Controlled reductive unfolding of BSA-α under pH stimuli and its supramolecular assembly with aminated gum acacia nanocarriers for targeted gemcitabine delivery in lung cancer therapy

Abstract

In this work, we developed a pH-responsive and biocompatible nanocarrier system for targeted lung cancer therapy using bovine serum albumin-α (BSA-α)-functionalized aminated gum acacia (Am-GA) as a carrier for gemcitabine (GEM). The nanocarriers were formed through electrostatic and hydrogen-bonding interactions between the amine groups of Am-GA, the protein matrix of BSA-α, and the polar functional groups of GEM, which resulted in high drug encapsulation efficiency and good formulation stability. FTIR spectra of the developed nanocarriers showed noticeable changes in the amide and polysaccharide regions when compared with the individual components, indicating the presence of intermolecular interactions after formulation. From the XRD patterns, it was evident that gemcitabine lost its crystalline nature following encapsulation and appeared mainly in an amorphous form, a change that could favour better dissolution behaviour. Particle size measurements obtained from DLS showed that most of the particles were distributed within the range of ~120–180 nm. Zeta potential measurements indicated that the dispersion was stable, with no visible aggregation. TEM images showed mostly spherical particles with consistent morphology across the observed fields. Drug release studies demonstrated a clear pH-dependent pattern. At pH 7.4, the release of GEM from the BSA-Am-GA system remained low. When the pH was reduced to 5.5, the amount of released drug increased sharply. This behaviour is consistent with acidic tumour-like conditions. In A549 cells, the nanocarrier-treated groups showed stronger cytotoxic effects than free GEM. Higher cellular uptake was also observed. Apoptotic cell population increased under acidic conditions. ROS measurements indicated a noticeable rise in intracellular levels (~82.4%) after treatment with BSA-α-Am-GA@GEM. These observations point toward oxidative stress–related mitochondrial damage contributing to cell death. Furthermore, in vivo studies in A549 tumour-bearing BALB/c mice showed marked tumour volume regression with minimal systemic toxicity. This was supported by stable body weights and the preservation of normal histoarchitecture in major organs. Taken together, these findings indicate that naturally derived polymer–protein conjugates, such as the BSA-Am-GA system developed in this study, offer a promising strategy for engineering intelligent, tumour-specific nanoplatforms for advanced chemotherapeutic intervention in lung cancer.

Supplementary information

The online version contains supplementary material available at 10.1186/s13036-026-00631-2.

Introduction

Lung cancer continues to be a major cause of cancer related death globally with non-small cell lung cancer (NSCLC) accounting for nearly 85% of all cases [1]. The poor survival outcome of NSCLC is primarily driven by its clinically silent progression during the early stages and the high incidence of late-stage diagnoses, at which point available therapeutic interventions are limited and exhibit reduced efficacy [2]. In spite of the widespread clinical use of frontline chemotherapeutics such as gemcitabine (GEM) in NSCLC treatment, their therapeutic effectiveness is substantially compromised by unfavourable pharmacokinetic and biodistribution profiles, such as low aqueous solubility, rapid enzymatic inactivation in the bloodstream, a short systemic half-life and non-specific tissue accumulation [3]. These limitations lead to poor tumor accumulation and significant off-target toxicity, emphasizing the need for drug delivery systems that improve GEM stability, enhance tumor selectivity, and minimize systemic side effects.

Nanotechnology-based platforms have emerged as promising strategies to overcome these challenges. Nanotechnology-driven drug delivery systems have emerged as promising strategies to overcome these therapeutic challenges. Due to their tunable size, high surface-area-to-volume ratio, and modifiable surface chemistry, nanocarriers enable prolonged circulation, enhanced permeability and retention (EPR)-mediated tumour accumulation, and controlled drug release profiles [4, 5]. However, many conventional synthetic nanomaterials are limited by related to long-term toxicity, poor biodegradability, and challenges in clinical translation. As a result, naturally derived polymers have attracted growing interest because of their inherent biocompatibility, biodegradability, low immunogenicity, and structural versatility [6, 7].

Gum acacia (GA) is a natural, water-soluble polysaccharide obtained from Acacia species and is widely used in biomedical applications because of its good emulsifying ability, colloidal stability, and proven safety [8]. Native GA has few functional groups, limiting its drug-loading capacity and stimulus-responsive release, which reduces its effectiveness as a carrier. To overcome this, GA was chemically aminated to form aminated gum acacia (Am-GA). The added primary amine groups improve electrostatic and hydrogen-bonding interactions with drugs and provide pH-responsive properties to the polymer matrix [9]. This modification is especially relevant for cancer therapy, as the mildly acidic tumour microenvironment (pH ~ 5.5–6.5) can be leveraged to trigger site-specific drug release while preserving carrier stability under physiological conditions (pH 7.4) [10].

Despite these physicochemical enhancements, polysaccharide-based nanocarriers frequently display limited biological recognition and suboptimal cellular uptake. To address this limitation, bovine serum albumin (BSA) was incorporated as a surface-functionalizing component. BSA is a biodegradable, non-immunogenic plasma protein with high drug-binding capacity and can exploit albumin receptor–mediated endocytosis, which is overexpressed in many cancer cells [11, 12]. Functionalization with reductively modified BSA (BSA-α) improves nanoparticle stability, drug encapsulation efficiency, and tumour-specific internalization via receptor mediated mechanisms. The synergistic integration of BSA-α with Am-GA thus overcomes both physicochemical and biological limitations commonly associated with conventional gum-based carriers.

In this study, we report the design of a pH-responsive protein–polysaccharide hybrid nanocarrier based on BSA-α–modified Am-GA for targeted delivery of GEM in lung cancer. The nanocarrier stays stable under physiological conditions, minimizing premature drug release, while enabling faster GEM release in acidic, tumour-like environments. Detailed physicochemical studies were carried out to confirm nanoparticle formation, stability, drug loading, and pH-dependent release behaviour. The therapeutic performance of the system was further examined through in vitro studies in A549 lung cancer cells and in vivo experiments using BALB/c tumour-bearing mice.

Gum-based hydrogels and polysaccharide nanocarriers reported previously for intravenous delivery often have limited pH responsiveness, low tumour selectivity, and poor long-term colloidal stability, particularly for hydrophilic anticancer drugs such as GEM [13, 14]. Furthermore, to the best of our knowledge, no previous study has reported a synergistic BSA-α–functionalized aminated gum acacia nanocarrier specifically optimized for lung cancer therapy. This study addresses these challenges through an integrated design that combines (i) amination-driven pH-responsive drug release, (ii) BSA-mediated receptor-assisted tumour targeting, and (iii) amorphous GEM encapsulation to improve solubility and bioavailability [15, 16]. Collectively these features improve the drug loading capacity, enhance anticancer efficacy, controlled drug release, reduce systemic toxicity and positioning the nanoplatform as a promising approach for lung cancer therapy.

Materials and methods

All chemicals were purchased from Sigma-Aldrich in Shanghai, China, including bovine serum albumin (BSA) selected for its structural flexibility and excellent biocompatibility. Then, Gum acacia (GA), sodium borohydride (NaBH₄), hydrochloric acid (HCl), ethylenediamine (EDA), acetone, sodium hydroxide (NaOH) and glutaraldehyde were obtained from Sigma-Aldrich Co. Ltd. (USA) and used for reduction, precipitation, pH adjustment, functionalization, and crosslinking of the polysaccharide matrix. Gemcitabine (GEM) was purchased from Wuhanvanz Pharma (Jingkai Future City, Hubei, China). 0.1 M Phosphate buffer solution (PBS) was prepared by dissolving proper amount of monosodium dihydrogen phosphate and disodium hydrogen phosphate, with the pH adjusted to 5.5 using NaOH or HCl. For cellular studies, the human lung adenocarcinoma cell line (A549) was obtained from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Minimum Essential Medium (MEM) supplemented with Earle’s Balanced Salt Solution (EBSS) from HyClone (Logan, Utah, USA) to provide a suitable nutrient environment for in vitro studies.

pH-tuned, reductively modified bovine serum albumin (BSA) fabrication

In a typical synthesis of pH-modulated, reductively modified BSA, 0.5 g of BSA was dissolved in 50 mL of deionized (DI) water, and the pH was adjusted to 5.5, 7.2, or 8.9 using HCl and NaOH. In that 5% sodium borohydride (0.2 g in 5 mL DI water) was dropwise added to each pH-adjusted solution, followed by continuous stirring for 3 hrs to reduce the disulfide bonds. This reduction process generates the additional functional groups and also high flexibility for further interactions. Modified BSA was precipitated with ice-cold acetone (1:3), centrifuged, rinsed, and lyophilized to obtain BSA. The pH-specific variants were designated as BSA-α (pH 5.5), BSA-β (pH 7.2), and BSA-γ (pH 8.9). Among these, BSA-α demonstrated the highest flexibility due to its preferential disulfide bond cleavage under acidic medium. This reduction process created the -COOH (carboxyl group) and -OH (hydroxyl group) functional groups, which interacted with the -NH (amine group) group of aminated gum acacia (Am-GA), improving electrostatic interactions and it will enhance the GEM encapsulation. Hence, the BSA-α was selected as the carrier for GEM encapsulation and nanocarrier fabrication to achieve efficient and targeted lung cancer treatment.

Preparation of aminated gum acacia (Am-GA)

In a typical synthesis, 1 g of gum acacia (GA) was dissolved into 250 mL of DI water and stirred continuously. Hereafter, ethylenediamine (15 mL) was added drop by drop into the solution, enabling the introduction of amine groups through interactions with the -OH and -COOH groups of GA. The reaction mixture was stirred overnight at room temperature to ensure effective amination. Finally, the solution was dialyzed against deionized water for 48 hrs to remove excess ethylenediamine and byproducts, and the purified product was dried using lyophilizer. The obtained Am-GA powder was used for further studies.

Synthesis procedure of pH-responsive BSA-α-Am-GA@GEM hydrogel

The optimized BSA-α-Am-GA@GEM hydrogel was prepared by dissolving 0.5 g of BSA in 50 mL of DI water, adjusting the pH to 5.5, and adding 5% sodium borohydride (0.2 g in 5 mL DI water) dropwise over 3 hrs stirring continuously to reduce disulfide bonds. The reduced BSA was precipitated with cold acetone (1:3, v/v), followed by centrifugation, washing, and lyophilization. Then, Am-GA was synthesized by dissolving 1 g of GA in 250 mL of DI water, adding 15 mL of ethylenediamine dropwise, and stirring overnight to introduce amine groups. After 48 hrs of dialysis and lyophilization, Am-GA was obtained. To prepare the hydrogels, BSA-α and Am-GA were dissolved in PBS (pH 5.5), followed by the addition of GEM and 0.2% glutaraldehyde as a crosslinker to trigger gelation. The resulting BSA-α-Am-GA@GEM hydrogel was fabricated as a pH-responsive, site-specific drug delivery system for lung cancer chemotherapy. It was thoroughly washed to remove unbound drug and stored for subsequent studies (Scheme 1).

Scheme 1.

Schematic illustration of the preparation and application of BSA-α-Am-GA@GEM hydrogel for lung cancer therapy

Instrumentation

X-ray diffraction (XRD) analysis was carried out on a Rigaku Smart Lab diffractometer with Cu Kα radiation (λ = 1.54 Å) at 40 kV and 20 mA and a scan rate of 2°/min. The diffraction pattern indicated the presence of crystalline domains within the hydrogel matrix. Raman spectroscopy was carried out 15 seconds with a STR 300 Raman spectrometer (Seki Technotron Corp., Japan) equipped with a 532 nm laser and a 1200 g/mm grating, the acquired vibrational spectra confirmed the material’s molecular composition. FTIR analysis was carried out using a Nexus 670 spectrometer (Thermo Nicolet, USA) over the range of 400–4000 cm^{− 1}. Surface morphology of the sample was studied by scanning electron microscopy (SEM, JEOL JSM-6490LA) displayed the porous structure morphology and its highly suitable for drug loading and release applications. Transmission electron microscopy (TEM, JEOL JEM-100CX II, Japan) confirmed the internal nano structural arrangement. The hydrodynamic diameter of the synthesized PB-NGs was measured using dynamic light scattering (DLS) with a Brookhaven BI-9000AT system (Brookhaven Instruments, USA), and their surface charge (zeta potential) was determined with a Brookhaven 90Plus Zeta Potential Analyser (Brookhaven Instruments). These analyses confirmed the formation of a structurally stable and chemically integrated hydrogel suitable for targeted drug delivery.

Swelling behaviour

The samples BSA-α-Am-GA and BSA-α-Am-GA@GEM were incubated 1 mL of DI water at 37 °C after being weighted with 0.1 g of dry hydrogels. The hydrogels were reweighed at the chosen time interval after the surplus water was removed using tissue paper. Then, followed by adding 1 mL of the new water to the hydrogels. The rate of swelling stopped changing and the measurement was determined.

Drug release in vitro IGF-1 release profiles for IGF-1-loaded

In vitro GEM release profiles for the GEM loaded BSA-α-Am-GA hydrogel we first tested to find out their differences in release rates. GEM 1 mg was taken, it was dissolved into 1 mL of DI water and loaded into the hydrogel. It was immersed in PBS at pH 5.5, 7.2, and 8.9 and incubated at 37 °C. Aliquots were collected at predetermined intervals and replaced with fresh PBS to maintain sink conditions. The cumulative drug release was measured over time to analyze the effect of different pH on drug release.

In vitro analysis

Cell culture

The A549 human lung adenocarcinoma cell line was purchased from ATCC. Then, it was cultured in minimum essential medium (MEM) enriched with Earle’s Balanced Salt Solution (EBSS), 50 U/mL penicillin, and 50 μg/mL streptomycin. The cell culture was maintained in a humidified atmosphere with 5% CO₂ at 37 °C. The medium was replaced every 48–72 hrs and the cells were subcultured frequently to preserve the viability and to prevent the over growth.

Cell viability

Cell viability was assessed by the MTT assay (3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). Cells were incubated in 96-well plates at 4000–5,000 cells per well and incubated overnight at 37 °C with 5% CO₂. Hereafter, cells were treated with the hydrogel formulation and incubated for 24 hrs. After treatment, 20 μL of MTT solution (5 mg/mL in PBS) was introduced to each well and incubated for 4 hrs to allow live cells to convert MTT to formazan. To dissolve purple formazan crystals, carefully aspirate the medium and add 150 μL of DMSO to each well. The formulation’s antiproliferative effectiveness was assessed by measuring absorbance at 570 nm and comparing cell viability to the untreated control group.

Assessment of intracellular ROS levels

A549 cells were treated with BSA, Am-GA, GEM, or BSA-α-Am-GA@GEM hydrogel and stained with the ROS-sensitive probe DCFH-DA. After treatment, cells were washed with PBS and incubated with 10 μM DCFH-DA in serum-free medium for 30 minutes at 37 °C in the dark. After internalization, DCFH-DA was enzymatically deacetylated to produce non-fluorescent DCFH, which was then oxidized by intracellular ROS to generate highly fluorescent DCF. Cells were harvested with trypsin, resuspended in PBS, and imaged using a fluorescence microscope (Ex: 488 nm/Em: 525 nm). Green fluorescence intensity was used to quantify ROS levels, allowing comparison of oxidative stress induced by each treatment.

Live/dead staining

The cytotoxic and anti-proliferative effects of BSA, Am-GA, GEM, and BSA-α-Am-GA@GEM hydrogel on A549 cells were evaluated using acridine orange/ethidium bromide (AO/EB) staining. Cells were seeded at 2 × 10⁴ cells/mL in 24-well plates and treated with the respective formulations. After 24 hrs of treatment, cells were trypsinized (20 μL/well), rinsed with PBS, and stained with 4 μL of AO/EB stain (100 μg/mL). The dyed cell suspensions were then put on glass slides and evaluated under a fluorescence microscope. The AO (green fluorescence) indicated early apoptotic cells, but the EB (red fluorescence) indicated late apoptotic. This approach distinguishes between viable and nonviable cells based on various staining and its allowing for a qualitative measurement of treatment induced cell damage.

Mitochondrial membrane potential assay (MMP)

The JC-1 fluorescent probe assessed MMP, where healthy mitochondria emit green fluorescence and compromised ones emit red-orange fluorescence. The cells were treated with fabricated samples and allowed to incubated for 24 hrs. After treatment, cells were stained with JC-1 (50 μg/mL) in the culture medium for 45 minutes. After treatment, cells were incubated with 2 μM JC-1 dye in PBS at 37 °C for 45 minutes to measure mitochondrial membrane potential (ΔΨm). Red fluorescence indicates healthy mitochondrial potential, while green fluorescence indicates depolarized mitochondria. The red-to-green fluorescence ratio was examined to identify ΔΨm disruption in treatment groups.

DAPI analysis

A549 cells were cultured in 96-well plates with DMEM media at a density of 1 × 10⁵ cells to study nucleus morphological alterations. After incubation, cells were stained with DAPI (4’,6-diamidino-2-phenylindole) for 10 minutes at 37 °C to detect the nuclear morphology. After labeling unwanted dye was removed by washing with PBS and the cells were under a fluorescence microscope for nuclear alterations that show the apoptosis and other cellular abnormalities.

Flow cytometry

The efficacy of anticancer fabricated samples was analyzed through quantification of apoptosis. The rate of apoptosis was assessed by Annexin V-FITC and PI dual staining. Cell lines were cultured in 96-well plates with 2 mL of DMEM medium. After 24 hrs of incubation the medium was replaced with 2 mL of fresh medium containing 50 µM various synthesized formulations. Untreated cells served as controls. Following another 24 hrs cells were stained and analyzed by flow cytometry using Dakewe EXFLOW-206 (Shenzhen, China).

Cellular uptake study (IF and FACS analysis)

The cells were seeded in 96-well plates and allowed to incubate for 24 hrs. Cells were treated with prepared samples at equivalent concentrations for 30 min, 2 hrs, or 24 hrs. For immunofluorescence, cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and nuclei stained with DAPI. Images were captured under identical settings, and intracellular fluorescence was quantified using ImageJ. For flow cytometry, cells were trypsinized, washed, and resuspended in FACS buffer. Fluorescence was recorded for at least 10,000 events per sample and analyzed with FlowJo. Experiments were performed in triplicate, and data are presented as mean ± SD.

In vivo studies

Animal grouping and treatment

After acclimatization, the animals were randomly divided into five groups (I–V) of ten mice each. Group I served as the untreated control and received normal saline. Group II received intravenous BSA-α, whereas Group III received Am-GA. Group IV received free GEM, while Group V received a mixed hydrogel formulation of BSA-α-Am-G A@GEM.

In vivo antitumor therapy

A xenograft tumour model was created by subcutaneously inoculating 1 × 10⁶ A549 or TE-1 cells into the right flank of 8-week-old male BALB/c mice. These mice have a defective T-cell immune response, which allows for continuous tumour formation and progression. After the tumours reached an average volume of ~ 150 mm³, the animals were randomly assigned into five experimental groups and treated intravenously with respective formulations every third day for 14 days. Tumor growth was measured twice weekly using digital calipers, and volume was calculated with the formula: V = (length × width²)/2. After treatment, mice were euthanized by CO₂ inhalation, and tumor tissues were collected for histological and molecular analyses. All experimental techniques were approved and carried out in strict conformity with the Animal Ethics Committee’s guidelines at Fujian Medical University in China.

In vivo toxicity

Blood was collected from the treated animals and incubated for 2 hrs at 36 °C to promote coagulation. Subsequently, the blood samples were centrifuged for 15 min at 1000 rpm and the serum was separated. The biochemical parametres, including SGOT, SGPT, ALP, urea, and creatinine were analyzed using commercial assay kits. This analysis

Histological assessment of tumour and organ tissues

Following treatment, major organs (liver, kidneys, heart, lungs, and spleen) were collected for histological examination. The tissues were fixed, embedded in formalin, sectioned and stained with hematoxylin and eosin (H&E). This analysis is for observation of morphological alterations, cellular damage or inflammatory responses. The histopathological analysis confirmed the safety profile of the prepared composite.

Wound scratch

A549 human lung cancer cells (5 × 10⁴ cells/well) were cultured in 6-well plates under standard conditions until reaching the logarithmic growth phase. To simulate a wound, a sterile pipette tip was used to create a uniform linear scratch (~2 mm wide) in the center of each well. Detached cells were removed by two PBS washes. Cells were then treated with BSA-α, Am-GA, GEM, or the combined BSA-α-Am-GA@GEM formulation (50 μg/mL). Untreated wells served as the negative control, and 5-fluorouracil (2 mg/mL) was used as the positive control. Wound closure was observed after 24 h using an inverted phase-contrast microscope. The degree of cell migration was measured by comparing the relative wound closure area of treatment groups.

qRT-PCR analysis

To investigate the transcriptional regulation of apoptosis-related markers, we evaluated the mRNA expression levels of Cyclin D1, PCDA, CDK6, and BCL-2 in tumour tissues from mice treated with BSA-α, Am-GA, GEM, and the combinatorial hydrogel BSA-α-Am-GA@GEM. Tumours were excised after eight weeks of in vivo exposure, homogenised in RNase-free conditions, and total RNA extracted using BioZol reagent (BioFlux). The genomic DNA was selectively removed using cDNA filtration columns, and the RNA samples were further purified with silica-based spin columns and centrifuged at 12,000 rpm for 30 seconds. Gene-specific primers were used to check the integrity and purity of the RNA before reverse transcription into complementary DNA (cDNA) (Vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was performed with the Quant Studio 1 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, USA) and SYBR Green chemistry, as well as a one-step qRT-PCR kit as indicated by the manufacturer. Primer specificity was validated, and gene amplification was monitored in real time. Relative gene expression was calculated using the 2^{− ΔΔCt} method and normalized to housekeeping genes such as GAPDH or 18S rRNA. This gave quantitative information on gene regulation across many treatment groups.

Statistical analysis

All experimental data were analysed statistically using GraphPad Prism 8.02 (GraphPad Software Inc., San Diego, CA, USA). Each experiment was conducted three times, and the data are presented as mean values with standard deviations (SD). Group comparisons were made using one-way or two-way ANOVA, depending on the study design. Intergroup differences were evaluated using Bonferroni’s post hoc multiple comparison test, with p < 0.05 considered statistically significant.

Result and discussion

XRD analysis

Structural characterization of BSA-α, Am-GA, GEM and BSA-α-Am-GA@GEM nanohydrogel was performed. The XRD patterns shown in Fig. 1A BSA-α displayed prominent diffraction peaks at 2θ ≈ 19.6°, 23.1°, 28.4°, and 32.7°, suggesting its semi-crystalline structure [17]. Then, the Am-GA exhibited high reflections at 2θ ≈ 14.8°, 21.4°, 27.6°, 30.2°, and 34.8°, indicating its crystalline polysaccharide structure [18]. The GEM exhibited a large diffuse halo centred about 2θ ≈ 20°-25°, reflecting the amorphous nature of the material [19]. The diffraction pattern of the BSA-α-Am-GA@GEM nanohydrogel retained some typical peaks of both BSA-α and Am-GA (e.g., at 2θ ≈ 20.1°, 28.2°, and 32.5°) but with considerably reduced intensity. The removal of strong GEM peaks and an overall reduction in crystallinity suggest that GEM has been successfully encapsulated within the polymeric matrix. This partial amorphization improves GEM solubility, uniform dispersion, and prolonged release.

Fig. 1.

(A) The XRD, (B) raman, (C) FTIR, and (D) UV-Vis spectra of BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM demonstrate crystalline GEM encapsulation, functional group interactions, and drug loading. (E) DLS reveals nanoscale particle size, while (F) zeta potential (∼ −35 to − 40 mV) suggests high colloidal stability

RAMAN

The Raman spectrum in Fig. 1B evidences structural information of the as-prepared samples. In the spectrum of BSA-α spectrum clearly shows the characteristic bands at 1650–1660 cm^{− 1} for amide I (C = O stretching), 1540 cm^{− 1} for amide II (N-H bending and C-N stretching), and 1240 cm^{− 1} for amide III, confirming the protein backbone structure [20]. The Am-GA exhibited a broad band centred between 1450 and 1600 cm^{− 1}, due to C-H bending and -OH vibrations of the polysaccharide, as well as a weaker band near 1100 cm^{− 1}, attributed to C-O-C stretching of the guar gum backbone [21]. Then, the pure GEM exhibited a crystalline structure, with characteristic peaks at ~ 1480 cm^{− 1} (C–N stretching of amine groups), 1340 cm^{− 1} (pyrimidine ring vibrations), and 780–800 cm^{− 1} (aromatic C–H bending) [22]. In the BSA-α-Am-GA@GEM hydrogel, these GEM peaks were present but weaker and slightly broadened, particularly at 1480, 1340, and 780 cm^{− 1}. The BSA amide bands and Am-GA polysaccharide backbone peaks were retained but slightly shifted, indicating hydrogen bonding and electrostatic interactions between GEM and the protein with polysaccharide matrix. These spectral changes confirm GEM encapsulation within the hydrogel and suggest partial molecular reorganization.

FTIR spectroscopy analysis

The interaction between the functional groups and molecular interactions of BSA-α, Am-GA, GEM and BSA-α-Am-GA@GEM nanohydrogel was performed by the FT-IR spectroscopy. The BSA-α spectrum displayed characteristic protein bands: a strong peak at ~ 1650 cm^{− 1} (amide I, C = O stretching), a peak near 1540 cm^{− 1} (amide II, N–H bending and C–N stretching), and a weaker band at ~ 1240 cm^{− 1} (amide III), confirming the polypeptide backbone [23]. The Am-GA exhibited broad stretching vibrations at ~ 3400 cm^{− 1}, corresponding to -OH groups, as well as bands at ~ 2930 cm^{− 1} (C-H stretching), ~1600–1400 cm^{− 1} (C-O-C and COO⁻ stretching of the polysaccharide backbone), and ~1020–1070 cm^{− 1} (C-O-C glycosidic linkages), which are typical of guar gum derivatives [24]. GEM has strong absorption peaks at ~ 3330 cm^{− 1} (N-H stretching), ~1670 cm^{− 1} (C = O stretching of amide group), ~1600–1500 cm^{− 1} (C = C and C-N stretching of heteroaromatic ring), and sharp bands at ~1250–1000 cm^{− 1} (C-F and C-O stretching), confirming its crystalline molecular structure [25]. The BSA-α-Am-GA@GEM composite hydrogel retained GEM’s distinctive peaks, albeit with reduced intensity and moderate broadening. The primary amide (BSA) and polysaccharide (Am-GA) bands also shifted slightly. The overlapping and intensity changes (especially at 1650–1540 cm^{− 1} and 3400 cm^{− 1}) indicate hydrogen bonding and electrostatic interactions between GEM and the BSA-α/Am-GA matrix. The spectrum shifts in Fig. 1C indicate efficient GEM encapsulation and the formation of a stable composite hydrogel [22].

UV–Vis spectroscopy analysis

UV-Vis spectra analysis of Fig. 1(D) shows the absorption spectrum of GEM within the BSA-α-Am-GA hydrogel system and investigated molecular interactions. BSA-α clearly shown in the absorption peak around 278 nm, coming from its aromatic amino acids (tryptophan, tyrosine, and phenylalanine) [26]. The optical absorption property of the Am-GA exhibits a broad absorption shoulder between 250 and 260 nm in which the former obviously confirms the transition of n→π* polysaccharide backbone. In GEM the main characteristic absorption peak at ~268–270 nm, confirms the conjugated π-system of the pyrimidine ring. While in the BSA-α-Am-GA hydrogel formation, GEM has been shifted and changes the peak intensity compared to free GEM, indicating that GEM was successfully encapsulated and may interact with protein–polysaccharide (π-π stacking) [27, 28]. This strong interaction is confirming GEM’s incorporation into the BSA-α-Am-GA hydrogel, including its electronic properties.

Dynamic light scattering (DLS)

The fabricated BSA-α-Am-GA@GEM nanohydrogel composite was subjected to DLS. The DLS analysis (Fig. 1E) shows that the nanohydrogel composite exhibited a desirable single hydrodynamic diameter in the range of 150–180 nm. The nanohydrogel composite materials make them ideal for medication delivery [29, 30]. The nanohydrogel composite particles in the 100–200 nm range can effectively enhance the cellular uptake, biological barriers and remain in circulation longer by avoiding rapid clearance. The nanohydrogel particles enable passive tumour targeting through the Enhanced Permeability and Retention (EPR) effect and it has more advantages in cancer therapy. The nanohydrogel’s high surface-area-to-volume ratio enriches the GEM encapsulation efficiency [31]. The DLS results indicate the GEM was successfully loaded without compromising the structural integrity of the nanohydrogel composite [32].

Table 1 shows the triplicate measurements (n = 3) provide consistent sizes of 150, 152, and 148 nm, with a mean particle size of 150 ± 2 nm. The minimal standard deviation confirms excellent reproducibility and is denoted by a narrow size distribution with even colloidal properties. The DLS measurement confirmed the BSA-α-Am-GA@GEM nanohydrogel has monodisperse nanoscale distribution, making it suitable for targeted controlled drug delivery applications [33].

Table 1.

DLS results

DLS (nm)
Sample	Rep 1	Rep 2	Rep 3	Mean ±SD
BSA-α-Am-GA@GEM	150	152	148	150± 2

Dynamic light scattering (DLS) analysis showing the hydrodynamic diameter of BSA-α-Am-GA@GEM nanoparticles. Measurements were performed in triplicate (n = 3), and the results are expressed as individual replicate values (Rep 1–Rep 3) and mean ± standard deviation

Zeta potential

Figure 1F shows the zeta potential analysis of the fabricated BSA-α-Am-GA@GEM nanohydrogel composite, which were found to have a moderately negative charge of −35 to −40 mV. This negative zeta potential provides electrostatic repulsion between particles, preventing aggregation and enhancing the long-term colloidal stability. The negative surface charge originates from carboxyl (-COO⁻) and hydroxyl (−OH) groups of aminated guar gum, as well as negatively charged residues on the BSA backbone [34]. This charge enhances biocompatibility by reducing nonspecific protein adsorption and limiting rapid opsonization and clearance by the reticuloendothelial system. It has significantly enhanced the stability, uniform particle size distribution and superior controlled drug release, consistent therapeutic effects [35].

Table 2 shows the triplicate measurements (n = 3) of zeta potential values of −35, −34, and −36 mV, with a mean of −35 ± 1 mV. The minimal standard deviation indicates high reproducibility and confirms a uniform surface area across the prepared nanoparticles. A zeta potential magnitude greater than ±30 mV is widely regarded as indicative of strong electrostatic repulsion, which effectively prevents particle aggregation in aqueous media. Accordingly, the observed negative surface charge confirms the good colloidal stability of the nanohydrogel system. In addition, the negatively charged surface promotes prolonged systemic circulation by reducing nonspecific protein adsorption and minimizing rapid clearance. Moreover, the zeta potential data indicate that the BSA-α-Am-GA@GEM nanohydrogel is stable, well-dispersed, and suitable for biomedical drug delivery [36].

Table 2.

Zeta potential results

Zeta Potential (mV)
Sample	Rep 1	Rep 2	Rep 3	Mean ±SD
BSA-α-Am-GA@GEM	−35	−34	−36	-35 ± 1

Zeta potential analysis of BSA-α-Am-GA@GEM nanoparticles. Measurements were carried out in triplicate (n = 3), and the data are presented as individual replicate values (Rep 1–Rep 3) along with mean ± standard deviation

SEM

The morphology of the fabricated samples was investigated using SEM analysis, as shown in Fig. 2. The SEM images of BSA-α with a heterogeneous nanosheet-like shape and irregular fragments, suggesting reductive unfolding resulting from selective disulfide bond cleavage clearly depicted in Fig. 2A. The, β-BSA (Fig. 2B) displayed wrinkled and layered nanosheets with increased surface roughness, whereas γ-BSA (Fig. 2C) showed aggregated globular morphologies, indicating conformational distinct variations among the different BSA forms [12]. Am-GA (Fig. 2D) showed a porous surface with nanoscale pits, confirming successful amination and the addition of -NH₂ groups. BSA-α was chosen as the nanocarrier for its enhanced structural flexibility and high drug-loading capacity. Acidic conditions selectively break disulfide bonds, unfolding the protein and exposing additional –OH and –COOH groups. Combined with Am-GA’s -NH groups, these features strengthen electrostatic and hydrogen bonding interactions, improving GEM encapsulation efficiency and stability. Nanosized BSA-α particles offer benefits for drug delivery, including higher cellular uptake, deeper tumour penetration, and preferential accumulation in the acidic tumour microenvironment. The BSA-α/Am-GA@GEM hydrogel was formed via supramolecular interactions between BSA-α and Am-GA, producing a unique 3D porous network (Fig. 2E & 2F). High-magnification images (Fig. 2E) showed an interwoven fibrous framework, while low-magnification images (Fig. 2F) revealed a sponge-like porous structure. This architecture allows effective GEM encapsulation, supports controlled, pH-responsive drug release, and promotes favourable cell-material interactions. Overall, the BSA-α-Am-GA@GEM hydrogel is a rationally designed nanoplatform with strong potential for targeted and effective lung cancer chemotherapy [37, 38].

Fig. 2.

(A) BSA-α showing heterogeneous nanosheet-like morphology with irregular fragments, indicating reductive unfolding; (B) β-BSA displaying wrinkled and layered nanosheets with roughened surfaces; (C) γ-BSA exhibiting aggregated globular nanostructures; (D) aminated gum acacia (Am-GA) presenting a porous surface with nanoscale pits, confirming successful amination; (E, F) BSA-α-Am-GA@GEM hydrogel showing a three-dimensional porous, sponge-like architecture at higher (E) and lower (F) magnifications, favorable for GEM encapsulation and sustained release

TEM

The surface morphology of the as prepared nanoparticles was characterized by TEM analysis displayed in Fig. 3. BSA-α exhibited reductive unfolding following selective disulfide bond cleavage, as seen in Fig. 3A. This unfolded conformation exposed reactive functional groups (-OH and -COOH), thereby enhancing its drug encapsulation capability [39]. Fig. 3B depicts aminated gum acacia (Am-GA), which contains generally round and irregular nanoparticles, with a limited degree of agglomeration. The observed morphology confirms successful amination, introducing –NH₂ groups that enhance reactivity and compatibility for supramolecular assembly [40]. The BSA-α/Am-GA@GEM hydrogel (Fig. 3C) exhibited a nanoporous structure with interconnected domains and evenly distributed GEM-loaded regions. This nanoscale architecture reflects strong supramolecular interactions between BSA-α and Am-GA via hydrogen bonding and electrostatic forces, enabling efficient gemcitabine encapsulation. The uniform nanostructure and porous matrix support controlled, pH-responsive GEM release, improving drug stability, tumour penetration, and overall therapeutic effectiveness [18].

Fig. 3.

(A) BSA-α exhibiting unfolded and irregular morphology; (B) aminated gum acacia (Am-GA) showing aggregated, particulate structures; (C) BSA-α-Am-GA@GEM hydrogel displaying nanoporous architecture with dispersed drug-loaded domains-indicative of supramolecular assembly conducive to controlled, pH-responsive chemotherapeutic delivery

The TEM micrographs (Fig. 3A–C) displayed the nanoscale particles exhibiting localized and partial agglomeration. This apparent aggregation is not a true limitation of the nanocarrier but results mainly from the dry-state conditions during TEM sample preparation. When deposited and dried on the carbon-coated copper grid, capillary forces along with van der Waals interactions and hydrogen bonding cause temporary particle clustering, giving the appearance of agglomeration.Moreover, the protein nature of BSA and the surface-active groups (-NH₂ and -OH) on aminated gum acacia enhance interparticle attraction under partially dehydrated conditions, further exaggerating the clustered morphology seen in the images. Importantly, this behaviour is an imaging artifact rather than a reflection of the true dispersion state in solution. Under aqueous and physiological conditions, the nanocarriers maintain stable colloidal dispersion, supported by electrostatic repulsion and hydration layers, confirming that the observed agglomeration has no adverse implications for the structural integrity, functionality, or biological performance of the system [41].

Swelling behaviour

Figure 4A shows that the swelling ratio of BSA-α-Am-GA and BSA-α-Am-GA@GEM hydrogels gradually increased and stabilized after 7 days under physiological conditions. The GEM-loaded hydrogel had a higher swelling capacity (~85% at day 24) compared with the unloaded hydrogel (~50%), indicating that drug incorporation enhances water absorption. This superior swelling is due to two synergistic factors: (i) the hydrophilic nature of GEM, which introduces additional polar domains into the hydrogel, promoting hydrogen bonding and water uptake, and (ii) the increased porosity and relaxation of the polymeric network caused by GEM loading, which allows for easier penetration and diffusion of water molecules into the gel matrix [42]. The hydrogel exhibited a prolonged and stable swelling profile, indicating it can maintain structural integrity over time, which is crucial for sustained and controlled drug release [43].

Fig. 4.

(A) Swelling behaviour of BSA-α-Am-GA and BSA-α-Am-GA@GEM hydrogels over 24 days. The drug-loaded hydrogel shows higher swelling capacity due to increased porosity and hydrophilic contacts. (B) in vitro GEM release profile of BSA-α-Am-GA@GEM hydrogel at three pH values (5.5, 7.2, and 8.9) shows sustained and pH-responsive release, with the maximum release recorded under mildly acidic circumstances (pH 5.5), simulating the tumour microenvironment

Drug release

The BSA-α-Am-GA@GEM hydrogel exhibited pH-responsive, sustained drug release, supporting its potential for targeted cancer therapy. Release followed a biphasic pattern: an initial burst over 3–5 days from rapid diffusion of loosely bound or surface GEM, followed by a prolonged, steady release up to 24 days, controlled by hydrogel swelling and gradual polymer network relaxation. The release rate of GEM was strongly influenced by the pH of the surrounding medium. At acidic pH 5.5, mimicking the tumour microenvironment, the hydrogel displayed maximal swelling and facilitated nearly complete GEM release (~100%), owing to protonation of functional groups, enhanced electrostatic repulsion, and expanded hydrogel pores. At physiological pH 7.2, drug release was slower (~85%), while at alkaline pH 8.9, it was most restricted (~70%) due to less ionization and tighter network packing shown in Fig. 4B. This pH-sensitive behavior indicates that the hydrogel releases more drug in acidic tumor environments while maintaining low, controlled release under normal or alkaline conditions. This selective response ensures drug accumulation at the target site, prevents premature systemic leakage, and minimizes off-target toxicity [44]. Overall, GEM release involves initial Fickian diffusion followed by anomalous (swelling-controlled) transport, creating a controlled, long-term, tumor-targeted delivery system with higher therapeutic efficacy than conventional formulations [45].

In vitro

Phase-contrast microscopy reveals hydrogel-induced morphological collapse in lung cancer cells

The cells were treated with BSA-α, Am-GA, GEM and BSA-α-Am-GA@GEM the morphological changes were observed under phase contrast microscopy. As shown in Fig. 5A, the control cells formed a dense, elongated, spindle shaped morphology reveals the health and undamaged growth of the cells. On the other hand, BSA-α treated A549 cells displays cell shrinkage and reduced spreading shows lessen cytoskeletal changes and retarded protein-cell interactions. Further, the cells exposed to Am-GA causes considerable changes in morphology including irregular boundaries, incomplete adherence of the cells, condensation of cytoplasm reveals the disintegration of membrane integrity and intracellular homeostasis. Besides, the GEM alone triggers cell shrinkage, membrane blebbing which leads typical apoptotic cell death. However, the cells subjected to BSA-α-Am-GA@GEM hydrogel group exhibits significant pathological changes such as cell detachment, cell shrinkage, accumulation of cell debris suggesting the advanced apoptosis and irreversible cell death [46]. The results displayed that, free GEM and other compounds cause minor cytotoxic stress, while the GEM loaded pH responsive hydrogel significantly enhances therapeutic efficiency of GEM due to the prolonged release and cellular uptake as depicted in Fig. 5B. Thus, it was observed that, the GEM loaded hydrogel induces cellular stress with sustained drug delivery producing a potent and targeted cytotoxic effect on A549 cancer cells [47].

Fig. 5.

(A) Microscopic pictures of cells after treatment with control, BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM (scale bar = 100 µm). (B) the BSA-α-Am-GA@GEM group had significantly lower cell viability (%) compared to the control and individual treatments

Apoptosis analysis by AO/EB dual staining

The pro-apoptotic activity of BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM on A549 cancer cells was investigated using AO-EB dual staining method and quantitative apoptosis analysis. As shown in Fig. 6A, control cells show predominantly green fluorescence suggesting intact membranes with high viable cells. The cells treated with BSA-α shows minimal apoptosis with very few red-orange cells observed. The cells exposed to Am-GA treatment displays reduced red fluorescence indicating minimal induction cancer cell apoptosis. In GEM treated cells, it was observed pronounced apoptotic response with emitting widespread red/orange staining over the cell population due to increased apoptosis. On the other hand, BSA-α-Am-GA@GEM hydrogel exhibits significant intensity in red/orange fluorescence, suggesting efficient intracellular drug delivery and increases apoptosis (Fig. 6A) [48]. Moreover, the quantitative analysis confirmed that, the control group did not show any apoptosis, and moderate apoptosis (15%) was observed in BSA-α treated group. Besides, Am-GA and Gem treated group shows moderate to increased apoptosis as 23 and 34% respectively. The cells treated with BSA-α-Am-GA@GEM hydrogel shows highest apoptotic rate (48%), indicating pronounced therapeutic potential of fabricated drug loaded hydrogel (Fig. 6B). This study found that BSA-α and Am-GA work together to stabilize and release GEM in a pH-responsive way, which enhances GEM-induced apoptosis in lung cancer cells [49].

Fig. 6.

(A) AO/EB dual staining images of living (green) and apoptotic/necrotic (red) cells after treatment with various formulations (scale bar = 100 µm). (B) Apoptosis percentage study reveals that the BSA-α-Am-GA@GEM group has the largest apoptotic cell population. (C) DCFH-DA staining images show intracellular ROS generation (green fluorescence) in various treatments (scale bar = 100 µm). (D) Quantitative fluorescence intensity analysis shows increased ROS generation in GEM and BSA-α-Am-GA@GEM-treated cells compared to controls

Synergistic induction of oxidative stress by hydrogel formulation

The ROS generation of A549 cells was assessed by DCFH-DA staining after treated with BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM. The results displayed that, the intensity of green fluorescence is due to the excess production of ROS which leads oxidative stress to the cancer cells (Fig. 6C). The cells in control or untreated group exhibits low level ROS production, while treatment with BSA-α resulted in a moderate generation of ROS, suggesting a protein related redox imbalance of the cells. Similarly, the A549 cells treated with Am-GA treatment shows slight increase in fluorescence intensity compared to control and BSA-α reveals the stimulation of polymer induced oxidative stress. Besides, GEM treated cells exhibits considerable increase in fluorescence intensity which is associated with marked elevation in ROS production due to its pro-oxidant cytotoxic mechanism. Nevertheless, BSA-α-Am-GA@GEM exposed cells showed highly intense fluorescence, demonstrating significant increase in ROS generation arsing from the synergistic effects of the hydrogel matrix and the drug [50]. The quantitative analysis of ROS production after treatment was displayed in Fig. 6D. It was observed that, amongst all tested groups, the BSA-α-Am-GA@GEM hydrogel treated cells exhibits enhanced ROS generation due to the oxidative damage which leads to cell death. Thus, the fabricated hydrogel with controlled release of GEM disrupts mitochondrial function and activates oxidative signalling which enables increased ROS production. Overall, the fabricated BSA-α-Am-GA@GEM hydrogel elevates the drug transport and oxidative stress, leading to increased apoptosis and therapeutic efficiency in lung cancer cells [51].

3.11.4 BSA-α-Am-GA@GEM hydrogel induces mitochondrial depolarization in lung cancer cells

Mitochondrial membrane potential of A549 cancer cells after exposed to BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM was assessed by JC-1 labelling. As depicted in Fig. 7A, the untreated control cells show increased red fluorescence with minimal reveals mitochondrial integrity. Alternatively, the cells subjected to BSA-α treatment exhibits diminished red fluorescence and slightly increased green fluorescence suggesting modest mitochondrial depolarization. Similarly, Am-GA treated cells show increased mitochondrial depolarization, resulting in enhanced green fluorescence indicating polymer induced mitochondrial stress. Further, GEM treated cells exhibits increased mitochondrial dysfunction as displayed by a red to green fluorescence shift. However, the cells exposed to BSA-α-Am-GA@GEM hydrogel caused the maximum shift of red to green fluorescence indicating significant loss of mitochondrial membrane potential and increased mitochondrial damage [52]. As displayed in Fig. 7B, the quantitative analysis of red/green fluorescence demonstrating that, control cells showed very lessen ΔΨm loss (⁓5%), where, BSA-α and Am-GA treated cells shows moderate effect in ΔΨm loss as ⁓15% and ⁓20% respectively. On the other hand, GEM treated cells shows ⁓30% loss in mitochondrial membrane potential and the highest ΔΨm loss was observed in BSA-α-Am-GA@GEM hydrogel treated cells. These findings confirms that, the drug loaded hydrogel elevates the mitochondrial targeting of GEM through sustained released and improved cellular uptake which leads mitochondrial membrane depolarisation [53].

Fig. 7.

(A) JC-1 staining images demonstrate mitochondrial depolarisation. Green fluorescence shows membrane potential loss, while red fluorescence indicates undamaged mitochondria (size bar = 100 µm). (B) Quantitative measurement of JC-1 fluorescence ratio (red/green) shows considerable mitochondrial depolarisation in GEM and BSA-α-Am-GA@GEM groups. (C) DAPI staining images indicate nuclear condensation and fragmentation in treated cells. Scale bar = 100 µm. (D) Quantitative fluorescence intensity analysis of DAPI staining reveals that the BSA-α-Am-GA@GEM group has the most apoptotic nuclear damage

Nuclear morphological alterations revealed by DAPI staining

Figure 7C highlights the impact of BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM on nuclear morphology and chromatin integrity in A549 cells. The control group cells show even distributed nuclei with minimal fluorescence reveals intact nuclear structure and lessen chromatin condensation. The cells exposed to BSA-α and Am-GA causes slight elevation in fluorescence intensity demonstrating early nuclear stress, and also displays that polymer alone cane stimulate the apoptotic signaling. while treated cells. In GEM alone treated cells, efficient nuclear disruption with enhanced fluorescence intensity and increased chromatin condensation suggesting strong apoptotic induction. On the other hand, BSA-α-Am-GA@GEM hydrogel exposed cells displays considerable fragmentation and condensation as revealed by pronounces emission of fluorescence intensity compared to others [54]. In addition, the quantitative analysis indicated that, gradual increase in nuclear damage was observed in the order of cells treated with control, BSA-α, Am-GA, GEM and BSA-α-Am-GA@GEM (Fig. 7D). The results displayed that, the hydrogel with drug significantly triggers the cytotoxicity by enabling sustained drug release and improved intracellular drug delivery. Hence, the synergistic mechanism if hydrogel and drug promote increased nuclear disruption demonstrating the potential of hydrogel to activate the potential of conventional chemotherapy into more reliable and focused technique for lung cancer therapy [55].

Flow cytometry

The A549 lung cancer cells were treated with BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM and the apoptotic cell death was determined by Annexin V-FITC/PI dual labelling (Fig. 8A). As depicted in Fig. 8A, the control cells remain viable exhibiting negligible apoptosis and normal cellular homeostasis. Besides, the cells exposed to BSA-α and Am-GA displays slight increase in early apoptosis reveals bio macromolecular carriers gently trigger the cells for apoptosis without causing significant damage. In addition, free GEM considerably increased the apoptotic percentage due to its chemotherapeutic activity. However, their therapeutic effect was limited by rapid metabolism and poor intracellular retention. In the case of BSA-α-Am-GA@GEM hydrogel treated cells, significant enhancement in apoptosis was observed in both early and late stages. This effect is likely due to the synergistic action of pH-responsive BSA unfolding which stimulates drug release in the acidic environment and Am-GA facilitates drug retention and cellular uptake. As shown in Fig. 8B, BSA-α-Am-GA@GEM hydrogel exhibits significantly enhanced apoptosis compared to BSA-α, Am-GA and free GEM. In addition to this, BSA-α-Am-GA@GEM hydrogel stimulates protein unfolding and polysaccharide stabilization to promotes apoptosis. Hence, the dual mechanism enhances the therapeutic potential by reducing the limitation of GEM highlights that the fabricated hydrogel can be used as a auspicious platform for targeted drug delivery to lung cancer therapy [56].

Fig. 8.

(A) Flow cytometric analysis using Annexin V-FITC/PI staining in control, BSA-α, Am-GA, GEM and BSA-α-Am-GA@GEM treated cells, showing populations of live (lower left), early apoptotic (lower right), late apoptotic (upper right), and necrotic cells (upper left). (B) The GEM and BSA-α-Am-GA@GEM groups had the highest proportion of total apoptotic cells (early + late apoptosis), indicating increased cytotoxicity. The data represents the mean ± standard deviation (n = 3)

Cell uptake efficiency

In the present study, the cellular uptake of BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM in A549 lung cancer cells was evaluated by immunofluorescence microscopy and flow cytometry analysis at 30 min, 2 hrs and 24 hrs time period. The cells in control group exhibits very limited fluorescence at 30 min, 2 hrs and 24 hrs. Besides, the cells treated with BSA-α, Am-GA displays moderate signal and increased the fluorescence intensity with enhanced time (Figure S1 A-E). Further, GEM alone shows weak fluorescence and it was observed that, the cellular uptake was lower even after 24 hrs. Meanwhile, BSA-α-Am-GA@GEM hydrogel displayed better cellular uptake than free GEM. In comparison, BSA-α-Am-GA@GEM demonstrates the enhanced intracellular fluorescence. After treatment with BSA-α-Am-GA@GEM, detectable fluorescence was observed at 2 hrs and the fluorescence was increased upto 24 hrs treatment. Similar findings were observed in flow cytometry analysis. In this context, cells treated with BSA-α-AM-GA@GEM displays increased fluorescence than the cells treated with BSA-α, Am-GA and Gem alone at the tested time points. Thus, significant increase in fluorescence reveals that the drug loaded hydrogel easily enter into the cells and remained inside the cell for a longer period which may facilitates the apoptosis in lung cancer cells [57].

Wound healing

As shown in Fig. 9A–C, the cells treated with BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM started with similar scratch width point at 0 hrs, suggesting similar baseline conditions. After 24 hrs treatment, the untreated control cells display rapid cell migration which leads significant gap closure. In BSA-α and Am-GA treated groups, only moderate quantity of cells have migrated. In the case of GEM treated cells, it was noticed that, cell migration was delayed with wider scratch appeared compared to control, BSA-α and Am-GA treated cells. Contrarily, the cells incubated with BSA-α-Am-GA@GEM hydrogel efficiently inhibits the cell migration over the period of 24 hrs and leaving minimal gap reduction. When the incubation time was increased upto 48 hrs, the scratch or gap was ultimately closed in control group. BSA-α and Am-GA treated group express only partial closure of the scratch. Meanwhile, the GEM treated cells shows minimal cell migration with wide gap. Amongst all tested group, BSA-α-Am-GA@GEM treated cells showed reduced cell migration with wide gap reveals the anti-migratory potential of fabricated hydrogel. Furthermore, BSA-α-Am-GA@GEM hydrogel enhanced anti-migratory effect than free GEM highlighting its potential to suppress invasion and metastasis. In addition, as shown in Fig. 9A–C the quantitative measurement of cell migration at 0, 24 and 48 hrs confirmed even scratch width at 0 hrs. After 24 hrs and 48 hrs incubation, the cells treated with BSA-α and Am-GA showed only partial inhibition. The cells exposed to GEM shows slowed wound closure suggesting effective inhibition of cell migration. However, BSA-α-Am-GA@GEM hydrogel exhibits the least closure at 24 hrs demonstrating its significant anti-migratory effect. Similar effect was observed after 48 hrs incubation reflecting that, the drug loaded hydrogel had the strongest effect over all treatment groups demonstrating sustained suppression of both cell migration and proliferation. Thus, both qualitative and quantitative analysis demonstrating that BSA-α-Am-GA@GEM hydrogel effectively inhibits the cell migration and exhibits their potential as pH responsive hydrogel to suppress cancer cells invasion and metastasis [58].

Fig. 9.

(A) Representative wound healing images of treated cells at 0, 24, and 48 hrs. The yellow dashed lines indicate the wound edges. Scale bar = 200 μm. Quantitative analysis of cell migration rates at (B) 24 hrs and (C) 48 hrs. Data are presented as mean ± SD (n = 3). The BSA-α-Am-GA@GEM treatment markedly inhibited cell migration compared with the control and individual treatments

In vivo

In vivo antitumor efficacy and biosafety evaluation

The therapeutic anticancer activity and systemic safety of the modified hydrogel formulations were determined in tumour-bearing mice. Figure 10A, the control groups (PBS and BSA-α) exhibited rapid tumor growth over 18 days, reflecting unchecked proliferation. Treatment with Am-GA slightly slowed tumor growth, likely due to partial inhibitory effects, but offered minimal extended benefit. Free GEM reduced tumor growth more effectively than the controls, though the effect was short-lived, likely because of rapid clearance, short half-life, and non-specific distribution. By contrast, mice treated with BSA-α-Am-GA@GEM hydrogel consistently showed smaller tumors throughout the study. This strong effect is attributed to two key features: (i) sustained, controlled GEM release that prolongs local drug availability, and (ii) pH-responsive release in the acidic tumour microenvironment, maximizing local cytotoxicity while limiting systemic exposure. The hydrogel’s porous, swelling-capable structure supports GEM retention and slow release, giving longer-lasting therapeutic effects compared with the burst release of free GEM.

Fig. 10.

(A) Antitumor efficacy of several formulations (PBS, BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM) in tumor-bearing mice, exhibiting relative volume (V/V₀) during 18 days. BSA-α-Am-GA@GEM hydrogel significantly reduced tumour development compared to free GEM and control groups. (B) Body weight profiles of treated mice during the trial period, which show no substantial weight loss across all groups, indicating that the hydrogel formulation is biocompatible and has low systemic toxicity

Systemic safety was monitored via body weight changes (Fig. 10B). No significant weight loss or adverse effects were observed in any group, including the GEM-loaded hydrogel group, exhibiting high biocompatibility and low systemic toxicity. This confirms the hydrogel’s ability to reduce the dose-limited toxicities of free GEM by limiting early systemic exposure and targeting drug delivery to the tumour. Collectively, the BSA-α-Am-GA@GEM hydrogel achieved higher effectiveness than free GEM and polymer-only controls, offering targeted, sustained therapy with low systemic side effects. This result validates the hydrogel as a very promising platform for localized and sustained chemotherapeutic intervention in cancer therapy [59, 60].

Systemic toxicity evaluation

Systemic safety was analyzed by measuring hepatic (SCOT, ALP) and renal (serum urea, creatinine) biomarkers in different treatment groups (Fig. 11A–D). The control group had much higher levels, reflecting organ stress from tumour growth. The BSA-α and Am-GA groups exhibited only slight reductions, as observed from limited protective effects. Free GEM treatment contributed to moderate improvement, although biomarker levels remained elevated, consistent with its established systemic toxicity. The BSA-α-Am-GA@GEM hydrogel significantly reduced all measured indicators (p < 0.05), demonstrating effective protection of liver and kidney function. This improvement is likely due to the hydrogel’s dual-responsive design, thereby enabling localized, sustained GEM release in the tumour microenvironment while limiting systemic exposure. Moreover, these findings reveal that the hydrogel platform outperforms free GEM or single-component carriers in terms of biosafety [45, 61].

Fig. 11.

Serum biochemical analysis of systemic toxicity markers, including (A) SCOT (U/L), (B) ALP activity (U/L), (C) serum urea (g/mL), and (D) serum creatinine (g/mL) across different treatment groups (control, BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM). The BSA-α-Am-GA@GEM hydrogel group exhibited significantly lower liver (SCOT, ALP) and kidney (urea, creatinine) toxicity markers compared with free GEM and Am-GA groups, demonstrating enhanced systemic safety and reduced organ toxicity. Data are expressed as mean ± SD (n = 3), and statistical significance was determined using one-way ANOVA followed by post-hoc testing

Histopathological evaluation and biodistribution analysis

The safety and organ-specific distribution of the formulations were evaluated using histopathology and biodistribution studies (Fig. 12A). As shown in the control group, all major organs exhibited normal architecture and cellular morphology, confirming baseline tissue integrity. Treatment with BSA-α and Am-GA did not induce significant pathological alterations, evaluating their high biocompatibility. By contrast, animals treated with free GEM exhibited clear histological abnormalities, predominantly in the liver and spleen, due to rapid systemic distribution and dose-dependent toxicity. The BSA-α-Am-GA@GEM hydrogel-treated group exhibited minimal structural damage and preserved organ architecture, underscoring the carrier system’s protective role in mitigating GEM-associated systemic toxicity. Biodistribution data (Fig. 12B) clearly showed that the BSA-α-Am-GA@GEM hydrogel preferentially accumulated in the lungs, with much lower levels in the liver, kidneys, and spleen compared to free GEM, which distributed non-specifically. This targeted delivery is driven by pH-responsive BSA unfolding and the polysaccharide carrier design, enabling controlled GEM release at the tumor site while minimizing off-target toxicity. The BSA-α-Am-GA@GEM system offers both organ protection and lung-targeted administration, representing a substantial advancement over traditional chemotherapy [59].

Fig. 12.

(A) Representative H&E-stained histological images of heart, liver, kidney and spleen tissues from different treatment groups (control, BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM). Free GEM-treated mice exhibited noticeable structural alterations in major organs, while the BSA-α-Am-GA@GEM hydrogel showed preserved tissue integrity, indicating reduced systemic toxicity. (B) Quantitative biodistribution analysis of GEM across major organs, revealing preferential accumulation of the BSA-α-Am-GA@GEM hydrogel in lung tissue, thereby confirming its targeted delivery potential and minimized off-target deposition

Lung tissues were evaluated histologically to analyze therapeutic effects and tissue changes (Fig. 13A). The control group showed normal lung architecture with intact alveoli. The BSA-α and Am-GA groups also maintained near-normal tissue structure, indicating good biocompatibility. Whereas, lungs from free GEM-treated mice exhibited disrupted alveoli and focal necrosis, likely due to GEM’s cytotoxicity and limited targeted retention. The BSA-α-Am-GA@GEM hydrogel group exhibited pronounced structural changes, such as condensed nuclei, thickened alveolar walls, and tissue necrosis, reflecting effective tumour cell elimination. Histopathological scoring (Fig. 13B) was observed significantly greater tumour-associated alterations in the hydrogel group compared to free GEM and other controls. The BSA-α-Am-GA@GEM system effectively suppresses tumours in the lungs while maintaining systemic biocompatibility and providing maximum therapeutic benefit [59, 60].

Fig. 13.

(A) H&E-stained sections at 10x and 20x magnifications exhibit tissue morphology across groups: control, BSA-α, Am-GA, free GEM, and BSA-α-Am-GA@GEM. Control lungs had normal architecture, while treatment with BSA-α and Am-GA resulted in little change. The BSA-α-Am-GA@GEM group showed significant tumour cell destruction and necrotic areas, indicating its therapeutic efficacy compared to free GEM, which produced tissue disruption. (B) The BSA-α-Am-GA@GEM group showed much more tissue damage than other treatments, indicating improved anticancer activity

Quantitative real-time PCR (qRT-PCR) analysis

To investigate the molecular effects of the developed formulations, qRT-PCR was used to evaluate the relative mRNA expression levels of Cyclin D, PCNA, CDK6, and BCL2, using GAPDH as the internal reference gene. Fold changes were calculated with the 2^{^-ΔΔCt} method. The BSA-α-Am-GA@GEM hydrogel group showed significant overexpression of Cyclin D, PCNA, CDK6, and BCL2 compared to the control, BSA-α, Am-GA, and free GEM groups (p < 0.05). Cyclin D and CDK6, which are key regulators of the G1/S transition in the cell cycle, were significantly enhanced, indicating that the hydrogel formulation considerably stimulates cell cycle advancement. PCNA, a marker of DNA replication and repair, was significantly higher in the hydrogel-treated group, indicating enhanced cell proliferation. The BSA-α-Am-GA@GEM group also had higher expression of the anti-apoptotic gene BCL2, suggesting activation of survival pathways that help control tumour growth. In comparison, the individual carriers (BSA-α and Am-GA) showed reduced expression of these genes, reflecting their limited therapeutic effect. Free GEM exhibited moderate regulation of target genes but was much less effective than hydrogel delivery. The BSA-α-Am-GA@GEM hydrogel, with its synergistic polymer-drug interactions and sustained release, depicted superior therapeutic effectiveness. The BSA-α-Am-GA@GEM hydrogel enriched expression of proliferation genes (Cyclin D, PCNA, CDK6) and influenced apoptosis resistance (BCL2), suggesting its potential as an advanced cancer therapy (Fig. 14 A-D). Results are shown as mean ± SD (n = 3), with statistical significance determined by one-way ANOVA tests [62]. To further benchmark the performance of the developed BSA-α-Am-GA@GEM system, a comparative analysis with previously reported polymeric and albumin-based nanoparticle platforms was conducted, as summarized in Table 3.

Fig. 14.

Relative mRNA expression of (A) Cyclin D, (B) PCNA, (C) CDK6, and (D) BCL-2 in different treatment groups (control, BSA-α, Am-GA, GEM, and BSA-α-Am-GA@GEM). The hydrogel-treated group (BSA-α-Am-GA@GEM) exhibited significantly higher expression of proli .3feration- and survival-associated genes compared to free GEM and other control groups, demonstrating its enhanced therapeutic potential. Data are expressed as mean ± SD (n = 3)

Table 3.

Comparison table

Author/Year	Nanoparticle System	Size (nm)	Encapsulation Efficiency (%)	Drug Loading (%)	IC50/Cytotoxicity	Blood Circulation Time	Tumor Type	Chemotherapy Drug	Reference
Akanksha Behl et al., 2022	PEG-PCL	128.66 ± 23	85.4	—	MCF-7: 22 nM	—	Breast	Gemcitabine	[63]
Hongdan Shen et al., 2021	mPEG-b-PCL-DOX	300	—	4	HCT116: 2.65 ± 0.29 μg/mL	—	Colorectal	Doxorubicin	[64]
Nasrullah Jan et al., 2021	Cytarabine-PCL	120.5 ± 1.18–341.5 ± 3.02	41.31 ± 0.49–62.28 ± 0.39	6–20	KG1 cells, 8.80 ± 0.48 μg/mL		Leukaemia/Breast	Cytarabine	[65]
Wufa Fan et al., 2022	OPDEA-PCL	45.4	—	7.3	NCI-H520: 2.52/0.62 μg/mL (CDDP/PTX)	> 24 h	Liver/Lung	CDDP/PTX	[66]
This work/2025	BSAα-AmGA@GEM	120–180	85–90 (example)	10–12	A549 cells: 8.5 μM	Stable in circulation	Lung	Gemcitabine	Present study

Comparative summary of previously reported polymeric and albumin-based nanoparticle systems for intravenous chemotherapy delivery, highlighting particle size, encapsulation efficiency, drug loading, in vitro cytotoxicity, blood circulation behaviour, tumour model, and chemotherapeutic agent, in comparison with the present BSA-α-Am-GA@GEM pH-responsive nanocarrier system

Conclusion

In summary, we have presented a new type of protein-polysaccharide nanocarrier by integrating reductively unfolded BSA-α and aminated gum acacia (Am-GA) to achieve efficient GEM delivery for lung cancer treatment. The controlled unfolding of BSA-α exposed thiol and amine groups, enhancing binding and conjugation. Functionalized Am-GA improved biocompatibility and pH responsiveness, allowing synergistic self-assembly and efficient GEM encapsulation via electrostatic and hydrogen-bond interactions. Combining a protein scaffold with a modified natural polysaccharide fabricated a highly stable, biocompatible, and stimuli-responsive nanoplatform that is novel. This is the first study to demonstrate that a BSA-α-Am-GA hybrid nanocarrier can efficiently encapsulate GEM, improve its water solubility, and release it selectively in the acidic tumour environment. Experiments in cells and animals confirmed its better performance, including higher cytotoxicity, increased ROS-driven apoptosis, effective tumour shrinkage, and reduced side effects compared with free GEM or single-component carriers. This study highlights the potential of BSA-α-Am-GA as an intelligent, naturally derived, and biocompatible material platform. Finally, the results suggest to its real potential for clinical translational promise as a next-generation nanoplatform for safe, precise and effective chemotherapeutic intervention in lung cancer treatment.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Y.L. and P.Z. contributed equally to this work and are co-first authors. Y.L. and P.Z. designed the study, performed the experiments, analyzed the data, and drafted the initial manuscript. Y.M. contributed to study conception, provided critical methodological guidance, and revised the manuscript for important intellectual content. C.J. supervised the overall study, coordinated the research activities, and finalized the manuscript. All authors read and approved the final manuscript.

Funding

No Funding was received.

Data availability

Data will be made available on request. All the data generated or analysed during the study are included in this published article.

Declarations

Ethical approval

This study was approved by Ethics committee of Shanxi Province Cancer Hospital/Shanxi Hospital Affiliated to Cancer Hospital (Approval Number: 202492).

Consent to publish

The approval of each author to publish the manuscript has been obtained. The manuscript is not currently under review by any other journal and has not been published before.

Competing interests

The authors declare no competing interests.