Exatecan-Loaded HSA Nanoparticles for Sustained Release and Enhanced Cancer Treatment

Abstract

Exatecan, a potent topoisomerase 1 inhibitor and a potent anticancer agent, is derived from naturally occurring camptothecin. Its clinical development has been halted due to dose-limiting toxicities and a shorter plasma half-life in humans. To overcome these challenges, we synthesized a novel human serum albumin nanoparticle (Exa-HSA-NPs) for selective cancer therapy. The Exa-HSA-NPs exhibited a uniform spherical morphology with an average hydrodynamic diameter of 147 nm, zeta potential of −24 mV, and a very low polydispersity index (PDI < 0.1). STEM-EDS mapping confirmed homogeneous distribution of exatecan within the albumin medium. In vitro release kinetics demonstrated pH-responsive, sustained drug release for up to 9 days, with faster release in acidic (tumor-like) conditions. Molecular docking of exatecan with HSA revealed the formation of a stable complex stabilized by hydrophobic interactions and hydrogen bonding. Exa-HSA-NPs displayed potent cytotoxicity in oral cancer (AW13516) and lung cancer (A549) cells, with significantly reduced toxicity in non-cancerous HEK293T cells. Cellular uptake analysis revealed preferential NPs internalization in cancer cells (70% in AW13516, 63% in A549) compared with normal cells (27%), consistent with gp60 and SPARC-mediated albumin uptake mechanisms. Apoptosis assays confirmed selective induction of cancer cell death with minimal impact on normal cells. The Exa-HSA-NPs exhibited excellent biodegradability and hemocompatibility, indicating favorable safety and potential for clinical translation. Overall, Exa-HSA-NPs offer a promising strategy for the sustained, selective tumor delivery of exatecan, with an improved therapeutic index and reduced systemic toxicity.

1. Introduction

Exatecan is a small molecule, a more potent analogue of camptothecin, a natural alkaloid extracted from the bark of Camptotheca acuminata. Exatecan has reached phase 3 of clinical trials. However, further advancement was halted since it failed to exhibit therapeutic benefit when combined with gemcitabine and also because of its dose-limiting side effects. Among the limiting factors of available small-molecule TOPO1 inhibitors (TOPO1i) is their relatively shorter half-lives. Specifically, exatecan has a half-life of approximately 7-8 h in humans. It has been established that the effective repression of TOPO1 requires the uninterrupted supply of the drug during the S phase of the cell cycle, and upon withdrawal of the drug, the enzyme TOPO1 is reverted back to its functional form. , As such, the briefer half-life of existing TOPO1i is inadequate for achieving therapeutic value, and there is a dire need for a better prolonged-release form of the current TOPO1i. In recent years, endogenous proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) have attracted significant attention as drug delivery platforms owing to their biocompatibility, long circulation half-life, and tumor-targeting ability, and the EPR effect. − HSA is a well-known intrinsic carrier that has a long circulation time in the blood. , HSA has displayed impressive potential as a carrier for anti-cancer agents. − HSA extends the circulation half-life of drugs that would otherwise be hastily cleared. HSA also enhances drug accumulation within tumor tissues. , HSA can protect the drug and deliver it to the tumor microenvironment because it is also a nutrient for rapidly growing, dividing tumor cells. , The present work was designed with two goals: (i) sustained release of exatecan to ensure continuous suppression of TOPO1, leading to a prolonged therapeutic effect; (ii) selective delivery of exatecan to tumor tissues for better efficacy and safety. To achieve these dual goals, we encapsulated exatecan within HSA nanoparticles (NPs) and successfully fabricated exatecan HSA NPs (Exa-HSA NPs). In this Exa-HSA-NP, HSA serves a dual purpose: it ensures the delivery of exatecan via the enhanced permeability and retention (EPR) mechanism and promotes preferential tumor accumulation. Multiple approaches have been attempted to develop longer-lived, tumor-specific TOPO1i’s. One approach is to covalently link TOPO1i as a payload in antibody-drug conjugates (ADCs). There are three ADCs containing TOPO1 inhibitors: 1) the sacituzumab deruxtecan (Trodelvy), specific for Trop2 delivering SN-38; 2) trastuzumab deruxtecan (Enhertu) targeting HER2; and 3) datopotamab deruxtecan targeting Trop2, carrying a similar analogue of exatecan - shows remarkable results in clinical trials and has been approved for metastatic HR-positive, HER2-negative breast cancer in 2025, and metastatic EGFR-mutated non-small cell lung cancer (NSCLC). However, there are a significant number of cancers that do not possess Trop2 or HER2 surface antigens identified by these ADCs and are not efficient targets for these ADCs. Likewise, these ADCs do not selectively target tumor subpopulations defined by defects in the DNA damage response to TOPO1 inhibitors, including BRCA or ATM-deficient tumors. Thus, there is a strong need to develop long-acting, tumor-specific drug delivery agents, such as Exa-HSA-NPs, that exert their antitumor effects independent of the presence of a specific tumor antigen. An alternative approach has been attempted to develop long-lived TOPO1i as antigen-independent prodrugs, in which TOPO1i is covalently linked to a synthetic carboxymethyl-dextran polyalcohol (a tumor-localizing macromolecular carrier) via a biodegradable linker. DE-310 is a conjugate in which exatecan is covalently attached to a 340 kDa carboxymethyl-dextran polyalcohol carrier through a cathepsin cleavable peptide linker. This DE-310 has a long systemic half-life and passively accumulates in xenografts, and cathepsin cleaves the linker to release exatecan. DE-310 has shown significant drug accumulation in tumor xenografts and suppresses tumor growth for a long period, but drug accumulation in human tumors was not observed. Hence, efforts with exatecan were diverted toward its use as a payload in ADCs. Subsequently, there is a great need for an innovative approach for the sustained release of exatecan as well as selective delivery to tumor tissues to achieve desirable safety and efficacy. HSA, in addition to being an excellent drug-protective agent with a longer half-life, is more readily taken up by rapidly growing, nutrient-starved cancer cells. It exhibits an excellent affinity for glycoprotein 60 (GP60) and secreted protein acidic and rich in cysteine (SPARC), both of which are overexpressed in many tumors. − Facilitating drug delivery via gp60 and SPARC-mediated endocytosis is one of the main mechanisms that ease drug penetration into cancer cells. , HSA has already demonstrated its ability to serve as a carrier for paclitaxel under the brand name Abraxane. Abraxane, an albumin-bound nanoparticle of paclitaxel, has been approved by the FDA for metastatic breast cancer, non-small cell lung cancer, , and late-stage metastatic pancreatic cancer and has superior efficacy or better response rate (21.5%) as compared to Taxol (11.1%). However, there remains a significant need for improved options with superior safety and efficacy for treating various types of cancer. Here, we present a novel Exa-HSA-NP that utilizes HSA NPs as a carrier to specifically deliver exatecan to tumor sites, thereby potentially reducing systemic toxicity while maintaining the therapeutic efficacy.

2. Materials and Methods

2.1. Materials

HSA (lyophilized powder), glutaraldehyde, ethanol, and phosphate-buffered saline (PBS) were of analytical grade. Exatecan with 98% purity was obtained from Novus ChemStrength, Ahmedabad, India. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent and Annexin V-FITC Apoptosis Detection Kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein isothiocyanate, isomer 1, 95% (FITC) was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, gentamicin solution, formaldehyde, Bradford reagent, doxorubicin hydrochloride (DOX), and 12-14 kDa molecular weight cut-off (MWCO) dialysis membrane were procured from Hi-Media Laboratories Pvt. Ltd. (Mumbai, Maharashtra, India). Culture flasks for cells (T-25 and T-75) and culture plates (6- and 96-well) were acquired from Tarsons Products Pvt. Ltd. (Kolkata, India). The AW13516 cell line was procured from the ACTREC, Tata Memorial Center, Navi Mumbai, India. The A549 and HEK293 cell lines were obtained from the National Center for Cell Science (NCCS, Pune, India).

2.2. Instrumentation

Dynamic light scattering (DLS), zeta potential, and polydispersity index (PDI) were analyzed using NanoPlus with NanoPlus Auto Titrator. Morphology, particle dispersion, and size of Exa-HSA-NPs were determined using a Field Emission Scanning Electron Microscope (FE-SEM-Quanta FEG 250). High-resolution TEM images were acquired using TALOS F200S G2. For the addition of the ethanolic exatecan solution during the synthesis of Exa-HSA-NPs, a New Era Instrument Syringe pump was used. To find out the percentage of exatecan entrapped, the maximum absorbance wavelength (λ_max) of exatecan in tris buffer was determined by scanning exatecan solution from 200 to 700 nm using a Thermo Scientific Orion AquaMate UV-vis spectrophotometer. Turbidimetry measurements for the biodegradation assay and absorbance for the hemolysis assay were performed using the Varioskan LUX Multimode Microplate Reader. The absorbance reading for the MTT assay was taken in a Thermo Scientific Multiskan FC 357 Microplate Photometer at 540 nm.

2.3. Synthesis of Exa-HSA-NPs

Exa-HSA-NPs were prepared by the desolvation method reported by F. Galisteo-González and J.A. Molina, with slight modifications. In brief, 80 mg HSA was solubilized in 5 mL of 5 mM NaCl buffer under stirring conditions at room temperature. 10 mg Exatecan was dissolved in a minimal amount of DMSO, and ethanol was used as a primary solvent for the ethanolic exatecan solution. The ethanolic exatecan solution was then added to the HSA solution under stirring conditions at a rate of 6 mL/min. Upon turbidity, 25 μL of 8% glutaraldehyde solution was added to the stirred reaction mixture. It was stirred for 24 h at room temperature, transferred to an Oak Ridge centrifuge tube, and centrifuged at 12,000 rpm for 30 min at 20 °C. The supernatant was set aside, and the pellet was resuspended in Milli-Q water, washed, centrifuged, sonicated between washes, and finally resuspended in Milli-Q water and stored at 4°C until further use.

2.4. Percentage Yield of Exa-HSA-NPs

The percentage yield of Exa-HSA-NPs was estimated using the Bradford assay. Different concentrations (20, 16, 12, 8, and 4 μg/200 μL) of the HSA standard were prepared in accordance with the manufacturer’s instructions, and the corresponding absorbance for each concentration was recorded at 595 nm. A standard curve was plotted using the obtained values. The amount of protein present in the supernatant was estimated as free HSA that wasn’t converted into nanoparticles, and the percentage yield was calculated using the following formula:

$$\mathrm{Yield}\mathrm{of}\mathrm{NPs}=\frac{[HSA]totaltakenforthereaction-[HSA]free}{[HSA]totaltakenforthereaction}\times 100$$ 1

2.5. SEM

The sample was diluted 50-fold in Milli-Q water, drop-cast onto a sterile coverslip, and air-dried. Following complete dehydration, the coverslip was sputter-coated with gold and examined by FE-SEM at magnifications of 30,000× and 60,000×.

2.6. TEM

For TEM, the diluted sample was applied by drop-casting onto a carbon-coated copper grid and air-dried, and 1% phosphotungstic acid was used as a negative stain. The sample was dried and washed and then viewed. Scanning transmission electron microscopy with energy dispersive X-ray spectroscopy (STEM-EDS) was used to evaluate the encapsulation of exatecan in Exa-HSA-NPs by elemental mapping of fluorine, which is unique to exatecan, and sulfur, which is unique to protein.

2.7. DLS and Zeta Potential

To determine the overall size, surface charge, and uniformity of Exa-HSA-NPs, DLS and ζ-potential analyses were carried out, respectively. For this, 5 mL of the diluted sample was sonicated and then analyzed.

2.8. Determination of Entrapment/Encapsulation Efficiency

To quantify the amount of exatecan entrapped within the NPs, enzymatic digestion was performed using proteinase K in 5 mM tris buffer. The absorbance maximum (λ_max) of exatecan was determined, and a calibration curve was plotted utilizing standard solutions of known concentrations. The concentration of the unknown sample was subsequently calculated from the calibration curve. For this, the maximum absorbance wavelength (λ_max) of exatecan was determined by scanning the exatecan solution from 200 to 700 nm. Then, a standard calibration curve was generated by preparing a series of exatecan dilutions in tris buffer at concentrations of 0.0001, 0.001, 0.01, and 0.1 mg/mL. The absorbance was recorded for these concentrations, and the graph was plotted. For sample analysis, 100 μL of the sample (80 mg/9 mL or 9 mg/mL) was digested with 1 mL of proteinase K in 5 mM tris buffer prepared in double-distilled Milli-Q water. After 1 h of digestion, absorbance was recorded, and the concentration of exatecan was estimated using the standard curve.

2.9. Drug Release Kinetics

The in vitro drug release profile of exatecan from NPs formulation was assessed using a dialysis bag (12-14 kDa MWCO) method. For this, 0.5 mL of the NP solution was diluted to 1.5 mL with 1× PBS. The study used two pH levels: physiological (pH 7.4) and tumor microenvironment (pH 5.5). At predetermined time intervals, aliquots were collected from the release medium, and readings were noted. The corresponding concentration was then derived from a known calibration curve by dissolving different concentrations (0.00066, 0.0066, 0.066, 0.66 mg/mL) of exatecan in 1×PBS buffer. After taking the absorbance, the sample aliquot was returned to the 50 mL flask (main stock) to prevent any change in the overall concentration of the stock solution.

2.10. Molecular Docking

The crystal structure of HSA (PDB ID: 1AO6) was obtained from the RCSB Protein Data Bank. PyMOL version 3.1.0 was utilized for removing co-crystallized water molecules and heteroatoms. The three-dimensional structure of exatecan was obtained from PubChem (CID 151115). Docking was achieved using PyRx version 0.8 with the AutoDock Vina scoring function. The pose with the lowest binding energy was selected as a representative docking conformation for further analysis. The docked complex was visualized by PyMoL, and non-covalent ligand interactions were analyzed using Protein Ligand Interaction Profiler (PLIP).

2.11. Biodegradation Test

The enzyme-catalyzed degradation of Exa-HSA-NPs was performed as per the protocol published by Langer et al. 1000 μg/mL of Exa-HSA-NPs was suspended in 1× PBS buffer at pH 7.4, and then it was blended with trypsin at a final concentration of 50 μg/mL. The degradation of Exa-HSA-NPs was measured using the turbidity results of the suspension at 565 nm at different time intervals (1, 2, 4, 6, 12, and 24 h). The exact amount of Exa-HSA-NPs dispersed in 1× PBS was used as a negative control. The concentration of Exa-HSA-NPs at various time points was determined by plotting a calibration curve for concentrations of 200, 400, 500, 800, and 1,000 μg/mL.

2.12. Hemolysis Assay

The hemolytic behavior of Exa-HSA-NPs toward erythrocytes was evaluated using a standard hemolysis assay. The study was approved by the Human Ethical Committee (HEC) (Ref No. HEC/BPGC/2025/013) of Birla Institute of Technology and Science, Pilani, K.K. Birla Goa Campus. The approval was issued on 03/06/2025. The experiment was conducted in accordance with the guidelines issued by the HEC. Informed consent was obtained from all participants. 5 mL of human blood from three healthy volunteers was collected in an anticoagulant tube and centrifuged at 5000 rpm for 5 min. The plasma was separated, and the pellet was washed twice with 0.01 M PBS to remove any residual plasma content. It was diluted to get a 2% suspension. Then, different concentrations (0.05, 0.5, and 1 mg/mL) of Exa-HSA-NPs or HSA NPs were incubated with a 2% RBC suspension for 4 h in a 37 °C CO₂ incubator. For the positive control, H₂O, and the negative control, 1× PBS, were utilized. After incubation with respective controls, the blend was centrifuged for at 3k rpm to pellet down the RBCs, and the optical density (OD) of the supernatant was measured at 540 nm. Hemolysis percentage was then calculated using the following formula, where A represents the UV absorption at 540 nm.

$$\mathrm{Hemolysis}(\%)=\frac{A_{Sample}-A_{Negative}}{A_{Positive}-A_{Negative}}\times 100$$ 2

2.13. Cell Viability Assay

Cell viability was determined using a patient-derived oral cancer cell line (AW13516), non-small cell lung cancer cell line (A549), and normal human embryonic kidney cells (HeK293T). − The assay was performed following an established protocol. All cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM) complemented with 10% (v/v) Fetal Bovine serum, 1% penicillin, streptomycin, and gentamicin solution. 10,000 cells were seeded per well in a 96-well plate. After 24 h, AW13516 was treated with different concentrations of Exa-HSA-NPs (1, 2, 6, 60, and 120 ng/mL) and exatecan (0, 0.5, 1, 3, 10, 30, and 50 nM). Similarly, A549 cells treated with different concentrations of Exa-HSA-NPs (200, 600, 1000, 1400, 1800, 2200 ng/mL) and or exatecan (0, 6.25, 12.5, 25, 50, 100, and 200 nM). HEK293T was treated with Exa-HSA-NPs (500, 1000, 1500, 2500, 3500, 4500, and 5500 ng/mL) and exatecan (0, 40, 80, 120, 160, and 200 nM). DOX was used as a positive control chemotherapeutic agent. Cells were treated with DOX at concentrations ranging from 0.01-25 μM for 48 h under identical conditions as described for Exa-HSA-NPs. After 48 h, the drug-containing media was removed, cells were washed with 1× PBS, and incubated with 0.5 mg/mL of MTT reagent for 4 h, and the formed formazan crystals were dissolved in 100% DMSO. Statistical analysis and IC₅₀ values were calculated using GraphPad Prism 10. One-way ANOVA, followed by Dunnett’s post hoc test, was used for multiple comparisons against the control group for MTT analysis. Nonlinear regression analysis was used to calculate the IC₅₀. The P values are denoted by asterisks as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

2.14. Conjugation of FITC with Exa-HSA-NPs

HSA was conjugated to FITC following Hungerford et al. (2007). 80 mg HSA was dissolved at 1 mg/mL in 0.1 M carbonate buffer (pH 9). FITC (17 mg) was dissolved in 1.7 mL DMSO (10 mg/mL) and added dropwise to the HSA solution with stirring. The mixture was incubated with gentle stirring in the dark for 4 h, then dialyzed against 0.01 M PBS (pH 7.4) using a 12-14 kDa MWCO membrane at 8 °C. The purified FITC-HSA conjugate was then utilized to obtain FITC-HSA-Exa-NPs by the desolvation method described in Section and subsequently characterized. To confirm successful conjugation, FTIR and UV absorbance were performed. FTIR spectra was recorded for FITC, FITC-HSA, and HSA. Pure FITC gave a pronounced peak at 2000 cm^–1, which corresponds to the vibrational stretch of the NCS group. The disappearance of this peak of FITC in the FITC-HSA indicated covalent bonding of FITC with the albumin NPs (Figure S1). Additionally, the appearance of the characteristic absorbance peak at 495 nm in the UV-visible spectrum further affirmed successful FITC conjugation (Figure S2).

2.15. Cellular Uptake Assay

FITC-Exa-HSA-NPs were utilized for the cellular uptake assay. 3 × 10⁵ cells were seeded per well in a culture plate. AW13516 was then treated with 8 ng/mL of FITC-Exa-HSA-NPs for 12 and 24 h. A549 and HEK293T cells were exposed to 1717 ng/mL of FITC-Exa-HSA-NPs for 12 and 24 h. After 12 or 24 h, cells were washed twice with 1× PBS, stained with Hoechst 33258, and analyzed by flow cytometry. The data were then processed using FlowJo software (version 10.10.0). A one-way ANOVA followed by Dunnett’s post hoc test was done for multiple comparisons against the control group. Significance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

2.16. Cell Death Assay

AW13516, A549, and HEK293T cells were plated in 6-well culture plates and incubated overnight. AW13516 was then treated with 8 ng/mL Exa-HSA-NPs or HSA NPs for 24 h. A549 and HEK293T were treated with 1717 ng/mL Exa-HSA-NP or HSA-NPs for 24 h. For the positive control, 70% ethanol was used. The cells were then trypsinized and collected. Cells were then stained with Annexin V-FITC and PI and analyzed for the percentage of apoptosis through a flow cytometer. The data were processed using FlowJo software (version 10.10.0). Two-way ANOVA was done to evaluate statistical significance, with p-values represented by asterisks indicating the levels of significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3. Results

3.1. Characterization of Exa-HSA-Nanoparticle (Exa-HSA-NPs)

DLS, Zeta potential analysis, SEM, and STEM were performed for characterization. Zeta potential analysis indicated an overall net surface charge of −24 ± 1 mV (Figure d), indicating good colloidal stability of Exa-HSA-NPs. DLS analysis indicated a mean hydrodynamic size of 147 ± 44 nm (Figure c), which falls within the optimal range of 100-200 nm, favoring tumor accumulation via the EPR effect. PDI of <0.1 indicated a low polydispersity and uniformity of particles. The surface was smooth without any porosity or roughness. The NPs displayed a smooth surface, uniform dispersion, and a spherical morphology, with well-dispersed particles and minimal agglomeration, as shown in Figure a and b, analyzed by SEM. STEM imaging revealed that the Exa-HSA-NPs exhibited a spherical morphology with proper dispersion, and the particles were in the size range of 100-200 nm (Figure a). EDS mapping of fluorine, unique to exatecan (Figure b), and sulfur (Figure c), which is present in native albumin in Exa-HSA-NPs, was carried out to validate the successful encapsulation of exatecan in Exa-HSA-NPs. The EDS mapping was to detect the presence of fluorine, which was unique to exatecan and not present in the native HSA protein. As depicted in Figure d, the elemental mapping clearly demonstrates the presence of fluorine signals (red) that are colocalized with HSA NPs, providing direct evidence of successful drug incorporation into the HSA-NPs. The uniform distribution of fluorine within the NPs indicates homogeneous drug distribution.

1.

Physicochemical characterization of Exa-HSA-NPs. (a) and (b) represent scanning electron microscope (SEM) images of Exa-HSA-NPs at 30,000× magnification and 60,000×, respectively, showing spherical morphology within a size range of 100-200 nm. (c) Dynamic Light Scattering (DLS) analysis of Exa-HSA-NPs shows an average size of 147 ± 44 nm and a PDI of 0.09, indicating low polydispersity. (d) Zeta potential of Exa-HSA-NPs measured at −24 mV, indicating good colloidal stability. Zeta potential and DLS size distributions were measured in triplicate for the same sample (blue, red, and black).

2.

STEM analysis. (a) STEM of Exa-HSA-NPs showing spherical structures with uniform density. (b) EDS elemental map showing sulfur (S). (c) EDS elemental map showing fluorine (F). (d) Merged image showing the elemental map of F and S of Exa-HSA-NPs.

3.2. Percentage Yield of Exa-HSA-NPs

The percentage yield of the NPs formed was estimated using the Bradford assay. A calibration curve with an R² value of 0.98 was prepared for the standards mentioned in the method section (Figure S3). The yield of Exa-HSA-NPs was estimated using the formula:

$$\mathrm{Yield}\mathrm{of}\mathrm{NPs}=\frac{[HSA]takenforthereaction-[HSA]free}{[HSA]total}\times 100=\frac{80mg-0.8mg}{80mg}\times 100=99\%$$

The yield was 99%.

3.3. Entrapment Efficiency and Drug Loading Efficiency

The λ_max of exatecan was obtained at 376 nm (Figure S4). The amount of exatecan present in the NPs was quantified using a calibration curve generated from a known concentration (as described in the method section), with a correlation coefficient of R² = 0.98 (Figure S5 and Table S2). The entrapment efficiency of exatecan in HSA NPs was 60 ± 0.09% (n = 3). For the synthesis, 10 mg of exatecan in an ethanolic solution was added to the reaction mixture. Based on the entrapment efficiency, the amount of exatecan entrapped in Exa-HSA-NPs is 60% of 10 mg, which is 6 mg. Total amount of Exa-HSA-NPs is 86 mg (80 mg HSA and 6 mg of exatecan).

$$\begin{array}{l}\mathrm{Weight}\%(\mathrm{drug}\mathrm{loading}\mathrm{efficiency})\mathrm{of}\mathrm{exatecan}\mathrm{present}\mathrm{in}\mathrm{Exa‐}\mathrm{HSA‐}\mathrm{NPs}=\frac{amountofexatecan}{totalamountof\mathrm{Exa‐}\mathrm{NPs}}\times 100\\ =\frac{6}{86}\times 100=6.98\%\end{array}$$ 3

Concentration of working Exa-HSA-NPs working stock = 9 mg/mL

Concentration of exatecan in working stock = 6.98% of 9 mg/mL = 0.63 mg/mL.

3.4. Drug Release Kinetics

Exatecan release from the NPs was monitored over 264 h (11 days). At each time interval, reading was noted, and the corresponding concentration was obtained from a known calibration curve (Figure S6 and Table S3). No exatecan release was observed in the initial 3 h at pH 7.4, whereas 24.9% release was observed at pH 5.5 after the 1^st hour (Figure ). At the 6^th hour, 4% and 27% of the drug were released at pH 7.4 and 5.5, respectively. Then, subsequent readings were taken at 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, and 264 h. Exa-HSA-NP exhibited gradual and prolonged drug release, with enhanced release at pH 5.5 compared withphysiological pH. Plateaus were reached at 168 h (7 days) and 216 h (9 days) at pH 7.4 and 5.5, respectively. The higher drug release under acidic conditions may offer a unique advantage to Exa-HSA-NPs for drug release in tumor tissues, which are known for their acidic environment. The slower release observed after 72 h at pH 5.5 may be due to interactions between exatecan and the albumin nanosystem, stabilization by glutaraldehyde-assisted cross-linking, and diffusion-restricted transport of exatecan within the NP system. This sustained release behavior can help ensure prolonged drug exposure within the tumor microenvironment. , This controlled drug release profile of Exa-HSA-NPs will help to continue suppression of TOPO1 without giving any opportunity for DNA religation. This sustained inhibition is crucial for improving the therapeutic efficacy while minimizing toxicity.

3.

Drug release kinetics graph. Drug release kinetics for exatecan at time intervals of 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 24, and 264 h. The red line represents pH at 5.5, and the black line pH 7.4. Exa-HSA-NPs shows sustained drug release. The formulation exhibited 56% cumulative drug release at pH 7.4 over 168 h (7 days) and 80% release at pH 5.5 over 216 h (9 days). The drug’s release kinetics favor less dosing frequency and lower toxicity. Results are expressed as mean ± SD (n = 3).

3.5. Molecular Docking Studies

Molecular docking was performed to investigate the binding interaction between exatecan and HSA and to identify the residues involved in stabilizing the HSA-exatecan complex. The crystal structure of HSA (PDB ID: 1AO6) was obtained from the Protein Data Bank (PDB), and the structure of exatecan was obtained from PubChem. It was converted to PDBQT format and docked using autodock Vina implemented in PyRx 0.8. The top-ranked binding pose exhibited a binding affinity of −10.4 kcal/mol, consistent with a thermodynamically favorable interaction. The binding site was found between the interface of subdomains IB and IIA of HSA. Interaction analysis via the Protein–Ligand Interaction Profiler (PLIP) identified five hydrophobic contacts (Pro110, Leu112, Arg145, Ala194, Arg197; 3.32–3.90 Å) and two hydrogen bondsone with the Pro110 backbone carbonyl (D-A distance 3.19 Å) and one with the Leu112 backbone amide (D-A distance 4.07 Å). The complete interaction dataset is provided in Table S4. These predicted non-covalent interactions, together with the covalent glutaraldehyde-mediated crosslinking of HSA lysine residues, are summarized in Figure .

4.

Schematic representation of Exa-HSA-NP formation. (a) Glutaraldehyde-mediated crosslinking of albumin via the ε-amino groups of lysine residues, resulting in imine (Schiff base) linkage. (b) Encapsulation of exatecan within the crosslinked albumin nanoparticle network. (c) Represents the predicted binding mode of exatecan within HSA (HSA, PDB: 1AO6). The docked ligand is shown as yellow sticks (fluorine substituent shown in its standard CPK color, pale cyan), with interacting residues shown as gray sticks and labeled by residue name and number. Hydrogen bonds are indicated by blue dashed lines; hydrophobic contacts are indicated by gray dashed lines. ChemSketch (ACD/Labs) was used for schematic illustration.

3.6. MTT Assay

The MTT assay was carried out to determine the antiproliferative activity of Exa-HSA-NPs and free exatecan in AW13516, A549, and HEK293T cell lines. The IC₅₀ value of AW13516 with exatecan was estimated to be 0.8 ± 0.17 nM at 48 h (Figure a), whereas the 48-hour IC₅₀ value of Exa-HSA-NPs in AW13516 oral cancer cell line corresponded to 8 ± 1.9 ng/mL (1.4 nM based on the amount of encapsulated exatecan in Exa-HSA-NPs (Figure a, b and Table ). For A549, the IC₅₀ obtained was 82 ± 3 nM for exatecan. Similarly, the IC₅₀ of Exa-HSA-NPs, in A549, was found to be 1716 ± 76 ng/mL (271 nM based on the amount of encapsulated exatecan in Exa-HSA-NPs) (Figure a, b, and Table ). To evaluate the comparative cytotoxicity and selectivity of exatecan and Exa-HSA-NPs in normal cells vs. cancer cell lines, MTT assay was performed in the noncancerous HEK293T cell line (Figure ). The IC₅₀ of free exatecan in normal HEK293T cell line was found to be 148 ± 17.5 nM, and for Exa-HSA-NPs, it was 5000 ng/mL ± 200 (801 nM with respect to total encapsulated exatecan (Table ). To validate assay responsiveness and provide a clinical chemotherapy benchmark, DOX was tested as a positive control across all three cell lines. DOX exhibited IC₅₀ values of 0.4 μM (HEK293T), 3 μM (A549), and 0.9 μM (AW13516) at 48 h (Figure S7). Blank HSA NPs across a concentration range of 500-8000 ng/mL maintained cell viability above 95% in all cell lines at 48 h, confirming their biocompatibility.

5.

Cell viability of exatecan vs. Exa-HSA-NPs in AW13516 cell line. (a) Cell viability data for AW13516 with exatecan, the x-axis represents different concentrations of exatecan from 0 to 50 nM and their corresponding cell viability on the y-axis. The IC₅₀ after 48 h of treatment was estimated to be 0.8 ± 0.17 nM, (b) represents the viability of Exa-HSA-NPs. The x-axis represents concentration of Exa-HSA-NPs from 0 to 120 ng/mL. The IC₅₀ was found to be 8 ± 1.9 ng/mL, corresponding to 6.9% encapsulation at a concentration of 1.4 nM. Results are expressed as mean ± SD from three independent experiments.

1. Showing IC₅₀ of Exa-HSA-NP.

Cell Line	Incubation Time (in h)	IC 50 in ng/mL for Exa-HSA-NPs	IC 50 for exatecan present in Exa-HSA-NPs (with respect to 6.98% w/w exatecan present in conjugate)
AW13516	48	8	0.6 ng/mL (1.4 nM)
A549	48	1716.6	118 ng/mL (271 nM)
HEK293T	48	5000	349 ng/mL (801 nM)

6.

Cell viability of exatecan vs. Exa-HSA-NPs in A549 Cell line. (a) Cell viability data of A549 cells treated with different concentrations of exatecan on the x-axis, ranging from 0 to 200 nM, and corresponding cell viability % in the y-axis, treated for 48 h. The IC₅₀ was found to be 82 ± 3 nM, (b) represents the cell viability data of A549 treated with different concentrations of Exa-HSA-NPs ranging from 0 to 2600 ng/mL. The IC₅₀ was estimated to be 1716 ± 76 ng/mL (with respect to 6.9 w/w of exatecan, which is 118 ng/mL or 271 nM). Results are expressed as mean ± SD (n = 3).

7.

Cell viability of exatecan vs. Exa-HSA-NPs in HEK293T cell line. (a) Cell viability data of HEK293T cells treated for 48 h with different concentrations of exatecan on the x-axis, from 0 to 200 nM, and corresponding cell viability in the y-axis. The IC₅₀ was found to be 148 ± 17.5 nM, (b) represents the cell viability data of HEK293T treated with various concentrations of Exa-HSA-NPs ranging from 0 to 5500 ng/mL. The IC₅₀ was estimated to be 5000 ± 200 ng/mL (with respect to 6.9 w/w of exatecan is 349 ng/mL or 801 nM). Results are expressed as mean ± SD (n = 3).

3.7. Biodegradation and Erythrocyte Toxicity of Exa-HSA-NPs

To assess the biodegradability of Exa-HSA-NPs, enzymatic digestion using trypsin was performed. Exa-HSA-NPs exhibits inherent turbidity under physiological conditions, allowing for quantitative measurement via UV absorbance at 565 nm. In Figure b, the red line indicates that the concentration of Exa-HSA-NPs displayed a time-dependent decrease with trypsin exposure. Post 6 h, 50% of NPs (1000 μg/mL) was digested in the presence of trypsin (50 μg/mL), whereas in the control, only 0.5% NPs digestion was observed in the absence of trypsin. After 24 h, 99% of NPs degraded in the presence of trypsin without a significant change in NPs incubated without trypsin. This confirms that the enzyme trypsin can digest and degrade Exa-HSA-NPs over time at physiological pH. The biocompatibility of Exa-HSA-NPs was measured using human erythrocytes (Figure a). In the RBC suspension incubated with Exa-HSA-NPs (1 mg/mL) for 4 h under agitation, less than 1% hemolysis was observed, and at a lesser concentration, no hemolysis was observed compared to 100% hemolysis when incubated in double-distilled water. Thus proving that the synthesized NPs are biocompatible in nature.

8.

Hemolysis and biodegradation assay. (a) Hemolysis% of human RBC quantified with absorbance at 540 nm after treatment with H₂O (positive control), PBS (negative control), 0.1 to 1 mg/mL of Exa-HSA-NPs, and HSA-NPs. (b) Biodegradation plot of Exa-HSA-NPs. The black line indicates incubation of Exa-HSA-NPs with PBS (negative control); the red line indicates incubation of Exa-HSA-NPs in the presence of trypsin. Results are expressed as mean ± SD (n = 3).

3.8. Cellular Uptake Assay

Cellular uptake was evaluated with the AW13516, A549, and HEK293T cell lines. FITC-Exa-HSA-NPs were used for this purpose. For this, 8 ng/mL of FITC-Exa-HSA-NPs was incubated with AW13516 for 12 and 24 h and 1716 ng/mL of FITC-Exa-HSA-NPs with A549 and HEK293T for 12 and 24 h. Flow cytometry analysis was performed using FlowJo version 10, employing a sequential gating strategy to precisely define cell populations that had internalized FITC-Exa-HSA NPs. The initial gate was used to obtain the primary population (P1), excluding cell debris, doublets, and triplets. Forward scatter height (FSH) vs forward scatter area (FSA) plots were then employed to select singlets, followed by side scatter height (SSH) vs side scatter area (SSA) to further refine this population. Then, Hoechst was plotted on the y-axis and FITC on the x-axis. The cell population was split into four quadrants (Figure ): Q1 for Hoechst-positive cells, Q2 for cells positive for both Hoechst 33258 and FITC (indicating uptake of FITC NPs by intact cells), Q3 for cells negative for both stains, and Q4 for cells positive for FITC only. We used dual staining to justify the assays. Hoechst 33258 is a nuclear stain to identify and confirm intact cells by staining nuclear DNA, thereby enabling the distinction between live cells and debris. FITC labeling marks the nanoparticles, allowing quantification of their intracellular uptake. The use of both dyes provides true and reliable discrimination between cells that have internalized FITC-tagged NPs and their viability status, improving assay accuracy for NP uptake studies. Flow cytometry analysis showed 57% and 70% cellular uptake after 12 and 24 h, respectively, for the AW13516 cell line. In the case of HEK293T, the cellular uptake was estimated to be 21% and 27%, and in A549, uptake was 31% and 63% after 12 and 24 h, respectively.

9.

Cellular uptake of FITC-Exa-HSA-NPs. Flow cytometry assessment of the cellular uptake of FITC-Exa-HSA-NPs. The cells were incubated with NPs for 12 and 24 h. Blue color in the dot plot in all cell lines represents cells incubated with free FITC control and Hoechst, and red represents the population of cells treated with FITC-Exa-HSA-NPs. The percentage of cells that were positive for Hoechst and FITC (Q2 quadrant) was considered as the number of live cells that had successfully taken up FITC-Exa-HSA-NPs. (a) A549, (b) AW13516, and (c) HEK293T. (d) Represents a graphical representation of the % uptake of FITC-Exa-HSA-NPs. Results are expressed as mean ± SD (n = 3).

3.9. Cell Death Assay

Apoptotic cell death following Exa-HSA-NP treatment was assessed by dual staining with Annexin V-FITC and propidium iodide (PI) in AW13516, A549, and HEK293T cell lines. AW13516 cells were exposed to Exa-HSA-NPs at a concentration of 8 ng/mL, whereas A549 and HEK293T cells were treated with 1716 ng/mL. Flow cytometry analysis was performed by using FlowJo version 10, employing a systematic gating strategy to ensure accurate identification of viable cell populations. The primary population (P1) was gated using forward and side-scatter parameters to eliminate the debris, doublets, and aggregates. FSH vs FSA plots were then used to isolate singlets, followed by SSH vs SSA to further purify the population. The singlet population was then analyzed by plotting PI on the y-axis against Annexin V-FITC on the x-axis, dividing the cell population into four quadrants (Figure ). Q1, representing dead cells which are positive for only PI, Q2 representing late apoptotic cells, double positive for FITC and PI, Q3 representing healthy cells, negative for both FITC and PI, and Q4 representing early apoptotic cells, which are only FITC-positive. AW13516 demonstrated significant apoptotic activity, with 44% of cells positive for annexin V, indicating an early apoptotic stage, and 39% showing dual positivity for both annexin V and PI, indicating a late apoptotic stage. 12% A549 cells were positive for both dyes in their late apoptotic stage, whereas 32% were positive for annexin in their early apoptotic stage. In contrast, only 7% of HEK293T cells were in late apoptosis and 12% in early apoptosis, further signifying the selectivity of Exa-HSA-NPs for cancerous cells. Treatment with HSA-NP alone resulted in minimal induction of apoptosis, with less than 2% of cells showing positive staining for either marker, comparable to that of the PBS negative control group. The group treated with 70% ethanol served as a positive control to validate the assay.

10.

Annexin V–FITC/PI apoptosis assay. Dot plot with histogram of annexin V FITC and PI staining of AW13516, A549, and HEK293T cell lines treated with Exa-HSA-NPs, HSA NPs, and 1× PBS. (a) AW13516, (b) A549, (c) HEK293T and their relative comparison of % apoptosis. Results are expressed as mean ± SD (n = 3).

4. Discussion

Herein, we have synthesized and characterized the novel Exa-HSA-NPs as an effective drug delivery platform to overcome the challenges associated with exatecan, which was withdrawn from the market due to its dose-limiting toxicity. The primary goal of this work was to achieve sustained exatecan release, enabling prolonged target engagement and improved therapeutic efficacy and safety. The Exa-HSA-NPs exhibited an average particle size of 147 nm, low polydispersity (PDI < 0.1), and uniform spherical particles along with very good colloidal stability (−24 mV zeta potential). The biodegradability profile of the Exa-HSA-NPs depicts a significant advantage for clinical translation. Unlike other carrier molecules, HSA, being a naturally occurring protein precursor for the synthesis of Exa-HSA-NPs, offers inherent biocompatibility and biodegradability. The degradation of HSA results in the subsequent formation of peptides and their constituent amino acids, which are then recycled within the cell. The biodegradable nature of Exa-HSA-NPs, verified by the enzymatic degradation, ensures that NPs will not accumulate in tissues, thereby minimizing immunogenicity and long-term safety concerns. Furthermore, the hemocompatibility observed in the erythrocyte toxicity test indicates minimal interaction with blood components, suggesting reduced risk of hemolysis or complement activation upon intravenous administration. This dual biocompatibility, as well as biodegradability, is crucial for clinical translation, as exemplified by the FDA-approved Abraxane and several other nanoformulations used in cancer chemotherapy. − To further assess stability under physiological conditions, Exa-HSA-NPs were incubated in PBS (pH 7.4) at 37 °C for 5 days. The hydrodynamic diameter increased by approximately 10% over the study period (Figure S8), while PDI remained below 0.2 throughout, confirming the colloidal integrity of the formulation under simulated physiological conditions. The prolonged drug-release characteristics of Exa-HSA-NPs represent the most significant finding of this study. The differential release profiles observed at physiological pH 7.4 and acidic pH 5.5 demonstrate the pH-responsive nature of the delivery system, which is required for drug accumulation in the tumor microenvironment. The extended release of exatecan over 7 days at pH 7.4 and 9 days at pH 5.5, as compared to a few hours for exatecan alone, indicates that this formulation can provide prolonged therapeutic effects while limiting the dosage frequency. This sustained release may be attributed to the size, stability, and rate of diffusion of the drug, as well as the slow degradation of nanoparticles and pH-sensitive chemical bonding, which reacts more rapidly in acidic conditions, thereby enhancing drug release in the tumor microenvironment. − Analyses of the docked complex by PILP revealed that exatecan occupies subdomain IB of HSA. Notably, the subdomain IB of HSA has also been reported to accommodate the structurally related parent compound camptothecin, lending added support to the plausibility of the predicted binding pose. The binding energy of −10.4 kcal/mol implies a stable non-covalent accommodation of exatecan which may contribute to the sustained release behavior observed experimentally, in addition to the factors discussed above. Additionally, a notable cause of the pH-responsive drug entrapment and release mechanism is the binding of HSA to the entrapped exatecan in Exa-HSA-NPs. Mi and Burke reported that exatecan exists in different forms at physiological pH and at mildly acidic pH. At physiological pH, exatecan exists in the open carboxylate form, which facilitates albumin binding during Exa-HSA-NP formulation. HSA has a marked 200-fold binding preference for the carboxylate form (K ≈ 1.2 × 10⁶ M^–1) over the lactone form (K ≈ 5.5 × 10³ M^–1), driving near-complete conversion of exatecan to the carboxylate form at physiological pH 7.4. In the acidic tumor microenvironment and lysosomal compartment, it is restored to the active lactone form, enabling intracellular drug release. This also explains the reason for the higher release of exatecan at acidic pH. The fabricated Exa-HSA-NPs have not only shown sustained drug release but also exhibited anticancer activity with lesser cytotoxicity in normal HEK293T cells (801 nM IC₅₀) as compared to free exatecan (148 nM IC₅₀).

This characteristic slow release is particularly critical since the free exatecan exhibited dose-limiting toxicity when administered systemically, making it difficult to maintain therapeutic plasma levels without inducing off-target adverse effects. This sustained drug release pharmacokinetic behavior is highly advantageous, as the mechanistic action of the drug requires continuous presence at the target site rather than transient high peak concentrations. Preferential intracellular uptake of FITC-Exa-HSA-NPs in cancerous cells provides compelling evidence of selective tumor accumulation, providing scope for future in vivo studies. This selective uptake may be due to the presence of a high number of gp60 and SPARC receptors on tumor cells. The higher NP uptake in AW13516 cells is consistent with published evidence of SPARC overexpression in OSCC, reported at approximately 2-fold relative to normal oral mucosa by Poomsawat et al. The lower uptake of NPs in HEK293T cells may be ascribed to the diminished expression of gp60 and SPARC receptors on their surface, as confirmed by the immunofluorescence studies of Meng et al. Additionally, they demonstrated relatively higher SPARC expression, but diminished gp60 expression, in A549 cells, suggesting SPARC-mediated internalization may represent the dominant uptake pathway in A549, even in the context of relatively lower gp60 expression. This is consistent with the enhanced cellular internalization of FITC-tagged Exa-HSA-NPs observed in A549 cells in the present study compared with HEK293T cells. However, direct mechanistic confirmation through receptor blocking or expression quantification in these specific cell lines remains to be established in future work. In essence, this selective uptake reduces the systemic toxicity of the drug molecule by limiting drug exposure to healthy cells, thereby lowering the adverse effects and enhancing the overall therapeutic efficiency of the drug, which otherwise would have been lethal to non-cancerous or normal cells. Additionally, the selective in vitro uptake of Exa-HSA-NPs by tumor cells establishes a strong foundation for future preclinical studies in animal models. Annexin V-FITC/PI double staining provides compelling evidence that the Exa-HSA-NPs effectively induce apoptosis in tumor cells. The lower number of apoptotic cells in non-cancerous HEK293T cells further confirms the selective induction of apoptosis in tumor cells by Exa-HSA-NPs. This proposed “selective execution” may be attributed to the preferential uptake of NPs by tumor cells, which is facilitated by the presence of specific receptors discussed previously. This differential susceptibility aligns with existing literature that substantiates nanoparticle-mediated drug delivery for selective cancer therapeutics, thereby improving treatment efficiency, overcoming drug resistance, and minimizing systemic toxicity. − The results obtained in this study collectively support the hypothesis that Exa-HSA-NPs significantly enhance the therapeutic potential of the drug while maintaining an excellent safety profile, making it a promising platform for advanced drug delivery applications.

5. Conclusion

The study demonstrates the successful design and evaluation of Exa-HSA NPs for the controlled and selective delivery of exatecan. This novel formulation has achieved dual objectives: one being sustained release and the other tumor-selective delivery, addressing key limitations of free exatecan. Exa-HSA-NPs exhibited optimal physicochemical characteristics, including a nanoscale size, high monodispersity, and strong colloidal stability, which enabled efficient tumor accumulation via an EPR effect. Their biodegradability and hemocompatibility further highlight physiological tolerance and biosafety. The Exa-HSA-NPs provided pH-responsive, extended drug release for up to 9 days, ensuring continuous inhibition of TOPO1 and minimizing systemic exposure. In vitro assays confirmed that Exa-HSA-NPs retained the anticancer activity of free exatecan in oral and lung cancer cell lines, while displaying markedly lower toxicity toward non-cancerous HEK293T cells. Enhanced cellular uptake and apoptosis in malignant cells further confirmed their tumor-selective actions. Collectively, our findings establish Exa-HSA-NPs as a next-generation drug delivery platform that prolongs the drug’s half-life, improves selectivity, and reduces dose-limiting side effects. By combining sustained pharmacokinetics with albumin-mediated selective tumor homing, this novel formulation paves the way for advancing exatecan-based therapy toward safer and more effective cancer treatment paradigms. Future work will focus on preclinical pharmacokinetic and efficacy studies to support the translation of these findings to clinical applications.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c02568.

• FTIR spectra of FITC, HSA, and FITC-tagged HSA (Figure S1); UV-vis absorption spectra of HSA-NPs and FITC-tagged HSA (Figure S2); standard curves for HSA quantification, exatecan entrapment efficiency, and drug release studies (Figures S3–S6); Hydrophobic contacts and hydrogen bonds between exatecan and HSA (Table S4), cell viability data for HEK293T, A549, and AW13516 cells treated with doxorubicin (Figure S7); physiological stability of nanoparticles (Figure S8) (PDF)

VHKP conceptualized and designed the study, performed all experimental work, analyzed the data, and wrote the original draft. VN provided conceptual guidance and supervised the overall project. RD provided technical input on viscosity measurements that assisted in nanoparticle characterization. All authors contributed to reviewing and editing the final manuscript and have approved the submitted version.

The work is funded by the Government of India under DBT Builder −BITS Pilani K K Birla Goa campus Interdisciplinary Life Science Programme for Advanced Research and Education (Level III) BT/INF/22/SP2543/2021.

The authors declare no competing financial interest.