significant decrease in the percentage of necrosis and an increase in apoptotic cells compared to groups 1 and 2.

Group 4 (albumin nanoparticles + 5FU + silver) showed the lowest percentage of necrosis and the highest number of apoptotic cells. This group showed better results than the other groups.

Group 5 (5FU) shows a number of apoptotic cells and a lower percentage of necrosis than the other treatment groups.

Group 6 (control) showed the highest percentage of necrosis and the highest degree of tumor differentiation.

These results indicate that the combination of albumin nanoparticles with silver and 5FU has a positive effect on reducing necrosis and increasing apoptosis in tumor tissue. Group 4 was identified as the most effective treatment in this study.

According to Akdogan et al.⁹³, BSA nanoparticles were synthesized by the desolvation method using acetonitrile as desolvating agent. Doxorubicin was successfully encapsulated in these nanoparticles without altering the main properties. The cytotoxic effect was reduced by loading with doxorubicin compared to doxorubicin alone, and studies on tumor tissue also showed a much lower percentage of necrosis and differentiation than other samples, which is consistent with the results of this study. The high necrosis observed in tumors could be due to the rapid proliferation of cancer cells, reduced nutrient supply and the resulting death of some cells. The high degree of necrosis in cancer cells indicates cancer progression and is a signal for entry into the malignant stage and metastasis⁹⁴. A high degree of necrosis is typically associated with the increased ability of cancer cells to proliferate rapidly, maintain themselves and not respond to normal cell death signals. This phenomenon leads to the formation of larger and scattered tumors that can also invade other tissues⁹⁵. Severe necrosis in cancer cells is usually associated with the activation of various signaling pathways, such as the phosphoinositide 3-kinase/ Protein Kinase B (PI3K/Akt) and Mitogen-Activated Protein Kinase (MAPK) pathways, which allow cells to proliferate indefinitely and not respond to death signals. The activation of these signaling pathways can be caused by genetic mutations, the activation of protein genes or changes in cell function. In addition, necrosis can trigger an immune response from the body, but in many cases cancerous tumors have developed mechanisms to evade the immune system. Consequently, the tumor’s ability to progress and resist harsh conditions may be enhanced in this way⁹⁴. Therefore, high necrosis in cancer cells is not only a sign of cancer progression but also an important factor for malignancy progression and metastasis⁹⁶. Identifying and treating factors that influence high necrosis may help to reduce tumor growth and control the disease. The results of our study indicate that treatment with albumin nanoparticles containing both nanosilver and drug combinations was more effective in reducing necrosis. This better efficacy indicates the increased effectiveness of the combination in inhibiting the growth of cancer cells.

Based on tumor grade, the results show that the lowest tumor grade is associated with the group treated with silver-containing albumin nanoparticles and 5FU.

The mechanisms of action of silver nanoparticles and their conjugates with antitumor agents include ROS (reactive oxygen species) production and oxidative stress, DNA damage, cell cycle arrest and increased cell apoptosis. The combination of these processes prevents malignancy and the phenomenon of metastasis^97–99. Our study shows increased apoptosis in both groups treated with silver-containing albumin and silver-containing albumin and 5FU. A large part of this effect is due to the effectiveness of silver nanoparticles.

On the other hand, 5FU can exert its anti-cancer effect in two ways: by inhibiting the enzyme thymidylate synthetase and interfering with DNA synthesis and by influencing the uracil metabolism, which leads to apoptosis (programmed cell death) of the cancer cells. Therefore, the effect of this drug on inducing apoptosis has been proven^100,101. The higher apoptosis rate and consequently lower tumor grade in the group treated with nanoalbumin containing both agents compared to the loading systems with each agent alone may be attributed to the different efficacy of the nanosilver and the agent 5FU with separate mechanisms to induce apoptosis and reduce tumor growth rate and progression to higher grades. The synergistic effect of compounds affecting apoptosis induction has been investigated and confirmed in similar studies^102–104.

Kidney histology results

In this section, the results of renal histology in the different groups are presented. The histologic evaluations include microscopic changes, renal complications, and the severity of renal tissue damage in each group (Fig. 8).

Fig. 8.

Comparison of renal histology in different groups treated with albumin nanoparticles (1), albumin nanoparticles containing silver nanoparticles (2), albumin nanoparticles containing 5-fluorouracil (3), albumin nanoparticles containing silver nanoparticles and 5FU (4), 5FU (5), and control (6).

Group 1: Albumin nanoparticles alone: No serious renal complications were observed in the group treated with albumin nanoparticles alone. Renal histology showed normal and healthy structure of the glomeruli and tubules. There were no tissue abnormalities or cell necrosis.

Group 2: Albumin nanoparticles with silver: Mild kidney damage was observed in this group. The tissue changes included an increased number of inflammatory cells in the kidney tissue and slight changes in the glomerular structure. However, these complications were significantly lower than in the treatment groups with the drug 5-fluorouracil. These results indicate that silver-containing albumin nanoparticles have fewer negative effects on the kidneys.

Group 3: Albumin nanoparticles + 5FU: This group showed moderate renal complications. Renal histology showed mild necrosis in the proximal tubules and an increased number of apoptotic cells. In addition, tissue changes including mild fibrosis and inflammation were observed in the renal tissue. These complications were more pronounced than in Group 2, but still less than in Group 5.

Group 4: Albumin nanoparticles + 5FU + silver: Group 4, which was treated with a combination of nanoparticles and the drug 5FU, had more renal complications than groups 2 and 3. Renal histology showed relative necrosis in the proximal tubules, increased inflammation and fibrosis in the renal tissue. The number of apoptotic cells was also increased in this group compared to the other groups. These results indicate the negative effects of the combination of albumin nanoparticles with 5FU and silver on renal tissue. However, the observed renal complications in this group were lower than in Group 5. Group 5 (5FU): Most renal complications occurred in this group. The histology of the kidneys showed extensive necrosis, severe inflammation and fibrosis in the kidney tissue. A large number of apoptotic cells and severe changes in the glomerular and tubular structure were observed. The complications in this group clearly show the negative effects of the drug 5-fluorouracil on the kidney tissue.

Group 6: Control group (normal saline): No serious renal complications were observed in this group. The histology of the kidneys showed normal and healthy structure of the glomeruli and tubules. There were no tissue abnormalities or cell necrosis, and this group was recognized as the control group with minimal renal complications.

These results indicate that treatment with albumin nanoparticles alone has the least side effects on the kidneys and in fact had no effect on kidney tissue. Kidney side effects in the groups receiving combination treatment with albumin nanoparticles, silver and the drug 5FU progressively increased from group 2 (albumin nanoparticles with silver) to group 3 (albumin nanoparticles + 5FU) and then to group 4 (albumin nanoparticles + 5FU + silver). Finally, group 5 (5FU only) showed the highest level of renal side effects and had more tissue damage compared to the other groups. These results emphasize the importance of studying the side effects of combination therapies on different tissues, particularly the kidneys, and show that nanoparticles could be investigated as potential strategies to reduce the side effects of chemotherapeutic agents.

The size of silver nanoparticles has a direct impact on tissue damage and silver accumulation in tissues. Recordaty et al.¹⁰⁵ reported that silver concentrations in the spleen, lungs, kidneys, brain and blood of rats treated with 10-nanometer silver nanoparticles were significantly higher than in rats treated with larger particles. Toxic effects on the kidneys and liver were observed in rats treated with 10-nanometer silver nanoparticles, while rats treated with 100-nanometer silver nanoparticles showed mild or negligible lesions. As mentioned above, the size of silver nanoparticles has a significant impact on their tissue accumulation. In our study, the results indicate appropriate synthesis of silver nanoparticles, which caused less damage to renal tissue due to their appropriate size.

5-Fluorouracil (5FU) can be associated with numerous side effects, with renal toxicity being one of the best known. The renal toxicity of 5-fluorouracil is due to the production of toxic metabolites in the body. These metabolites can damage kidney tissue and disrupt the function of this vital organ. One such metabolite, fluoroacetic acid, can damage the epithelial cells of the renal tubules. 5-Fluorouracil-induced renal toxicity can lead to hemodynamic disturbances in the glomerular circulation, tubule cell damage and necrosis, nephropathy due to chemotherapeutic crystallization, renal damage due to rhabdomyolysis, and thrombotic thrombocytopenic purpura (TTP/HUS)^106,107. In our study, the highest renal toxicity was observed in the group treated with free drugs.

In our study, lower toxicity of 5FU was observed for the nano-based formulation compared to the free formulation. Based on related studies with 5FU, this lower toxicity can be explained as follows: A controlled-release formulation reduces the exposure of renal cells to high doses and ultimately decreases the adverse effects associated with the dose. In a similar study, Hagag et al. developed a nano-based dosing of 5FU using PLGA nanoparticles. These researchers demonstrated that delivery of 5-fluorouracil through nano-drug carriers such as PGLC can potentially reduce the associated adverse effects, particularly renal toxicity, due to the aforementioned reason¹⁰⁸. Liu et al. reduced the renal toxicity and increased the antifungal activity of amphotericin B by encapsulating it in albumin nanoparticles. The results of their study, which are consistent with our work, confirmed the lower renal toxicity of nanocarrier-based drugs compared to free drugs¹⁰⁹. Our study also confirmed these results. On the other hand, a significant part of this nephrotoxicity may be due to the accumulation and formation of unmetabolized residues of 5FU in these tissues. The incorporation and use of several essential amino acids and other acids with adequate capacity for antioxidant activity in protein-coated nanocarriers could significantly reduce the extent of this type of side effect through their role as antioxidants. These rationales also apply to the observed lower toxicity of albumin nanocarriers with silver nanoparticles compared to nanocarriers alone and of silver and 5-fluorouracil nanocarriers compared to 5FU alone.

A study by Rashid et al.¹¹⁰, which confirmed our findings, substantiated the ability of protein engineered nanocarriers to act as potential antioxidants and anti-apoptotic agents. All in all, for the reasons mentioned above.

Blood parameters results

Table 7 provides a comprehensive analysis of blood parameters, including red blood cell (RBC) count, hemoglobin (Hb) level and white blood cell (WBC) count, for all treatment groups at baseline and after 21 days of treatment. This table with associated p-values provides a key insight into the systemic effects and potential toxicities of the different formulations. Red blood cell (RBC) count and hemoglobin (Hb) level: The data show different effects on erythropoiesis in the different groups. Groups 1 (albumin nanoparticles only) and 2 (albumin nanoparticles with silver) showed remarkable stability in RBC count and Hb levels from the start of treatment to day 21, with no statistically significant changes observed (p > 0.05). This suggests that these formulations alone do not induce anemia or adverse effects on red blood cell production, highlighting their biocompatibility. In stark contrast, groups 3 (albumin nanoparticles with 5-FU), group 4 (albumin nanoparticles with 5FU + silver) and group 5 (free 5-FU) all showed a highly statistically significant decrease in red blood cell counts and Hb levels (p < 0.001) at day 21. This substantial decrease suggests the induction of anemia by 5FU, either in its free form or in encapsulated form. Of note, Group 4 (ANP-Ag-5FU), despite its strong anticancer effect, showed a less pronounced decrease in RBC count and Hb levels than Group 5 (free 5-FU) (e.g., Hb levels decreased to 9.8 g/dL in Group 4 vs. 9.0 g/dL in Group 5). This suggests that encapsulation in albumin nanoparticles, even in a double-loaded system, helps to attenuate some of the systemic hematologic toxicity (anemia) associated with free 5FU monotherapy through a controlled release mechanism and potentially less direct interaction with red blood cells. These results are consistent with the lower rates of hemolysis observed in the in vitro studies for nano-encapsulated forms^111–115. White blood cell (WBC) count: An increase in white blood cell counts was observed in all treatment groups from the start of treatment through day 21, generally indicating an immune response to the treatments, tumor burden, and stress associated with surgery and chemotherapy. This increase was statistically significant for most active treatment groups. Group 1 showed no significant change (p > 0.05), but group 2 (ANP-Ag) showed a statistically significant increase (p < 0.05), consistent with the known immunostimulatory properties of silver nanoparticles. Groups 3 (ANP-5FU), 4 (ANP-Ag-5FU) and 5 (free 5-FU) all showed a statistically highly significant increase in leukocyte count (p < 0.01 or p < 0.001). This leukocytosis can be attributed to the body’s immune response to the therapeutic agents and the inflammatory response due to tumor regression and tissue damage. The highest increase was observed in group 4, which also had the strongest anti-tumor effect, indicating a strong engagement of the immune system in response to the combination therapy. Albumin, an important multifunctional macromolecule in the human body, primarily determines oncotic pressure in plasma and modulates capillary permeability and fluid distribution between compartments. Its multiple binding sites and molecular flexibility make it suitable for the binding of several chemotherapeutic agents, which affect the distribution, metabolism, half-life and toxicity of the drug and are therefore crucial for its success in the bloodstream¹¹⁶. To reduce the cellular toxicity of anticancer drugs, nanoparticles can be combined with the drug, which is crucial to prevent the reduction of the drug’s interaction with the red blood cell surface. The results of the reduced effect of Nanoform on red blood cells could be due to a reduced interaction of the drug with the surface of the red blood cells. The results obtained in hematology showed changes in blood and red blood cell parameters. These changes in red blood cells could indicate the influence of the nanoparticles on hemoglobin synthesis during the maturation of red blood cells in the bone marrow¹¹⁷. White blood cells play a crucial role in immunity and may indicate infection, allergic reaction or toxicity to drugs or chemical substances¹¹⁸. An increase in white blood cells was observed in all treatment groups. This increase is due to the stimulation of the immune system against the active substances. At the same time, the level of red blood cells decreased in the treated animals compared to the control group. The occurrence of anemia is due to hypoxia, and the reduction in the number of red blood cells indicates increased destruction of red blood cells by the blood toxicity of silver nanoparticles and the drug 5FU¹¹⁹. Studies have shown that both silver nanoparticles and 5FU can suppress the hormone erythropoietin (a glycoprotein that stimulates erythropoiesis), leading to a slight reduction in red blood cells and hemoglobin¹¹⁷. In summary, the analysis of blood parameters confirms the systemic effects suggested by the hemolysis and renal toxicity results. While 5FU in its free or encapsulated form induces hematologic changes such as anemia and leukocytosis, the albumin nanoparticle-based delivery system, particularly the double-loaded ANP-Ag-5FU, appears to have a protective effect leading to less red blood cell depletion compared to monotherapy with free 5FU. This reduced cellular toxicity of anticancer drugs in combination with nanoparticles is crucial in preventing the reduction of drug-red blood cell surface interaction and represents a key advantage of nanoformulations. These results highlight the potential of the nanocarriers we have developed to improve the therapeutic window by increasing efficacy while mitigating some of the severe systemic side effects of conventional chemotherapy.

Table 7.

Comparison of blood parameter analysis results in different groups treated with albumin nanoparticles, albumin nanoparticles containing silver nanoparticles, albumin nanoparticles containing 5-fluorouracil, and albumin nanoparticles containing silver nanoparticles and 5-fluorouracil.

Group	Blood Collection Time	Red Blood Cell Count (×10⁶ cells/µL) ± SD	p-value (RBC)	Hemoglobin Level (g/dL) ± SD	p-value (Hb)	White Blood Cell Count (×10³ cells/µL) ± SD	p-value (WBC)
Albumin nanoparticles only	Treatment start	5.7 ± 0.5	–	13.2 ± 1.1	–	7.8 ± 1.0	–
	Day 21	5.8 ± 0.6	> 0.05	13.5 ± 1.2	> 0.05	8.0 ± 1.0	> 0.05
Albumin nanoparticles with silver	Treatment start	5.6 ± 0.4	–	12.9 ± 1.0	–	8.0 ± 1.1	–
	Day 21	5.9 ± 0.5	> 0.05	13.0 ± 1.2	> 0.05	9.0 ± 1.5	< 0.05
Albumin nanoparticles with 5-FU	Treatment start	5.7 ± 0.6	–	13.1 ± 1.0	–	8.2 ± 1.2	–
	Day 21	4.5 ± 0.5	< 0.001	10.5 ± 1.0	< 0.001	10.5 ± 1.8	< 0.01
Albumin nanoparticles with 5FU + silver	Treatment start	5.8 ± 0.5	–	13.0 ± 1.1	–	8.5 ± 1.0	–
	Day 21	4.0 ± 0.4	< 0.001	9.8 ± 0.9	< 0.001	11.0 ± 2.0	< 0.001
5-FU	Treatment start	5.6 ± 0.5	–	12.8 ± 1.2	–	8.3 ± 1.1	–
	Day 21	4.2 ± 0.3	< 0.001	9.0 ± 0.8	< 0.001	10.0 ± 1.5	< 0.01

This research makes several distinct contributions to the existing literature. While previous studies have independently investigated protein nanoparticles for drug delivery and silver nanoparticles for cancer therapy, this work represents the first comprehensive evaluation of a dual-loaded system combining green-synthesized AgNPs with 5FU in albumin carriers specifically for the treatment of colorectal cancer. In contrast to existing approaches that typically focus on encapsulation of a single drug or chemically synthesized metallic nanoparticles, our study introduces an environmentally benign synthesis method for AgNPs that simultaneously addresses the dual challenge of improving therapeutic efficacy and minimizing toxicity. In addition, this research provides the first in vivo evidence of the synergistic potential between green-synthesized silver nanoparticles and 5FU when co-administered via albumin nanoparticles. It thus offers a novel therapeutic strategy that could bridge the gap between nanotechnology and sustainable cancer treatment approaches. The comprehensive evaluation, which includes synthesis, characterization, in vitro biocompatibility and in vivo efficacy in a well-established animal model, provides a complete translational framework that expands the field beyond current single-component or unsustainable nanoformulations.

Conclusion

The results of this study indicate that the delivery system designed based on albumin nanoparticles with simultaneous loading of silver nanoparticles and 5FU can be effective and beneficial in treating colon cancer. This system has the ability to provide slow and controlled drug release, which can help reduce the required dosage and decrease the side effects of chemotherapy drugs.

Author contributions

M.D. conducted the laboratory experiments, including synthesis and characterization of nanoparticles, drug loading, release kinetics studies, and in vitro cytotoxicity assessments, and drafted the initial manuscript text. H.Z.Z. and H.E. conceptualized the study design, oversaw the research process, and provided critical revisions to the manuscript. H.Z.Z. and H.E. also supervised the animal model experiments, performed histopathological analyses, and contributed to data interpretation. M.A. assisted with nanoparticle characterization techniques and contributed to in vitro experimental procedures. F.F. performed blood analysis, assisted with animal experiments, and contributed to data collection and analysis. H.Z.Z. prepared all figures and tables. All authors reviewed and approved the final manuscript.

Data availability

All data are available in the manuscript.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This study was approved by the Ethics Committee of Shahid Sadoughi University of Medical Sciences, Yazd, Iran, and was conducted in accordance with the ethical guidelines for the treatment of animals (Ethics Code: IR.SSU.REC.1402.140). All efforts were made to minimize animal suffering and reduce the number of animals used. This study is reported in compliance with the ARRIVE guidelines (https://arriveguidelines.org), ensuring transparency and reproducibility in the reporting of in vivo experiments.