Targeted Immunoliposomal Delivery of a 5-Fluorouracil Analog in EGFR-Expressing Pancreatic Cancer Models

Abstract

Introduction

Dysregulated epidermal growth factor receptor (EGFR) signaling is a key mechanism driving cancer progression and metastasis. Owing to its frequent overexpression in pancreatic cancer (PCa), EGFR has become a desirable molecular target for targeted therapies. XYZ-I-73 (N-(5-fluoro-2-oxo-1-(tetrahydrofuran-2-yl)-1,2-dihydropyrimidin-4-yl) dodecanamide), a structural analog of 5-fluorouracil (5-FU), has been previously synthesized and shown to exhibit cytotoxicity against PCa cells.

Methods

XYZ-I-73 was entrapped in liposomes via thin-film hydration and subsequently conjugated to EGFR antibodies to produce an immunoliposome formulation- Ab-XYZ-I-73LnP (where ‘Ab’ denotes antibody-conjugated and ‘LnP’ denotes liposomal nanoparticle). In vitro efficacy was assessed by measuring cell viability and apoptosis in MiaPaCa-2 and PANC-1 cells, while pharmacokinetics and antitumor efficacy were determined in a cell line-derived xenograft (CDX) mouse model.

Results

Ab-XYZ-I-73LnP exhibited a mean particle size of 143nm ± 2.3, PDI (0.37), and zeta potential -46.2 ± 1.3mV. In MiaPaCa-2 cells, Ab-XYZ-I-73LnP showed remarkably higher cytotoxicity than 5-FU in both 2D (IC₅₀ = 2.5 ± 0.9μM vs 13.2 ± 1.1μM) and 3D cultures (IC₅₀ = 8.1 ± 1.1μM vs 26.7 ± 1.1 μM). Similarly, in PANC-1 cells, Ab-XYZ-I-73LnP showed lower IC₅₀ values compared to 5-FU;2D (IC₅₀ = 2.9 ±1.1 μM vs 20.4±1.2 μM), 3D (IC₅₀ = 12.9 ± 0.6μΜ vs 37.1±0.9 μM). Pharmacokinetic analysis revealed a prolonged half-life for Ab-XYZ-I-73LnP compared with free 5-FU (t1/2 = 1.62 ± 0.03 h vs 0.49 ± 0.01 h, p < 0.001). There was about a 2-fold increase in the area under the curve (AUC) for Ab-XYZ-I-73LnP compared to 5-FU (AUC= 0.32 ± 0.04µg/(L*hr) vs 0.15 ± 0.02µg/(L*hr), p<0.01).

Conclusion

Overall, the study supports the formulation of an immunoliposome of modified 5-FU, which may significantly enhance drug bioavailability and therapeutic potential for the treatment of PCa.

Introduction

Despite studies on novel biomarkers and therapeutic advancements in pancreatic cancer (PCa) treatment, the rapidly progressing malignancy is projected to become the second deadliest cancer in the US by 2030.^1,2 The 5-year survival rate for about 90% of patients diagnosed with PCa is a meager 8%. ³ Late diagnosis continues to plague PCa survival, as over 80% of patients present when the disease is either metastatic or unresectable, leading to poor survival outcomes. ⁴ Risk for pancreatic cancer increases with age (>55years), sex (male), obesity, cigarette smoking, genetic mutations, ethnicity, and chronic pancreatitis.^5-7 To facilitate early diagnosis and prevention of pancreatic cancer, imaging-based screening (magnetic resonance imaging and endoscopic ultrasound) is recommended in groups with high familial risk.^8,9

Major therapeutic breakthroughs in PCa treatment have been hindered by the emergence of cancer stem cells, the high mutation burden resulting from the malignancy’s aggressive nature, and the complex tumor microenvironment. ¹⁰ Nanoparticles have been utilized for several years in cancer research and treatment due to their unique size, composition, and potential for targeted drug delivery. ¹¹ Among nanoparticles, liposomes are particularly useful as drug-delivery systems because their hydrophilic core and an outer lipid bilayer allow for the entrapment of both lipophilic and hydrophilic compounds. ¹² Liposomes significantly enhance drug pharmacokinetics, reduce the need for high doses, reduce adverse effects, and enhance efficacy. ¹³ Their small size, biocompatibility, biodegradability, and low toxicity and immunogenicity support their utility in cancer therapy.^14,15 The FDA recently approved Onivyde (liposomal irinotecan) in combination with oxaliplatin, fluorouracil, and leucovorin for the treatment of metastatic PCa, further validating the prospects of liposomal formulations in PCa therapy. ¹⁶ Despite liposomes being highly advantageous as a delivery system, their utility can be marred by low encapsulation efficiency, leakage of entrapped drugs, oxidation, target accessibility and expression, and formulation stability.^17,18

Antibody-conjugated liposomes, known as immunoliposomes, are formulated to target cells that express specific antigens. Immunoliposomes enhance the targeted delivery of chemotherapeutic agents and are valuable in imaging, immunotherapy, and immunoassays. ¹⁹ Some advantages of immunoliposomes over conventional liposomes include targeted delivery, enhanced drug accumulation, improved pharmacokinetics, potential for personalized medicine, and stimulation of the immune response, thereby improving therapeutic efficacy.^20-23

EGFR, one of four receptor tyrosine kinases (RTKs) in the ErbB family, plays a significant role in both normal physiological processes and cancer formation and progression.^24,25 Amplification of the EGFR gene and mutations in the tyrosine kinase domain are evident in several cancers. ²⁶ EGFR overexpression is observed in about 30%-89% of pancreatic ductal adenocarcinoma (PDAC) cases.^26-28 The prevalence of EGFR overexpression and its contribution to disease pathogenesis and aggressiveness position this potent oncogene as a promising target for PCa therapy. ²⁹ Although EGFR inhibitors have improved health outcomes in some cancer patients, their benefits have been marred by resistance, underscoring the need for novel agents that offer sustainable benefits for EGFR-expressing tumors. ³⁰ The proven efficacy of liposomes and the increasing relevance of monoclonal antibodies in cancer therapy make immunoliposomes a valuable strategy in PCa therapy.

Fluorouracil analogs, such as 5-FU, Fluorodeoxyuridine (FdUR), capecitabine, and tegafur, treat gastrointestinal cancers by acting as antimetabolites that disrupt DNA/RNA synthesis.^31,32 They enter cells primarily via nucleoside transporters (e.g., hENT1) or uracil-like transport mechanisms. ³² Once inside, they undergo phosphorylation, leading to the misincorporation of active metabolites into DNA and RNA, leading to apoptosis.^33,34 XYZ-I-73(N-(5-fluoro-2-oxo-1-(tetrahydrofuran-2-yl)-1,2-dihydropyrimidin-4-yl) dodecanamide), a newly synthesized analog of 5-FU, formed by chemical modification of 5-fluorocytosine with lauroyl chloride, has demonstrated anticancer effects in PCa cells. ³⁵ Our previous study showed that XYZ-I-73 demonstrated a remarkable cytotoxic effect, inhibition of cell migration, and improved in-vitro metabolic stability over 5-FU. ³⁵ Building on these findings, the present work focused on developing an immunoliposome delivery system by conjugating pegylated XYZ-I-73 liposomes with an EGFR monoclonal antibody and evaluating its efficacy in in vitro and in vivo PCa models. The study aimed to determine the cytotoxicity and apoptotic effects in PCa cells, as well as the pharmacokinetics and the antitumor efficacy of the immunoliposome in a cell line-derived xenograft (CDX) mouse model.

Materials and Methods

Materials

5-FU, analytical-grade reagents, and anti-EGFR antibodies were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Soy was purchased from EMD Millipore Corp. (Billerica, MA, USA). Dipalmitoyl phosphatidylcholine (DPPC), 1-Myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC), 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG2000), and DSPE-PEG2000 carboxy NHS lipids were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Labrasol was purchased from Gattefosse Sas, Saint-Priest, France. Pancreatic cancer MiaPaCa-2 and PANC-1 cells were bought from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The Pierce™ micro–BCA Protein Assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). EGFR (Cat. # 4267S), VEGF (Cat. # 2479S), and HER-2 (Cat. # 2165S) proteins were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Prior Synthesis, Characterization, and In Vitro Evaluation of XYZ-I-73

The design, synthesis, characterization, and in vitro anticancer effect of XYZ-I-73 in PCa cells have been comprehensively reported in our previous study. ³⁵ Briefly, XYZ-I-73 was synthesized by introducing a tetrahydrofuran ring onto 5-fluorocytosine (a precursor of 5-FU) and conjugating it to lauroyl chloride. ³⁵ The biological activity of the novel analog was evaluated in MiaPaCa-2, PANC-1, and BxPC-3 PCa cells. The cytotoxicity, inhibition of cell migration, in vitro metabolic stability, and inhibition of pro-apoptotic protein expression were investigated. All detailed experimental procedures, results, and in-depth discussion of these foundational studies are available in our earlier publication.

Western Blot Study

Western blot was performed as previously described.^36,37 Briefly, MiaPaCa-2 cells were seeded in a 6-well plate containing DMEM supplemented with 10% FBS at a density of 2.5× 10⁵ cells per well. Upon reaching 70% confluency, cells were treated with IC₅₀ and 2*IC₅₀ concentrations of XYZ-I-73 and 5-FU for 12 hours. After treatment, cells were rinsed with phosphate-buffered saline (PBS) and lysed with RIPA buffer supplemented with protease and phosphatase inhibitors (1:100) to obtain whole-cell lysate (Sigma-Aldrich, St. Louis, MO, USA). Following that, the lysate was incubated on ice for 30 min, then centrifuged at 10,000 rpm for 15 min, and the supernatant was collected. Protein estimation was subsequently conducted via the Bicinchoninic Acid (BCA) protein assay. ³⁸ A sample containing 40 µg of equivalent proteins was run on Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) and then transferred to a Polyvinylidene fluoride membrane (PVDF). The membrane was blocked with 5% BSA in Tris-buffered saline with Tween 20 (TBST). The membrane was incubated with primary antibodies: EGFR, VEGF, HER-2, and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (each at a 1:1000 dilution), prepared in TBST, and incubated overnight at 40°C on a shaker. GAPDH served as the loading control. After washing, membranes were incubated with Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:20,000), and chemiluminescence was visualized using a Fusion-FX chemiluminescence imager (Vilber Lourmat, Germany). The relative band densities were quantified using ImageJ 1.36 software (Wayne Rasband, National Institutes of Health, MD, USA).

Preparation of XYZ-I-73 Liposomal Nanoparticles (XYZ-I-73LnP)

Different liposomal nanoparticles containing XYZ-I-73 (XYZ-I-73LnP) (Figure 1) were prepared by the thin film hydration method.^39,40 XYZ-I-73, together with lipids (Soy, DPPC, MPPC) and DSPE-PEG 2000, was measured at different molar ratios with Labrasol, Compitrol, or Tween80, and the mixtures were placed in round-bottom flasks. The molar ratios were chosen to maintain a higher proportion of structural lipids (e.g., DPPC) to ensure the formation of a stable bilayer, which is essential for efficient drug encapsulation. A relatively lower proportion of DSPE-PEG2000 was used to prolong circulation without hindering cellular uptake. Similarly, surfactants were incorporated at lower ratios to enhance and improve homogeneity.

Figure 1.

Structure of XYZ-I-73

The lipids were dissolved in chloroform, vortexed, and then the solvent was removed under reduced pressure using a rotor evaporator to generate a uniform lipid film. This film was rehydrated with 10 mL of PBS at 60°C, a temperature exceeding the lipid phase transition. The suspension was then agitated by brief vortexing for 1 min and bath-sonicated for 5min to facilitate vesicle formation. Finally, the resulting multilamellar vesicles were passed through a 200nm polycarbonate membrane to obtain uniformly sized unilamellar liposomes.

Preparation of XYZ-I-73 Immunoliposomes (Ab-XYZ-I-73LnP)

Conjugation of the EGFR antibody to the surface of XYZ-I-73LnP was performed following the manufacturer’s protocol.^41-43 In addition to DSE-PEG 2000 used in formulating XYZ-I-73LnP, DSE-PEG (2000)- Carboxy NHS was used in immunoliposome preparation. The direct-coupling process allowed for the covalent coupling of free carboxylic groups on the liposomes’ surfaces to primary amines of antibodies through activation of the carboxyl groups with EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide). 0.4mg of 2mM EDC was added to XYZ-I-73LnP. Subsequently, 2mg of DSE-PEG (2000) Carboxy NHS was added to the EDC-activated liposomes. The resultant solution was mixed thoroughly and allowed to react for 15 min at room temperature before the EGFR antibody(cetuximab) was added. Excess EDC was removed by size exclusion. 1mg/mL of the EGFR antibody was dissolved in PBS, pH 7.4, and added to the EDC/Sulfo-NHS-activated pegylated liposomes at a 1:1000 antibody-to-lipid molar ratio. The solution was mixed thoroughly and allowed to react for 2h at room temperature. Subsequently, the unconjugated proteins and free XYZ-I-73 were removed by dialysis overnight using a dialysis bag against phosphate buffer (pH 7.4). The resulting immunoliposome was denoted by Ab-XYZ-I-73LnP, where ‘Ab’ denotes antibody-conjugated and ‘LnP’ denotes liposomal nanoparticle.

Characterization of XYZ-I-73 Liposomal Nanoparticles (XYZ-I-73LnP)

Particle Size Measurement

The particle size, polydispersity index (PDI), zeta potential of the XYZ-I-73 liposomal formulations, and XYZ-I-73 immunoliposomes were determined by dynamic light scattering using the NICOMP™ 380 ZLS particle sizer. Deionized water was added to the samples at a 1:10 ratio before measurements were taken.

Entrapment Efficiency (EE%)

100 μL of 30% Triton X-100 was used to disrupt the XYZ-I-73 liposomal suspension. The disrupted liposomes were gently mixed for 2 min and then centrifuged at 6,000 rpm at room temperature for 5 min. The supernatant was collected and analyzed for XYZ-I-73 using reverse-phase high-performance liquid chromatography (HPLC). The mobile phase solution consisted of 95% acetonitrile (ACN) and 5% acidified water, as previously described. ⁴⁴ The sample injection volume was 10 μL, with a flow rate of 1.0 mL/min at room temperature. XYZ-I-73 was detected at 264 nm using a photodiode-array detector. The entrapment efficiency was calculated according to the following equation:

$$\text{EE}\hspace{0.17em}(\%)=\frac{\text{Amount}\hspace{0.17em}\text{of}\hspace{0.17em}\text{drug}\hspace{0.17em}\text{entrapped}\hspace{0.17em}\text{in}\hspace{0.17em}\text{nanoparticles}}{\text{Initial}\hspace{0.17em}\text{amount}\hspace{0.17em}\text{of}\hspace{0.17em}\text{drug}}\times 100\%$$

Confirmation Conjugation of EGFR Antibody to XYZ-I-73LnP

Fourier Transform Infrared Analysis (FTIR)

FTIR analysis was conducted on the Ab-XYZ-I-73LnP to confirm the conjugation of the anti-EGFR to the XYZ-I-73LnP surface. XYZ-I-73LnP, Ab-XYZ-I-73LnP, 5-FU, and Labrasol were analyzed in the spectra range of 450-4000 cm^-1 using an FTIR spectrophotometer (PerkinElmer Life and Analytical Sciences, Connecticut, USA). Triplicate measurements were taken, and the presence of conjugation between the EGFR antibody and the liposomal nanoparticles was determined from the acquired spectra.

Fluorescence Microscopy

Conjugation of the EGFR antibody to the surface of the XYZ-I-73LnP was performed as previously described. ⁴⁵ Briefly, FITC-conjugated EGFR antibody was added to XYZ-I-73 liposomes labeled with rhodamine. The excitation spectrum was scanned from 450–500 nm and the emission spectrum from 500–550 nm for fluorescein, while for rhodamine, excitation was scanned from 520–570 nm and emission from 550–650 nm. Images were captured using a Nikon Ti Eclipse microscope to show co-localization between rhodamine and fluorescein.

In-Vitro Cell Viability Studies of Ab-XYZ-I-73LnP Against MiaPaCa-2 and PANC-1 Pancreatic Cancer Cells

2D- Cell Viability Studies

MiaPaCa-2 and PANC-1 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM with high glucose and L-glutamine), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PenStrep) before conducting cell viability studies. ⁴⁶ Following this, MiaPaCa-2 and PANC-1 cells were plated in 96-well plates at a density of 6 x 10³ cells per well, with each drug concentration tested in triplicate. The plates were incubated at 37 °C and 5% CO₂. Once the cultures reached approximately 70% confluence, they were exposed to Ab-XYZ-I-73LnP, XYZ-I-73LnP, or 5-FU. Different concentrations of the modified 5-FU liposomal formulations were prepared by diluting the stock solution stepwise in growth medium. ⁴⁷ A separate 5-FU stock was prepared in PBS and subsequently diluted to final concentrations of 3, 6, 12, 25, and 100 µM. Each well received 200 μL of each drug, and the cells were incubated for 48 hours. At the end of the treatment period, 20 μL of a 0.05% resazurin sodium salt solution (Alamar Blue) was added to each well, followed by a 4 h incubation under standard culture conditions (37°C, 5% CO₂). ⁴⁸ Fluorescence measurements were then obtained using excitation at 560/580 nm and emission at 590/610 nm, and cell viability percentages were determined for each concentration.

3D- Cell Viability Studies

10 × 10³ cells per well of MiaPaCa-2 and PANC-1 cells were seeded in Nunclon Sphera® 96-well plates using 100 μL of complete media. The plates were then incubated at 37 °C with 5% CO₂ for 48 hours to allow the formation of 3D spheroids. ⁴⁰ For treatment, 100 μL of each drug formulation, prepared as described for the 2D viability assays, was added to the wells. At the end of the exposure period, 50 μL of a 0.15% resazurin sodium salt solution (Alamar Blue) in growth medium was added to each well and incubated for an additional 4hours. Fluorescence measurements were subsequently obtained following the previously described procedure.

3D Apoptosis Study

To determine the effects of Ab-XYZ-I-73LnP on 3D spheroids, MiaPaCa-2 and PANC-1 cells (10x10³ cells per well) were seeded into 96 U round-bottom Nunclon Sphera plates and incubated for 48 hours for spheroid formation. The cells were treated with Ab-XYZ-I-73LnP, XYZ-I-73LnP, and 5-FU at 24 and 48 hours, respectively. After 72 hours of the spheroid treatment, the spheroids were stained with 5 µg/mL of an acridine orange/ethidium bromide (AO/EB) solution. ⁴⁹ Images of the spheroids were captured using a Nikon Ti Eclipse microscope. The ratio of AO to EB fluorescent intensities for each treatment concentration was computed and plotted in GraphPad Prism 10.

Animal Studies

Ethics statement: Eight-week-old female NOD/SCID (Non-Obese Diabetic/severe combined immunodeficiency) mice weighing 22-25g were obtained from the Jackson Laboratory (Bar Harbor, ME). Upon arrival, the animals were kept in a temperature-controlled facility for 1 week with unrestricted access to food and water before beginning the treatment phase. All experimental procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. ⁵⁰ The reporting of this study conforms to Animal Research Reporting of In Vivo Experiments (ARRIVE) 2.0 guidelines. ⁵¹ Approval (Reference # 023-07) for the study and protocol was granted by the Animal Care and Use Committee.

Subcutaneous Cell-Line Derived Xenograft (CDX) Tumor Implantation

MiaPaCa-2 (2.5 × 10⁶ cells injected per mouse) was used to form subcutaneous tumors in the left flank of the mice. NOD/SCID mice have reduced NK cell activity and severely impaired B and T cell function, supporting engraftment with human tissues and cells. ⁵² The mice were observed for 4 weeks until a sizeable tumor developed, and treatment was initiated. Upon reaching a tumor volume of approximately 60-80 mm³, the mice were randomly divided into four groups of eight: control, Ab-XYZ-I-73LnP, XYZ-I-73LnP, and 5-FU. Randomization was achieved using computer-generated random numbers. Group size (n=8) was selected based on prior studies showing detectable differences in tumor volume with similar sizes. Animals with tumor volumes < 60 mm³ or showing signs of illness prior to treatment were excluded from the study. The control group comprised mice that developed tumors 60-80mm³ but received no treatment. The mice in the control group received normal saline. Carbon dioxide (CO₂) inhalation was used for euthanasia. The study used a parallel-group design to evaluate therapeutic efficacy and pharmacokinetics. The primary endpoint was tumor growth inhibition, and the secondary endpoint was acute toxicity.

Tumor-Efficacy Studies

After baseline tumor volumes were established, treatment began with mice receiving 12.5mg/kg of 5-FU, XYZ-I-73LnP, and Ab-XYZ-I-73LnP (5-FU equivalent dose) intraperitoneally twice weekly for 5 weeks. The 5-week treatment period enabled assessment of long-term tumor growth inhibition, relapse, and metabolic changes, bridging the gap between acute efficacy and survival studies. Studies indicate that 12.5mg/kg of 5-FU provides moderate to significant antitumor effects without causing severe side effects, such as significant body weight loss.^53,54 Tumor width (W) and length (L) measurements were taken every other day using calipers, and the tumor value was calculated using the following equation: V(mm3) = (L*(W)2)/2. Investigators measuring tumor volumes and performing statistical analyses were blinded to treatment allocation until data analysis was completed. Animals were monitored daily for signs of distress and changes in body weight. Humane endpoints included >20% body weight loss. All efforts were made to minimize the number of animals used and to reduce suffering.

Pharmacokinetics Studies

Pharmacokinetics was conducted as previously described by Wang et al. ⁵⁵ Mice were grouped into 3 groups of 5 (control, 5-FU, and Ab-XYZ-I-73LnP). Mice in the control group developed tumors 60-80mm³ but received no treatment. They were injected intraperitoneally with a bolus dose of 12.5 mg/kg of 5-FU, 37mg/kg of Ab-XYZ-I-73LnP (equivalent to 5-FU at 12.5 mg/kg), or normal saline. Blood samples were collected at 5, 15, 30, 1, 2, 4, 8, 12, and 24 hours. The extraction solvent, 1 mL of 15% isopropyl alcohol in ethyl acetate, was used to treat the blood samples, and the mixture was vortexed for approximately 30 seconds. The samples were then centrifuged at 3,000 rpm for 15 min. The supernatant from the blood samples was evaporated in a water bath and placed in a vacuum chamber overnight to remove residual solvent. The mobile phase (500 μL of 5% acetonitrile in acidified water was used to reconstitute the dried samples, which were then centrifuged. The supernatants were filtered, and the filtrates were analyzed for 5-FU and XYZ-I-73 using HPLC (Agilent Technologies, 1290 Infinity). The extraction method using isopropyl alcohol and ethyl acetate was validated through recovery studies, demonstrating high and consistent recovery with minimal variability across replicates. HPLC analysis of 5-FU and XYZ-I-73 was performed using the Eclipse Plus C18 column (5 µm, 4.6 × 250 mm, Agilent, Santa Clara, CA) with a diode-array detector. The mobile phase consisted of acetonitrile (ACN) and acidified water (90:10, v/v). Standard solutions of 5-FU were prepared in ACN and acidified water (90:10) at concentrations of 1–1000 ppm (1, 10, 100, 250, 500, and 1000 ppm). Samples were injected at 10 µL and eluted at 1mL/min, with detection at 264nm and a retention time of approximately 5.2 minutes. A calibration curve was plotted using the peak areas of the standard solutions versus their respective concentrations. Linear regression analysis of the calibration data was performed, and the amount of 5-FU and XYZ-I-73 from plasma samples was determined by comparing the peak areas from the samples with those of the standard solutions.

Statistical Analysis

Data was analyzed for significance by one-way ANOVA, followed by Tukey’s Multiple Comparison Test using GraphPad Prism 10 Software, and IC₅₀ values were determined. Results are presented as mean ± SEM. Significance is defined as p<0.05.

Results

Western Blot Study

The levels of EGFR, VEGF, and HER-2 protein expression decreased with increasing drug treatment concentration for both 5-FU and XYZ-I-73. However, XYZ-I-73 significantly reduced EGFR expression in the MiaPaCa-2 treated cells at IC₅₀, and 2*IC₅₀ concentrations compared to 5-FU treated cells (Figure 2A). A similar reduction in VEGF and HER-2 expression was observed in MiaPaCa-2 cells treated with a 2*IC₅₀ concentration of XYZ-I-73 in comparison with 5-FU (Figure 2B and C. This indicates the potential role of XYZ-I-73 in inhibiting key proteins involved in tumor growth and proliferation.

Figure 2.

MiaPaCa-2 cells protein expression after 12h treatment with IC₅₀ & 2*IC₅₀ concentrations of XYZ-1-73 and 5-FU

Figure 2 shows a blot of the protein expression after treating MiaPaCa-2 with XYZ-I-73 and 5-FU for 12h. IC₅₀ and 2*IC₅₀ concentrations of the treatments are indicated as L and H, respectively. Untreated MiaPaCa-2 cells (Control) were used as the negative control, while 5-FU-treated cells served as the positive control. For protein band expression, the blots were cut prior to hybridization with primary antibodies. a) Quantitative expressions of EGFR, b) Quantitative expressions of VEGF, and c) Quantitative expression of HER-2. Full-length blots are presented in Supplementary Figure 1.

XYZ-I-73 Liposomal Nanoparticles (XYZ-I-73LnP) and Ab-XYZ-I-73LnP Characterization

Particle Size, Zeta Potential, and Polydispersity Index Measurement

The XYZ-I-73 liposomal nanoparticles were prepared by varying the amounts of the lipids and surfactants and maintaining the XYZ-I-73 amount at 0.05%w/v across all formulations, as shown in Table 1. The XYZ-I-73LnP₁ to XYZ-I-73LnP₇ particle sizes were observed to be between 105nm and 208nm. XYZ-I-73 LnP₅ was found to have an optimum particle size (105± 2.1nm) and stability (zeta potential of -34.30± 0.3 mV and PDI of 0.24).

Table 1.

Characterization of XYZ-I-73 Liposomal Nanoparticles

Formulation	Amount of drug (mg)	Lipid composition	Molar ratio	Mean particle size (nm)	Zeta potential (mV)	Polydispersity index (PDI)
XYZ-I-73 LnP1	5	DPPC: SoyPC: DSPE-PEG2000	70:20:10	169.6 ± 2.3	-8.9 ± 4.1	0.35
XYZ-I-73 LnP2	5	DPPC: COMP: DSPE-PEG2000	50:20:30	208.2 ± 3.4	4.9 ± 3.7	0.46
XYZ-I-73 LnP3	5	DPPC: MPPC: SoyPC: DSPE-PEG2000	60:20:10:10	166.8 ± 2.5	3.1 ± 3.9	0.41
XYZ-I-73 LnP4	5	DPPC: TWEEN 80: DSPE-PEG2000	60:10:30	150.0 ± 1.8	1.4 ± 1.1	0.38
XYZ-I-73 LnP5	5	DPPC: LBR: DSPE-PEG2000	60:10: 30	105.1 ± 2.1	-34.3 ± 0.3	0.24
XYZ-I-73 LnP6	5	DPPC: DSPE-PEG2000	60:40	125.8 ± 1.1	-17.7 ± 0.1	0.29
XYZ-I-73 LnP7	5	DPPC: MPPC: DSPE-PEG2000	70:20:10	136.7 ± 1.7	-13.9 ± 0.2	0.31

Data expressed as mean ± SEM, n=3. Dipalmitoyl phosphatidylcholine (DPPC), 1-Myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC), 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG₂₀₀₀), Labrasol (LBR), Compitrol (COMP).

Entrapment Efficiency

Overall, XYZ-I-73 LnP₅ had the highest entrapment efficiency (84.31±4.8%), as shown in Table 2. The incorporation of a surfactant and amounts of the lipids and/or the surfactants used contributed significantly to the XYZ-I-73LnP particle size and entrapment efficiency. The incorporation of Labrasol into XYZ-I-73LnP₅ produced liposomes with reduced particle size (105.1 ± 2.1nm), PDI (0.24), and zeta potential (−34.3 ± 0.3 mV) relative to that obtained for the formulation without Labrasol (XYZ-I-73LnP₆: particle size (125.8 ± 1.1nm), PDI (0.29), and zeta potential (−17.7 ± 0.1 mV)). This could be due to Labrasol’s amphiphilic properties, which enhance interfacial stabilization and efficient lipid bilayer packing, thereby increasing drug-loading capacity. Our findings suggest that the Labrasol-containing liposomal nanoparticles was a more effective delivery system for XYZ-I-73 than other formulations incorporating different surfactants.

Table 2.

Entrapment Efficiency of XYZ-I-73 Liposomal Nanoparticles

Formulation	Amount of drug (mg)	Lipid composition	Molar ratio	Mean particle size (nm)	Zeta potential (mV)	Entrapment efficiency (%)
XYZ-I-73 LnP5	5	DPPC: LBR: DSPE-PEG2000	60:10: 30	105.1±2.1	-34.3 ± 0.3	84.3 ± 4.8
XYZ-I-73 LnP6	5	DPPC: DSPE-PEG2000	60:40	125.8±1.1	-17.7 ± 0.1	69.5 ± 7.1
XYZ-I-73 LnP7	5	DPPC: MPPC: DSPE-PEG2000	70:20:10	136.7±1.7	-13.9 ± 0.2	75.2 ± 3.1
Ab-XYZ-I-73LnP	5	EGFR + DPPC: LBR: DSPE-PEG2000	60:10: 30	143.0±2.3	-46.2 ±1.3	73.0 ± 3.9

Data expressed as mean ± SEM, n=3.

Immunoliposome Characterization

The antibody-conjugated liposome (Ab-XYZ-I-73LnP) was successfully formulated after coupling EGFR to the surface of XYZ-I-73 LnP₅ (DPPC: LBR: DSPE-PEG 2000), as depicted in Figure 3. Characterization of the immunoliposomes showed a mean particle size of 143nm ± 2.3, PDI (0.37), and zeta potential -46.2±1.3mV. The entrapment efficiency of Ab-XYZ-I-73LnP was 73 ± 3.9% (Table 2).

Figure 3.

Scheme showing the conjugation of the antibody to the surface of the liposomal nanoparticle. EDC - (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide); Sulfo-NHS- N-hydroxysulfosuccinimide sodium salt

Antibody Conjugation to Liposomal Nanoparticles

Fluorescence Microscopy

Antibody conjugation to the surface of XYZ-I-73 LnP was examined using the Nikon Ti Eclipse fluorescent microscope. In this study, a FITC-conjugated secondary antibody was added to rhodamine-labeled XYZ-I-73 LnP. Images of the labeled secondary antibody and the liposomes were obtained. The images obtained showed a significant co-localization between rhodamine (XYZ-I-73 LnP), indicated as red, and fluorescein (secondary antibody), indicated in green, as shown in Figure 4. The formation of Ab-XYZ-I-73LnP was confirmed by co-localization, which demonstrated the conjugation of the antibody to the surface of the liposomal nanoparticles (XYZ-I-73 LnP).

Figure 4.

Fluorescence microscopy images confirming conjugation of EGFR monoclonal antibody to the surface of liposomes

Fluorescence microscopy was used to examine the binding of EGFR monoclonal antibody-conjugated liposomes to MiaPaCa-2 cells. The cells were labeled with DAPI (blue), the liposomes with rhodamine (red), and the secondary antibody with FITC (green). In the top row, liposomes conjugated with the antibody show surface binding. In contrast, the bottom row shows liposomes without EGFR antibody conjugation, in which no binding of the FITC-labeled secondary antibody was detected.

Fourier Transform Infrared Analysis (FTIR)

EGFR-antibody conjugation to XYZ-I-73 liposomal nanoparticles was subsequently investigated by using FTIR spectroscopy. The spectrum of XYZ-I-73 showed prominent peaks at 3314 cm⁻¹ (-C(=O)-NR') and 1005 cm⁻¹ (C-O-C), shown in Figure 5A. Characteristic Labrasol peaks also occurred at 3442 cm⁻¹ and 1736 cm⁻¹ (Figure 5B). As shown in the FTIR spectrum, XYZ-I-73LnP (DPPC: LBR: DSPE-PEG 2000) exhibited prominent peaks at 3279 cm⁻¹ and 1623 cm⁻¹ (Figure 5C). The Ab-XYZ-I-73LnP (XYZI-I-73LnP + EGFR) showed prominent peaks at 3278 cm⁻¹ and 1644 cm⁻¹ (Figure 5D). These observations may be linked to the –OH stretching and –NH2 stretching vibrations present in the formulation. The distinct sharp peaks characteristic of XYZ-I-73 at 1005 cm^-1, 2817 cm^-1, and 2930 cm^-1 were not significantly visible in the spectra of XYZ-I-73LnP and Ab-XYZ-I-73LnP containing Labrasol. Instead, these formulations exhibited a broad band between 2000 cm⁻¹ and 2500 cm⁻¹ with no distinct sharp peaks, suggesting an interaction between XYZ-I-73 and the lipid-Labrasol matrix (LnP). When comparing the XYZ-I-73LnP and Ab-XYZ-I-73LnP spectra, the O–H stretching region (3000–3500 cm^-1) appeared broader with a deeper trough in the antibody-conjugated formulation, accompanied by a shift in the carbonyl peak from 1623 cm^-1 to 1644 cm^-1. These spectral changes indicate successful binding of the antibody to the liposomal nanoparticle.

Figure 5.

FTIR spectra of (A) XYZ-I-73, (B) Labrasol, (C) XYZ-I-73LnP, and (D) Ab-XYZ-I-73LnP

In-Vitro Cell Viability Studies of Ab-XYZ-I-73LnP Against MiaPaCa-2 and PANC-1 PCa Cells

Ab-XY-Z-I-73LnP showed good cytotoxicity against MiaPaCa-2 and PANC-1 2D cultures, as depicted in Figure 6A–C. The IC₅₀ value of Ab-XY-Z-I-73LnP in MiaPaCa-2 and PANC-1 was 2.5±0.9µM and 2.9±1.1µM, respectively, as shown in Table 3. On the other hand, 5-FU had IC₅₀ values of 13.2 ± 1.1 µM and 20.4 ± 1.2 µM in MiaPaCa-2 and PANC-1, respectively. Our findings show that Ab-XY-Z-I-73LnP had about a 5-fold increase in cytotoxicity over the standard drug (5-FU) in MiaPaCa-2 cells and over a 6-fold higher cytotoxicity in PANC-1 cells over 5-FU.

Figure 6.

Cytotoxicity of Ab-XYZ-I-73 LnP against MiaPaCa-2 and PANC-1 cells (2D & 3D)

Table 3.

Comparison of IC₅₀ (µM) of 5-FU, XYZ-I-73LnP and Ab-XYZ-I-73LnP

Compound	MiaPaCa-2	PANC-1	MiaPaCa-2	PANC-1
	2D		3D
5-FU	13.2 ± 1.1	20.4 ± 1.2	26.7 ± 1.1	37.1 ± 0.9
XYZ-1-73 LnP	3.1 ± 0.4	3.3 ± 0.5	12.6 ± 0.8	22.6 ± 1.3
Ab-XYZ-I-73LnP	2.5 ± 0.9	2.9 ± 1.1	8.1 ± 1.1	12.9 ± 0.6

Data expressed as mean ± SEM, n=3.

A 3D cytotoxicity assay was also conducted to determine the cytotoxicity of XYZ-I-73 LnP in a model that provides a more in vivo-like environment and better predicts drug response. A similar cytotoxicity trend was observed with Ab-XYZ-I-73LnP in 3D spheroids of MiaPaCa-2 and PANC-1 cells. Ab-XYZ-I-73 LnP showed improved cytotoxicity over XYZ-I-73LnP and the unmodified drug (5-FU) in Figure 6D–F. Ab-XYZ-I-73 LnP exhibited a 3-fold higher cytotoxicity over 5-FU (8.1±1.1µM vs 26.7±1.1 µM) in MiaPaCa-2 cells. In PANC-1 cells, the half-minimal inhibitory concentration of Ab-XYZ-I-73 LnP (12.9 ±0.6 µM) was significantly lower than that of XYZ-I-73LnP (22.6 ±1.3µM) and 5-FU (37.1±0.9µM).

2D Cytotoxicity of Ab-XYZ-1-73LnP, XYZ-I-73LnP, and 5-FU on (a) MiaPaCa-2 cells (b) PANC-1 cells (c) Comparison of Ab-XYZ-I-73LnP cytotoxicity against MiaPaCa-2 and PANC-1 cells, 3D Cytotoxicity of Ab-XYZ-1-73LnP, XYZ-I-73LnP and 5-FU on (d) MiaPaCa-2 spheroids (e) PANC-1 spheroids (f) Comparison of Ab-XYZ-I-73LnP cytotoxicity against MiaPaCa-2 and PANC-1 spheroids. Data represent ± SEM, n = 3 (***p = 0.001; **p = 0.01; *p = 0.05 ns = not significant).

3D Spheroids Apoptosis Study (MiaPaCa-2 & PANC-1)

The apoptotic effects of Ab-XYZ-I-73 LnP were investigated in 3D spheroids, which better mimic the tumor microenvironment than 2D models. As shown in Figure 7, the rate of apoptosis increased with increasing drug concentration. After obtaining the mean fluorescent intensity ratios, the extent of apoptosis was determined. Ab-XYZ-I-73LnP showed significant spheroid destruction, as indicated by merged AO and EB images, which represent live and dead cells, respectively. 48 h AO/EB staining of MiaPaCa-2 cells showed a significant number of dead cells for Ab-XYZ-I-73 LnP compared to the XYZ-I-73LnP and 5-FU (Figure 7A–D).

Figure 7.

MiaPaCa-2 &PANC-1 spheroid culture showing apoptosis

A similar trend in Ab-XYZ-I-73 LnP-induced apoptosis was observed in PANC-1 spheroids, as shown in Figure 7E and F. A concentration-dependent cell death was observed over the 48-hour period in Ab-XYZ-I-73 LnP. The relative mean cell viability was significantly lower in Ab-XYZ-I-73 LnP at 12, 25, and 50µM concentrations (0.65, 0.38, and 0.22, respectively) in comparison to XYZ-I-73LnP (0.76, 0.57, and 0.39) and 5-FU (0.90, 0.72, and 0.59).

Spheroids apoptosis on MiaPaCa-2 and PANC-1 spheroids treated with (a) 5-FU, (b) XYZ-I-73LnP, (c)Ab-XYZ-1-73LnP, and (d) Relative mean cell viability of MiaPaCa-2 spheroids after treatment with Ab-XYZ-1-73LnP, XYZ-I-73LnP, and 5-FU, Spheroids apoptosis on PANC-1 spheroids treated with (e) 5-FU, (f) XYZ-I-73LnP, (g)Ab-XYZ-1-73LnP, and (h) Relative mean cell viability of PANC-1 spheroids after treatment with Ab-XYZ-1-73LnP, XYZ-I-73LnP, and 5-FU. The results are presented as the mean ± SEM (n = 3). Statistical significance (* p < 0.05, ** p < 0.01, and *** p < 0.001) was determined with two-way ANOVA with Tukey’s Multiple Comparison Test.

Animal Studies

Pharmacokinetics

To confirm that chemically modifying 5-FU to XYZ-I-73 and subsequent entrapment into a targeted nano-delivery system improved systemic stability, bioavailability, and reduced clearance, a pharmacokinetics study was conducted to describe the kinetics of XYZ-I-73 in vivo. To compare the pharmacokinetics of 5-FU to Ab-XYZ-I-73 LnP, 12.5mg/kg of 5-FU was administered intraperitoneally, and 37mg/kg (equivalent dose of 5-FU) was injected into the Ab-XYZ-I-73LnP group. Ab-XYZ-I-73LnP showed a significant increase in bioavailability compared to 5-FU (0.32 ± 5.4µg/(L*hr) vs 0.15 ± 0.02, p-value < 0.01) as shown in Table 4. There was about a 3-fold increase in half-life for Ab-XYZ-I-73 LnP (1.62 ± 0.03 hr) compared to 5-FU (0.49 ± 0.01 hr), p-value < 0.001. Furthermore, clearance was significantly reduced in Ab-XYZ-I-73LnP-treated mice in comparison to 5-FU-treated mice (0.88 ± 0.17L/hr vs. 2.30 ± 0.29L/hr, p-value< 0.01). We determined that the reduction in clearance observed in mice treated with the entrapped, monoclonal antibody-conjugated treatment could have resulted from an increase in half-life, leading to a prolonged duration of action compared to the unmodified drug. We further observed that antibody conjugation to the liposome surface significantly improved stability, reduced degradation, prolonged half-life, and enhanced bioavailability compared to the unconjugated liposome.

Table 4.

Pharmacokinetics of Ab-XYZ-I-73 in CDX Mice

Parameter	Unit	5-FU	XYZ-I-73LnP	Ab-XY-I-73LnP	p-value
K10	1/hr	1.31 ± 0.19	0.57 ± 0.03	0.43 ± 0.04	0.001
t1/2	hr	0.49 ± 0.01	1.21 ± 0.01	1.62 ± 0.03	0.001
Vd	L	1.75 ± 0.24	1.89 ± 0.46	2.04 ± 0.35	ns
CL	L/hr	2.30 ± 0.29	1.10 ±0.15	0.88 ± 0.17	0.001
AUC (0-t)	µg/(L*hr)	0.15 ± 0.02	0.24 ± 0.01	0.32 ± 0.04	0.01
MRT	hr	0.76 ± 0.16	1.74 ± 0.25	2.33 ± 0.42	0.04

t_1/2, half-life.

K₁₀, elimination rate constant.

V_d, volume of distribution.

CL, Drug clearance is the volume of plasma completely cleared of a drug per unit of time.

AUC, area under the plasma concentration-time curve.

MRT, mean residence time.

Data expressed as mean ± S.D, ns = not significant.

Tumor Efficacy

The tumor volume of MiaPaCa-2 CDX subcutaneous tumors was measured throughout the 35-day treatment period to study the efficacy of Ab-XYZ-I-73LnP on pancreatic tumors. The Ab-XYZ-I-73LnP significantly suppressed tumor growth in the CDX mice compared to the mice treated with 5-FU, as shown in Figure 8A. After about 2 weeks of treatment, the mean tumor volume of the Ab-XYZ-I-73LnP-treated mice was significantly lower than that of 5-FU and the control (172 mm³ vs 386 mm³ vs 567 mm³, p-value < 0.001), Figure 8B. The change in tumor volume for the Ab-XYZ-I-73LnP was steady after day 15. The tumor volumes of the mice in the control and 5-FU groups, however, showed a significant increase over time. Acute toxicity was assessed in mice by measuring body weight changes throughout the study. As depicted in Figure 8C, there was no significant change in mouse weight (<10%), suggesting minimal toxicity of the immunoliposome. No unexpected adverse effects or treatment-related mortality were observed during the study period.

Figure 8.

Tumor efficacy of Ab-XYZ-I-73LnP in the MiaPaCa-2 tumor-bearing mice model

In vivo efficacy of Ab-XYZ-I-73LnP in the CDX mice and acute toxicity testing. (a) Tumor growth curves of 5-FU and Ab-XYZ-I-73LnP-treated mice bearing CDX tumor, (b) tumor-volume measurements of 5-FU and Ab-XYZ-I-73LnP-treated CDX mice, (c) body weight measurements during the treatment period. All data represent mean ± SD (n = 8/group). Level of significance between the control and treatment group (** p < 0.01, *** p < 0.001).

Discussion

A major drawback of current PCa chemotherapy is its low metabolic stability, leading to a short duration of action and reduced therapeutic efficacy. ¹³ This necessitates high doses of administration and results in severe adverse effects. ⁵⁶ Despite high doses, only a small fraction of the drug reaches the tumor site to exert its anticancer effects, thereby reducing patient outcomes in PCa. Nanodelivery systems enable enhanced drug accumulation in tumor cells, facilitate targeted delivery, and ultimately enhance efficacy.^57,58

XYZ-I-73(a novel 5-FU analog) has previously been shown to demonstrate enhanced cytotoxicity, inhibit cell migration, inhibit pro-apoptotic proteins, as well as enhanced metabolic stability over 5-FU in PCa cells. ³⁵ In the current study, treatment with XYZ-I-73 resulted in substantial decreases in EGFR, VEGF, and HER-2 protein expression in MiaPaCa-2 cells, as determined by Western blot analysis. The downregulation of these oncogenic proteins indicated that developing a targeted formulation could interfere with essential signaling pathways that drive tumor growth and angiogenesis. ⁵⁹ Previous research has documented similar results when EGFR-targeted liposomes were used on various cancer cell lines. Blocking of EGFR signaling pathways results in higher cytotoxicity and diminished tumor growth and spread. ⁵³ This demonstrates the potential of EGFR-targeted immunoliposomes to enhance the effectiveness of chemotherapeutic agents in treating pancreatic cancer. The overexpression of EGFR in pancreatic tumors enables immunoliposomes to deliver drugs selectively, thereby increasing therapeutic effectiveness and reducing systemic toxicity.

Building on the findings on the anticancer effect of XYZ-I-73 and the strong evidence for the use of liposomes in cancer therapy, we formulated XYZ-I-73 into an immunoliposome (Ab-XYZ-I-73LnP) targeting EGFR, which is highly expressed in pancreatic cells and tumors. Successful conjugation of the EGFR-antibody to the liposome (XYZ-I-73LnP) was subsequently confirmed by particle size analysis, FTIR, and fluorescence microscopy. Results from the in vitro cytotoxicity assay demonstrate that Ab-XYZ-I-73LnP substantially decreased cell viability across the 2D and 3D spheroid cultures of MiaPaCa-2 and PANC-1 PCa cells in comparison to 5-FU.

The liposomal formulation without the antibody (XYZ-I-73LnP) also showed lower IC₅₀ values than 5-FU; however, the most significant cytotoxic effect was observed with the immunoliposome formulation, with a 3-fold improvement over 5-FU and a 2-fold improvement over XYZI-73LnP in the 3D spheroid model. These results suggest that the targeted formulation had enhanced anticancer activity compared to the unmodified drug. Previous studies have shown that EGFR-targeted liposomes enhance the antitumor activity of encapsulated drugs in EGFR-overexpressing PCa cells, as demonstrated by Jeong et al. ⁶⁰ Another study highlighted the enhanced cellular uptake, toxicity, and targeting of EGFR-thermosensitive liposomes in lung cancer and epidermoid carcinoma cell lines and tumor models. ⁶¹ EGFR conjugation to liposomes triggers receptor-mediated endocytosis, increasing liposome internalization into cancer cells. ⁶² Following endocytosis, endosomes and lysosomes act on the liposome, destabilizing it and releasing XYZ-I-73. This leads to enhanced cellular uptake, higher bioavailability, contributing to the enhanced cytotoxic effect of the immunoliposome compared to free 5-FU.

Furthermore, the apoptotic analysis revealed that cell death increased in a concentration-dependent manner following treatment with Ab-XYZ-I-73LnP. Ab-XYZ-I-73LnP treatment produced a more substantial apoptotic effect in comparison to 5-FU. The enhanced apoptotic response likely results from EGFR-mediated endocytosis, which promotes targeted delivery and intracellular uptake of the immunoliposomes.^63-65 The mechanism likely increases the intracellular concentration of the active cytotoxic compound, thereby enhancing the therapeutic effect.

Pharmacokinetic results indicated that Ab-XYZ-I-73LnP had a prolonged duration of action compared to 5-FU due to slower clearance rates. Chemical modification of the XYZ-I-73 liposomal carrier by PEGylation may have reduced opsonization and RES clearance, allowing the liposomes to circulate far longer than free 5-FU, which is rapidly eliminated due to its small size and rapid metabolism. The improved half-life and reduced clearance increase systemic exposure (AUC) and enhance tumor accumulation through both prolonged circulation, controlled drug release, and improved targeting. ⁶⁶

Targeted delivery systems improve drug localization at tumor sites and reduce exposure to non-target tissues. ⁶⁷ In a similar study, Daram et al demonstrated that EGFR-targeting immunoliposomes showed potential to enhance antitumor efficacy and improve drug release in non-small cell lung cancer. ⁶⁸ Tumor volumes in the MiaPaCa-2 CDX mice treated with Ab-XYZ-I-73LnP were significantly lower than those of XYZ-I-73LnP and 5-FU. Ab-XYZ-I-73LnP showed enhanced antitumor efficacy compared with XYZ-I-73LnP and 5-FU, owing to EGFR-targeting that improved and selectively enhanced cellular uptake. Receptor-mediated endocytosis improved intracellular delivery of the immunoliposome, resulting in more potent tumor growth inhibition than passive uptake. ⁶² The observed enhanced tumor accumulation with Ab-XYZ-I-73LnP backs this conclusion. Additionally, minimal body weight changes in the mice indicate low acute toxicity, suggesting a favorable safety profile of the immunoliposome formulation.

The current research demonstrates that EGFR-directed immunoliposomes containing the novel 5- FU analog XYZ-I-73 (Ab-XYZ-I-73LnP) showed enhanced anticancer effect over the standard 5-fluorouracil (5-FU) in 2D and 3D pancreatic cancer models.

The study results demonstrate increased cytotoxic effects, enhanced apoptotic induction, improved pharmacokinetic properties, and greater tumor accumulation. The combined findings demonstrate that EGFR-specific nanocarriers may offer a solution to the limitations of traditional chemotherapy. Future research should assess whether combining this targeted therapy with immunotherapy or radiotherapy yields synergistic effects. Using combined treatment approaches can provide stronger and longer-lasting responses, resulting in improved patient outcomes for patients with PCa. Also, the in vivo metabolic stability and potential mechanisms of resistance can be investigated.

Limitations

Long-term toxicity of Ab-XYZ-I-73LnP and histological analysis to assess potential changes in tumor morphology following treatment were not evaluated. Additionally, the release kinetics of the immunoliposome formulation was not determined, limiting insight into its drug-release behavior. These gaps should be addressed in future studies.

Conclusion

This study highlights the therapeutic potential of EGFR-targeted immunoliposomes encapsulating the novel 5-FU analog, XYZ-I-73 (Ab-XYZ-I-73LnP), for the treatment of pancreatic cancer. Compared with 5-FU, the targeted formulation demonstrated enhanced cytotoxicity, greater apoptosis induction, improved pharmacokinetics, and increased tumor accumulation. These outcomes support the role of receptor-mediated drug delivery in addressing critical limitations of conventional chemotherapy, including low tumor selectivity and systemic toxicity.

ORCID iDs

Esther Frimpong https://orcid.org/0000-0001-6908-823X

Edward Agyare https://orcid.org/0000-0002-6905-5136

Ethical Considerations

The Florida A&M University Institutional Animal Care and Use Committee approved the experimental procedures used in this study (approval no. 023-07) on July 31, 2023.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.*