Hyaluronic acid- bovine serum albumin coated nanostructured lipid carriers for targeted combinational delivery of fisetin and flavokawain A in non–small cell lung cancer

Abstract

Lung cancer remains the leading cause of cancer-related mortality worldwide, accounting for approximately 2.2 million new cases and 1.8 million deaths annually. Non–small cell lung cancer (NSCLC) constitutes nearly 85% of all cases, underscoring its substantial global burden. This study reports the development of hyaluronic acid–bovine serum albumin (HA–BSA)-coated nanostructured lipid carriers (NLCs) for targeted combinational delivery of fisetin and flavokawain A in NSCLC using a Quality-by-Design (QbD) approach. The optimized formulation demonstrated a particle size of ~ 115 nm with a low polydispersity index (< 0.3) and high zeta potential (~–40.8 mV), indicating excellent colloidal stability. Drug entrapment efficiency reached ~ 85.5–89.5%, while cumulative drug release exhibited a sustained profile (~ 90–93.8% over 9 h). Solid-state characterization confirmed reduced crystallinity and successful encapsulation. Stability studies showed negligible changes in particle size (162.5 to 165.1 nm) and drug content (> 98%) under refrigerated conditions over 6 months. In vitro cytotoxicity studies revealed significantly enhanced anticancer activity, with the formulation achieving up to ~ 70.32% inhibition of A549 cells compared to lower activity of free drug. Statistical optimization confirmed model significance (p = 0.0385) and good reproducibility (CV < 3%). Overall, the developed HA–BSA-coated NLCs offer a robust, stable, and efficient nanoplatform for improved flavonoid delivery in NSCLC therapy.

Graphical abstract

Introduction

Lung cancer represents the leading cause of cancer-related mortality worldwide, with non–small cell lung cancer (NSCLC) comprising 80–85% of cases. Chemotherapy remains the standard treatment for metastatic cancer; however, it presents several challenges, such as severe side effects, rapid clearance from the body, targeting difficulties, and occasionally restricted dosages due to toxicity. Multidrug resistance (MDR) in NSCLC poses a significant challenge as it reduces the intracellular concentration of anticancer drugs [1]. The mediation of drug resistance frequently arises from the overexpression of specific proteins, such as P-glycoprotein (P-gp), which expel drugs from cancer cells. These challenges underscore the urgent necessity to enhance drug delivery systems to improve drug retention within cells and achieve more precise targeting of tumors [2].

Flavonoids such as Fisetin and Flavokawain A have been extensively studied due to their diverse biological activities [3]. Polyphenolic compounds, which are naturally occurring and closely related to flavonoids, possess the ability to inhibit cellular growth. Furthermore, certain flavonoids can inhibit P-glycoprotein (P-gp), thereby enhancing the sensitivity of cancer cells to chemotherapeutic agents. Despite their significant pharmacological potential, flavonoids encounter challenges such as poor water solubility, low oral bioavailability, rapid metabolic degradation, and physicochemical instability. To address these issues, various nanocarrier systems, including polymeric nanoparticles, liposomes, and micellar systems, have been investigated [4]. Despite their potential, practical applications are often hindered by challenges such as drug leakage, insufficient drug-loading capacity, difficulties in scaling up, and inconsistencies between batches. Consequently, there is an urgent demand for a nanocarrier system that not only enhances flavonoid delivery but also remains stable, reproducible, and scalable, all while meeting regulatory standards [5].

Lipid-based nanocarriers have become significant instruments for drug solubilization. Solid lipid nanoparticles (SLNs), considered the first generation of lipid carriers, consist of a solid lipid matrix stabilized by surfactants [6]. Self-assembled nanocarriers (SLNs) are systematically organized particles that provide the advantage of biocompatibility and controlled release. Nevertheless, their highly ordered crystalline structure restricts their drug-loading capacity [7]. Additionally, they experience drug expulsion during storage. To address these challenges, nanostructured lipid carriers (NLCs) have been developed as a next-generation lipid nanocarrier. NLCs incorporate both solid and liquid lipids, leading to a more imperfect and less-ordered crystalline structure. This alteration allows for increased space for drug accommodation, thereby reducing the likelihood of drug expulsion and enhancing both efficiency and long-term stability compared to conventional solid lipid nanoparticles (SLNs) [8]. Nanostructured lipid carriers represent significant advancements in drug delivery systems, employing various mechanisms to improve therapeutic outcomes. Their nanoscale dimensions enhance the enhanced permeability and retention (EPR) effect, thereby increasing drug accumulation in tumors. The lipid composition of these carriers facilitates interactions with cellular membranes, promoting endocytic uptake. Additionally, surface functionalization can enable targeted delivery. Hyaluronic acid (HA), a naturally occurring polysaccharide, exhibits affinity for the CD44 receptor, which is often overexpressed in non-small cell lung cancer (NSCLC) cells and cancer stem-like populations [9]. Nanoparticles containing HA were internalized via CD44-mediated endocytosis, thereby enhancing intracellular drug delivery and reducing off-target effects. Concurrently, BSA, serving as a biocompatible stabilizing agent, inhibits nonspecific protein adsorption, enhances systemic circulation, and improves colloidal stability [10]. The dual-functional platform, developed through receptor-mediated targeting and steric stabilization by HA and BSA, may represent an effective strategy for targeted drug delivery [11].

Lipid-based nanocarriers demonstrate considerable efficacy. However, challenges persist due to formulation variability and issues related to scalability. The development of these carriers according to quality-by-design (QbD) principles represents a systematic and science-based approach to pharmaceutical development. This approach involves the establishment of predetermined quality target product profiles (QTPPs), the identification of critical quality attributes (CQAs), and the control of critical material and process parameters [18–20]. Incorporating Quality by Design (QbD) principles in the development of nanocarriers is expected to improve the rational optimization, reproducibility, and regulatory compliance of these systems, thereby increasing their likelihood of successful translation [12].

This study aims to develop nanostructured lipid carriers, optimized through Quality by Design (QbD) principles and coated with hyaluronic acid–bovine serum albumin (HA–BSA), to enable the dual delivery of fisetin and flavokawain A for the treatment of non-small cell lung cancer (NSCLC) [13] This study sought to develop a stable and scalable nanoplatform through the application of lipid nanoengineering, receptor-mediated targeting, and systematic process optimization. The objective was to improve intracellular drug retention and enhance therapeutic efficacy in lung cancer treatment, with a future focus on oral drug delivery [12].

Materials and methods

Materials

Fisetin and flavokavain A got from Yucca Enterprises, Mumbai. Paclitaxel(Control & comparative anticancer drug) the inclusion of paclitaxel as a positive control and reference standard during comparative characterization studies, Liquid Lipid & Solid Lipid, Hyaluronic acid, Bovine Serum Albumin Bought from Sigma Aldrich, India.

The A549 cell line was obtained from the National Centre for Cell Science (NCCS), Pune. We cultured the cells in minimal essential medium supplemented with 10% fetal bovine serum (FBS), penicillin at 100 U/ml and streptomycin at 100 µg/ml. The cultivation environment was maintained in a humidified atmosphere containing 50 µg/ml CO2 at 37 °C.

Quality-by-design (QbD)–determined construction strategy

A systematic framework based on the concept of quality by design has been employed for the design and optimization of HA–BSA coated nanostructured lipid carriers. To begin, the Quality Target Product Profile (QTPP) was defined for nanoscale particle size, high drug entrapment efficiency, sustained drug release, physical stability, and suitability for targeted lung cancer treatment. The particle size, polydispersity index (PDI), zeta potential, drug entrapment efficiency (EE%), and in vitro drug release behavior are identified as Critical Quality Attributes (CQAs) [14]. Based on the risk assessment and previous formulation knowledge, lipid concentration, surfactant concentration and drug-to-lipid ratio have been chosen as Critical Material Attributes (CMAs) [15]. The pressure of homogenization and the number of homogenization cycles are Critical Process Parameters (CPPs) having a substantial impact on nanoparticle size and uniformity. The Design of Experiments (DoE) was performed by means of a factorial or central composite design to evaluate how Critical Material Attributes (CMAs) and Critical Process Parameters (CPPs) affect the nominated Critical Quality Attributes (CQAs). The statistical analyses for the experimental data were performed to determine the respective main effect, interaction effect, and quadratics responses. The optimization criteria were set to achieve a minimum particle size, narrow size distribution, maximum drug entrapment efficiency, and sustained drug release. The formulation that was deemed suitable was selected making use of desirability function analysis.

Preparation of HA–BSA coated nanostructured lipid carriers

Nanostructured lipid carriers were synthesized using a high-pressure homogenization technique, a method recognized for its scalability and acceptance in the industry for lipid nanoparticle production [16]. In summary, the selected solid and liquid lipids were melted above their respective melting points and combined with flavonoids under continuous stirring to form the lipid phase. The aqueous phase containing surfactants was heated to the same temperature and subsequently integrated into the lipid phase to form a coarse pre-emulsion. This pre-emulsion underwent high-pressure homogenization at an optimized pressure and cycle number to yield nano-sized lipid carriers. The resulting dispersion was then cooled to room temperature, allowing recrystallization of the lipid matrix and establishment of stable NLCs. BSA was adsorbed onto the surface of NLCs through electrostatic and hydrophobic interactions to enhance colloidal stability and biocompatibility. Subsequently, hyaluronic acid was adsorbed onto the BSA-coated NLCs to facilitate CD44-mediated active targeting [9]. Excess unbound BSA and HA were removed by centrifugation or dialysis. The final HA–BSA coated NLCs were stored under refrigerated conditions until further characterization and evaluation [17].

The optimized free and loaded NLCs were prepared by a modified high-pressure homogenization (HPH). Integrated with a Design of Experiments (DoE) approach to ensure systematic and efficient formulation development. Briefly, mention in Table 1 material methods.

Table 1.

Composition of Lipid Phase and Aqueous Phase of HA-BSA Coated NLCs Fisetin & Flavokawain A

Ingredient	F1	F2	F3	F4	F5	F6	F7	F8	F9
Lipid Phase
Fisetin	2 mg	2 mg	2 mg	2 mg	2 mg	2 mg	2 mg	2 mg	2 mg
Flavokawain-A	3 mg	3 mg	3 mg	3 mg	3 mg	3 mg	3 mg	3 mg	3 mg
Precirol ATO 5	200 mg	250 mg	300 mg	350 mg	400 mg	250 mg	300 mg	350 mg	300 mg
Oleic acid	100 mg	150 mg	200 mg	250 mg	300 mg	150 mg	200 mg	250 mg	200 mg
Soya lecithin	20 mg	20 mg	25 mg	25 mg	25 mg	20 mg	25 mg	25 mg	25 mg
Aqueous phase
Tween 80	0.5%	0.75%	1.0%	1.25%	1.5%	0.75%	1.0%	1.25%	1.0%
BSA (Bovine Serum Albumin)	1 mg/mL	1.5 mg/mL	2 mg/mL	2.5 mg/mL	3 mg/mL	1.5 mg/mL	2 mg/mL	2.5 mg/mL	2 mg/mL
Hyaluronic Acid (HA)	0.25 mg/mL	0.5 mg/mL	0.75 mg/mL	1.0 mg/mL	1.25 mg/mL	0.5 mg/mL	0.75 mg/mL	1.0 mg/mL	0.75 mg/mL
Distilled Water(q.s.)	Up to 10 mL	Up to 10 mL	Up to 10 mL	Up to 10 mL	Up to 10 mL	Up to 10 mL	Up to 10 mL	Up to 10 mL	Up to 10 mL
Tween 80	0.5%	0.75%	1.0%	1.25%	1.5%	0.75%	1.0%	1.25%	1.0%

Physicochemical characterization

A comprehensive evaluation was conducted on the physicochemical properties of both uncoated and HA–BSA-coated nanostructured lipid carriers, focusing on the particle size, surface charge, drug loading, solid-state characteristics, and in vitro drug release behavior. These parameters are essential for determining the stability, cellular uptake, and therapeutic efficacy of the nanoparticles [18].

Particle size, polydispersity index, and zeta potential

Mean particle size, polydispersity index (PDI), and zeta potential were assessed using dynamic light scattering (DLS) and electrophoretic light scattering methodologies. The samples were suitably diluted with deionized water to mitigate multiple scattering effects and were analyzed at ambient temperature. The particle size distribution was represented as the Z-average diameter, whereas PDI served as an indicator of size uniformity. Zeta potential measurements were conducted to assess the surface charge and colloidal stability both prior to and following surface functionalization [18].

Drug entrapment efficiency and drug loading

The efficiency of drug entrapment (EE%) and the extent of drug loading were evaluated through an indirect approach. Nanostructured lipid carrier (NLC) dispersions were subjected to high-speed centrifugation to effectively separate the free drug from that associated with the nanoparticles. The concentration of unentrapped flavonoids in the supernatant was determined using a validated analytical method. Subsequently, the EE% and drug loading were calculated based on the initial drug content and quantity of drug encapsulated within the nanocarriers.

Fourier transform infrared spectroscopy (FTIR)

Fourier transform infrared spectroscopy (FTIR) was used to assess the potential chemical interactions among flavonoids, lipid excipients, and surface modifiers. The FTIR spectra of the pure drugs, individual excipients, physical mixtures, and optimized formulations were meticulously recorded across a suitable wavenumber range. The retention of the characteristic functional group peaks served as evidence of compatibility and the absence of chemical degradation throughout the formulation development process.

Differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC) was employed to investigate the thermal characteristics and physical state of the flavonoids within the lipid matrix. Thermograms of the pure drugs, lipids, physical mixtures, and optimized formulations were obtained under controlled heating conditions. The analysis of changes in melting endotherms facilitated the evaluation of drug encapsulation, reduction in crystallinity, and amorphous dispersion within nanostructured lipid carriers [19].

Powder X-ray diffraction (PXRD)

Powder X-ray diffraction (PXRD) analysis was conducted to further investigate the crystalline or amorphous characteristics of the flavonoids in the NLC formulations. Diffractograms were obtained over a specified 2θ range. A reduction or absence of characteristic crystalline peaks was interpreted as indicative of successful drug encapsulation and diminished crystallinity within the lipid matrix [20].

In-vitro drug release studies

In vitro drug release studies were conducted utilizing a dialysis membrane diffusion technique to assess flavonoid release from NLCs [21]. An appropriate release medium was selected to maintain sink conditions throughout the experiment. Samples were collected at predetermined intervals and replaced with fresh medium. Drug content was quantified using a validated analytical method, and the release profiles of the free drug and HA–BSA-coated NLCs were compared to evaluate sustained release behavior [22].

Results & conclusion

Physicochemical characterization: preformulation studies

Preformulation study appears to be a crucial component in the appropriate formulation of pharmaceutical dosage forms for the medication. Preformulation studies are primarily conducted to generate relevant data for the development of stable, compliant, and effective dosage forms.

The organoleptic properties of fisetin, flavokawain A, were evaluated using standard analytical methods to confirm their purity and identity. The assessment included parameters such as color, odor, physical appearance, and melting point. Fisetin was observed as a yellow to pale yellow, odorless, crystalline powder with a melting point in the range of 314–316 °C, which is consistent with reported standard values [23]. flavokawain A appeared as a yellow, odorless, crystalline powder and exhibited a melting point between 154 and 156 °C, confirming its purity and characteristic properties. All observed parameters were found to be within the acceptable range as per literature values, indicating that the drugs used in the study were in their pure and unadulterated form [24].

The solubility studies revealed that fisetin, flavokawain A are practically insoluble in water, indicating their hydrophobic nature. However, drugs showed good solubility in organic solvents such as methanol and ethanol, and were freely soluble in dimethyl sulfoxide (DMSO), while exhibiting variable solubility in chloroform. These findings are essential for selecting suitable solvents during the formulation of nanostructured lipid carriers (NLCs). Preliminary Liquid Lipid screening studies were conducted to select suitable formulation components based on drug solubility and compatibility. Among the solid lipids evaluated, Precirol ATO 5 exhibited the highest solubility for all three drugs, making it the most suitable lipid for NLC formulation. Surfactant screening revealed that Tween 80 provided the highest solubilization capacity, followed by Tween 20 and Poloxamer 188, while Span 80, despite lower solubility, was selected to achieve the required hydrophilic–lipophilic balance (HLB) and improve formulation stability. In oil screening, oleic acid demonstrated superior drug solubility compared to other oils, such as Caproyl 90 and Labrafac PG. Overall, the screening results confirm that the combination of Precirol ATO 5 (lipid), Tween 80 and Span 80 (surfactants), and oleic acid (liquid lipid) is optimal for developing stable and efficient HA–BSA coated NLCs [25].

Preliminary Solid lipid screening was conducted to identify suitable solid lipids, surfactants, and formulation parameters for the development of nanostructured lipid carriers. Among the solid lipids evaluated, Precirol ATO 5 exhibited the highest solubility for all three drugs, with values of 8.5 ± 0.3 mg/mL (fisetin), 9.2 ± 0.4 mg/mL (flavokawain A), compared to Compritol 888 ATO, glyceryl monostearate, and stearic acid, which showed comparatively lower solubility. This superior solubilizing capacity makes Precirol ATO 5 the most suitable lipid for formulation development [26]. Similarly, surfactant screening revealed that Tween 80 exhibited the highest drug solubility (15.6–18.2 mg/mL), followed by Tween 20 and Poloxamer 188, while Span 80 showed lower solubility values (2.8–3.6 mg/mL). Despite its lower solubility, Span 80 was selected as a stabilizer to achieve the required hydrophilic–lipophilic balance (HLB) and enhance formulation stability. In oil screening, oleic acid demonstrated the highest solubility (12.4–13.1 mg/mL for fisetin and flavokawain A, and 9.6 mg/mL for paclitaxel), followed by Capryol 90 and Labrafac PG, whereas coconut oil showed minimal solubility [27]. Overall, the screening study confirms that Precirol ATO 5, Tween 80, Span 80, and oleic acid are the most suitable components for NLC formulation due to their superior drug solubilization and stabilization properties [28].

Pre-formulation analysis confirmed that both drugs are crystalline in nature and remain stable under dry conditions without visible degradation. Due to their poor aqueous solubility and limited bioavailability, particularly in the case of fisetin and paclitaxel, an appropriate formulation strategy is required to enhance their solubility and therapeutic effectiveness. Overall, these characteristics justify the need for advanced drug delivery systems to improve their performance and efficacy.

QbD optimization and design space

A Quality-by-Design (QbD) strategy was applied to systematically optimize formulation variables governing the performance of HA–BSA coated nanostructured lipid carriers. Based on preliminary risk assessment and prior reports on lipid-based nanocarriers, the concentrations of Precirol ATO 5 (X₁) and Tween 80 (X₂) were identified as critical material attributes, while particle size and drug entrapment efficiency were selected as critical quality attributes due to their direct impact on formulation stability, drug loading, and cellular uptake. The result of the main composite design was statistically evaluated with the aid of Design Expert software. Precirol ATO 5 (X1) and Tween 80 (X2) were investigated as independent variables, and their effects on Entrapment Efficiency and Drug Release the effectiveness of entrapment were examined as dependent variables. The 3^2factorial result in 9 runs [29].

Optimization of the variables (fisetin with flavokawain A)

The response surface, contour, perturbation, and normal probability plots collectively demonstrate the effect of formulation variables, namely Precirol ATO 5 (X₁) and Tween 80 (X₂), on entrapment efficiency (Y₁) and drug release (Y₂) of fisetin and flavokawain A loaded HA–BSA coated nanostructured lipid carriers (NLCs) (Fig. 1). The response surface and contour plots for entrapment efficiency indicate a positive relationship with both variables, showing that increasing the concentration of lipid and surfactant enhances drug encapsulation. This behavior can be attributed to improved solubilization of fisetin and flavokawain A within the lipid matrix and stabilization of the nanocarrier system. The formulation table shows 3² full factorial design comprising nine experimental runs further supports this observation, where batch F4 exhibited the highest entrapment efficiency (85.5%) at optimized concentrations of Precirol ATO 5 and Tween 80. The perturbation plot confirms that both variables significantly influence entrapment efficiency, while the normal probability plot indicates that the residuals follow a normal distribution, confirming the adequacy of the model. The Table 2 illustrates the outcome of different formulations for %EE and % drug release.

Fig. 1.

3^2 full factorial design was employed to optimize the formulation of Fisetin and Flavokawain a loaded nanostructured lipid carrier (NLCs)( 3² full factorial design comprising nine experimental runs)

Table 2.

Formulation table with responses (Fisetin & Flavokawain A)

Formulation Code	Precirol ATO 5% w/v X1	Tween 80, % w/v X2	Entrapment Efficiency (%) (Y1)	Drug Release (%) (Y2)
F1	803.553	1.0	78.32	88.32
F2	450	1.70711	79.1	86.68
F3	96.4466	1.0	80.07	85.39
F4	200	1.5	85.5	93.80
F5	200	0.5	66.86	92.01
F6	450	0.292893	82.1	83.2
F7	450	1.0	80.4	83.45
F8	700	0.5	74.68	89.24
F9	200	1.5	70.3	91.49

In contrast, the response surface and contour plots for drug release reveal an inverse relationship with formulation variables, indicating that increasing concentrations of Precirol ATO 5 and Tween 80 lead to a reduction in drug release. This suggests the formation of a dense lipid matrix, which restricts the diffusion of fisetin and flavokawain A and promotes sustained release behavior. The ANOVA results confirm that the model is statistically significant (F = 5.88, p = 0.0385), with Tween 80 showing a more significant effect (p = 0.0140) compared to Precirol ATO 5. The R² value (0.6622) and adequate precision value (5.0431) indicate acceptable model predictability and reliability. Additionally, the low coefficient of variation (2.89%) confirms good reproducibility of the experimental data.

The overlay plot clearly identifies the optimized design space, where both responses meet the desired criteria of maximum entrapment efficiency and controlled drug release. Based on the optimization criteria (Precirol ATO 5: 200–400 mg; Tween 80: 50–150 mg), the optimized formulation (F4) was selected as the best formulation, exhibiting high entrapment efficiency (~ 85.5%) and sustained drug release (~ 93.8%). Overall, the results demonstrate that the QbD-based optimization approach successfully developed a stable and efficient HA–BSA coated NLC system for the combinational delivery of fisetin and flavokawain A, ensuring enhanced drug loading, controlled release, and improved therapeutic potential [30, 31].

Particle size, polydispersity index, and zeta potential, entrapment efficiency

The particle size, polydispersity index (PDI), and zeta potential of HA–BSA coated nanostructured lipid carriers (NLCs) were evaluated to assess their suitability for targeted drug delivery applications. Dynamic light scattering analysis confirmed that all formulations were within the nanometric range, indicating successful formulation of nanosized carriers.

The fisetin + flavokawain A loaded system showed particle sizes in the range of 115 to 325 nm, with batch F4 demonstrating the smallest particle size of ~ 115 nm (Fig. 2A). These nanoscale dimensions are considered optimal for enhanced tumor targeting via the enhanced permeability and retention (EPR) effect, as well as for improved cellular internalization. The particle size distribution curves obtained from intensity-based measurements displayed narrow and well-defined peaks, indicating uniform size distribution and minimal aggregation. The PDI values for all formulations were within acceptable limits (< 0.3), confirming homogeneity and good dispersion stability of the nanocarriers.

Fig. 2.

A- Particle Size & (B)- Zeta Potential of Formulated Batches

Surface functionalization with bovine serum albumin (BSA) followed by hyaluronic acid (HA) resulted in a slight increase in particle size compared to uncoated systems, which confirms successful coating of the nanoparticle surface. Similar observations have been reported for protein- and polysaccharide-coated nanocarriers, where surface adsorption contributes to an increase in hydrodynamic diameter without compromising nanoscale characteristics.

Zeta potential analysis revealed that all formulations possessed a negative surface charge, which was attributed to the presence of ionizable groups in HA and lipid components (Fig. 2B). The optimized fisetin-loaded formulation (F4) showed a higher negative value of − 40.8 mV. These values indicate strong electrostatic repulsion between particles, thereby preventing aggregation and ensuring good colloidal stability.

Additionally, entrapment efficiency was found to be influenced by formulation composition, fisetin-based system demonstrated slightly higher entrapment efficiency of 89.5%. This suggests better compatibility of flavonoids with the lipid matrix, contributing to improved drug loading. Overall, the results demonstrate that HA–BSA coated NLCs possess desirable physicochemical properties, including nanoscale particle size, narrow size distribution, adequate surface charge, and high drug entrapment [32].

Solid-state characterization

Fourier transform infrared spectroscopy

FTIR analysis of the optimized HA–BSA-coated NLCs demonstrated retention of the characteristic functional group peaks of flavonoids, with no emergence of new peaks or significant shifts. This observation confirms the chemical compatibility between flavonoids, lipid excipients, and surface modifiers, indicating that drug degradation did not occur during the formulation process [33].

The FTIR spectrum of fisetin (Fig. 3A) exhibits characteristic peaks confirming its polyphenolic structure. Broad O–H stretching bands (3680–3518 cm⁻¹) indicate phenolic hydroxyl groups, while peaks at 3347–3248 cm⁻¹ correspond to hydrogen-bonded O–H/N–H vibrations. The strong carbonyl (C = O) stretching at 1740 cm⁻¹ and aromatic C = C bands at 1613–1593 cm⁻¹ confirm its flavonoid backbone. Additional C–O and C–O–C stretching vibrations further validate the presence of phenolic and ether functionalities, confirming the structural integrity and purity. The FTIR spectrum of flavokawain A (Fig. 3B) shows distinct peaks corresponding to its chalcone structure. Broad O–H stretching bands (3899–3509 cm⁻¹) indicate hydroxyl functionalities, while peaks at 2925 and 2857 cm⁻¹ confirm aliphatic C–H stretching. A strong carbonyl (C = O) peak at 1738 cm⁻¹ and aromatic C = C stretching at 1559–1458 cm⁻¹ validates the conjugated system. Additional bands for C–O and C–N vibrations confirm the presence of functional groups essential for its biological activity. The FTIR spectrum of paclitaxel (Fig. 3C) demonstrates multiple characteristic functional groups. Broad O–H/N–H stretching bands (3576–3206 cm⁻¹) indicate hydrogen bonding, while aromatic C–H stretching appears at 3069 cm⁻¹. The prominent carbonyl (C = O) peak at 1710 cm⁻¹ confirms ester functionality. Aromatic C = C vibrations at 1644 and 1535 cm⁻¹, along with C–O stretching bands, validate its complex diterpenoid structure. These peaks confirm the chemical identity and structural integrity of paclitaxel. The FTIR spectrum of the fisetin–flavokawain A mixture (Fig. 3D) retains all major characteristic peaks of both drugs without significant shifts. Broad O–H/N–H stretching bands (3908–3249 cm⁻¹), carbonyl stretching at ~ 1739 cm⁻¹, and aromatic C = C vibrations confirm structural preservation. The absence of new peaks or peak disappearance indicates no chemical interaction between the components. This suggests good compatibility and stability of the drug combination, supporting its suitability for formulation development [34].

Fig. 3.

FTIR spectra of (A)-Fisetin, (B)- Flavokawain A and (C)- Paclitaxel, (D)- Drug Mixture of Fisetin and Flavokawain A

The FTIR spectrum of paclitaxel and flavokawain A mixture (Fig. 4A) shows retention of key functional group peaks from both compounds. Characteristic O–H/N–H stretching (3882–3311 cm⁻¹), carbonyl stretching at ~ 1735 cm⁻¹, and aromatic C = C bands confirm structural integrity. No significant peak shifts or new peaks are observed, indicating absence of chemical interaction. This confirms compatibility between the drugs, suggesting that their combined incorporation into nanocarriers will not compromise stability. The FTIR spectrum of bovine serum albumin (BSA) (Fig. 4B) displays characteristic amide bands confirming its protein structure. The broad Amide A band (3300–3500 cm⁻¹) corresponds to N–H stretching, while Amide I (1650–1630 cm⁻¹) and Amide II (1540–1520 cm⁻¹) represent C = O stretching and N–H bending, respectively. Additional peaks for C–N and carboxylate groups further confirm protein integrity. These features validate the structural stability of BSA used for nanoparticle surface modification. The FTIR spectrum of hyaluronic acid (Fig. 4C) shows characteristic polysaccharide peaks. Broad O–H stretching (3400–3300 cm⁻¹) indicates extensive hydrogen bonding. The presence of carboxylate groups is confirmed by peaks at 1620–1600 cm⁻¹, while amide II bands at 1550–1530 cm⁻¹ reflect N–H vibrations. Glycosidic linkages (C–O–C) and alcohol groups are evident from peaks in the 1150–1020 cm⁻¹ range. These features confirm the structural integrity of HA for targeted drug delivery applications. The FTIR spectrum of Precirol ATO 5 (Fig. 4D) exhibits characteristic lipid peaks. Strong aliphatic C–H stretching bands (2920–2850 cm⁻¹) indicate long hydrocarbon chains. The ester carbonyl (C = O) peak at 1750–1735 cm⁻¹ confirms triglyceride structure. Additional C–O–C and C–O stretching bands validate ester linkages. Peaks in the lower region (900–720 cm⁻¹) correspond to long-chain lipid vibrations. These features confirm the suitability of Precirol ATO 5 as a solid lipid matrix for NLC formulation.

Fig. 4.

FTIR Spectrum of (A)- Drug Mixture of Paclitaxel and Flavokawain A, (B)- BSA (Bovine Serum Albumin), (C)- Hyaluronic Acid (HA), (D)- Precirol ATO 5

Differential scanning calorimetry

Differential scanning calorimetry thermograms showed a substantial reduction or complete disappearance of the sharp melting endotherms associated with pure flavonoids in the optimized formulations. This finding suggests a decrease in crystallinity and successful encapsulation of the drugs within the lipid matrix, indicating an amorphous or molecularly dispersed state, which is known to enhance drug dissolution and bioavailability. Furthermore, powder X-ray diffraction analysis corroborated these results by revealing a significant reduction in the intensity of the crystalline diffraction peaks characteristic of the pure flavonoids. The prevalence of diffuse diffraction patterns confirmed the transformation of the drugs from a crystalline to an amorphous form within the nanostructured lipid carriers.

The DSC thermograms of the pure drugs exhibited sharp endothermic peaks corresponding to their crystalline nature; however, these peaks were significantly reduced or completely absent in the optimized formulations. This suggests the successful encapsulation of the drugs within the lipid matrix and their transformation into an amorphous or molecularly dispersed state. Such amorphization is known to enhance drug dissolution and bioavailability.

The DSC thermogram reveals the important thermal transitions of the drug, polymer, and formulation. The sharp melting peak of the pure drug indicates its crystalline nature. A reduced or shifted peak in the formulation suggests partial amorphization and improved drug-polymer interaction. Thermogram obtained for FIS sample is shown in Fig. 5. The sharp endotherm observed at 321.3 ^o C indicate the purity of drug sample. The FIS’s reported melting point is 330 °C (Fig. 5). The absence of new peaks confirms the compatibility and thermal stability of the system. Red Line Represents the pure drug (API), showing a sharp endothermic peak at its melting point, confirming its crystalline nature. Blue Line: Represents the polymer (e.g., PCL), showing a broader thermal transition, typically associated with its glass transition or melting behavior. Green Line: Represents the PCL- API formulation, where the drug’s peak is either shifted or reduced, indicating molecular dispersion and compatibility with the polymer matrix.

Fig. 5.

DSC thermogram showing reduced drug crystallinity and successful encapsulation of fisetin in HA–BSA-coated nanostructured lipid carriers

Scanning electron microscopy

SEM was employed to examine the surface morphology and microstructural characteristics of the prepared formulation. The SEM reveals a relatively uniform and well-defined architecture with distinct particle distribution. The surface appears heterogeneous surface with porous and irregular structures, indicating efficient formulation and stabilization. No significant aggregation or structural collapse is observed, suggesting good physicochemical integrity. The particle size observed in the SEM micrograph is consistent with prior characterization results. Additionally, the porous nature may facilitate enhanced drug loading and release behavior, supporting its potential applicability in targeted drug delivery systems. SEM images of HA-BSA Coated NLCs showed morphological characteristics of nanocarriers. For efficient delivery nanocarriers must be spherical in shape thus surface morphology showed that the particles are spheroidal in shape and smooth. shows the surface morphology of HA-BSA coated nano carriers (Fig. 6).

Fig. 6.

SEM micrographs illustrating the spherical morphology and smooth surface characteristics of HA–BSA coated nanostructured lipid carriers indicating successful formulation and uniform particle distribution

Powder X-ray diffraction

Powder X-ray diffraction (PXRD) analysis was performed to investigate the crystalline nature and structural modifications of flavonoids following their incorporation into nanostructured lipid carriers (NLCs). The diffractograms of pure fisetin and flavokawain A exhibited sharp and intense characteristic peaks, confirming their highly crystalline nature. In contrast, the optimized HA–BSA-coated NLC formulations showed a significant reduction in peak intensity, along with peak broadening or complete disappearance of the characteristic diffraction peaks. PXRD measurements were conducted over a 2θ range of 5° to 60° using Cu Kα radiation under standard operating conditions. The marked attenuation of crystalline peaks and the appearance of diffuse halos in the formulation indicate a transition of the drugs from a crystalline to a partially or completely amorphous state within the lipid matrix. This reduction in crystallinity can be attributed to the formation of an imperfect lipid lattice in the NLCs, which accommodates drug molecules in a disordered arrangement. Such structural transformation confirms successful drug encapsulation and molecular dispersion within the carrier system. The amorphization of flavonoids is advantageous, as it enhances drug solubility, dissolution rate, and ultimately bioavailability. PXRD findings corroborate the results obtained from DSC analysis, confirming the successful incorporation of flavonoids into the lipid matrix and the formation of a stable amorphous nanostructured system (Fig. 7). [35]

Fig. 7.

Graph of XRD nanostructured lipid carrier’s formulation

In-vitro drug release and release kinetics

In vitro drug release investigations demonstrated that flavonoids exhibit a sustained release profile when administered via HA–BSA-coated nanostructured lipid carriers, contrasting with the rapid release observed for the free drug (Fig. 8). Each formulation displayed an initial mild release phase, followed by an extended-release period throughout the study, indicating controlled diffusion from the lipid matrix. The drug release behavior was inversely proportional to the lipid concentration, with formulations containing a higher lipid content exhibiting slower release rates due to increased diffusion resistance within the lipid matrix. The optimized formulations achieved a balanced release profile, which was advantageous for maintaining prolonged therapeutic exposure and effective drug levels at the target site. Kinetic modeling of the release data revealed the strongest alignment with diffusion-controlled models, suggesting that drug diffusion through [9]the lipid matrix and surface coating layers dictated the release mechanism [36].

Fig. 8.

In vitro release profile of fisetin and flavokawain A from HA–BSA coated nanostructured lipid carriers demonstrating sustained and controlled drug release compared to free drug

The optimized HA–BSA-coated NLC formulations were able to release nearly 90–92% of the embedded drug within a time-frame of 9 h, however, this release pattern was considerably slower and more controlled as compared to the free drug which more often than not gets released rapidly by exhibiting a burst release pattern. The pattern of release showed an initial burst effect followed by gradual and sustained diffusion from lipid matrix. In the treatment of lung cancers, the release over several hours is advantageous as it may help maintain the drug at effective concentrations within the tumor microenvironment following nanocarrier accumulation and cellular uptake by CD44. Also, the HA–BSA coating can create an extra diffusional barrier which will afterwards control the release of the drug and minimise early leakage of the drug. As such, the release behavior observed can be deemed sustained as compared to the free drug and suitable for targeted anticancer therapy.

The HA–BSA coating further modulated the release by providing an additional diffusion barrier, thereby extending the duration of drug release (Table 3).

Table 3.

% cumulative drug release of nanostructured lipid carriers F1-F9 formulation

Time in hr.	% Cumulative Drug release
	F1	F2	F3	F4	F5	F6	F7	F8	F9
0	0	0	0	0	0	0	0	0	0
1	7.45	9.45	10.24	9.46	10.64	11.22	9.91	10.68	12.77
2	10.31	12.63	13.10	14.33	15.66	17.90	15.08	18.43	22.08
3	16.23	17.49	18.18	20.86	21.54	25.31	21.20	27.29	31.83
4	23.22	27.22	24.35	25.75	28.03	29.95	30.9	34.38	41.65
5	33.38	38.81	40.20	35.17	35.51	38.40	45.73	47.4	52.9
6	44.22	46.04	48.94	48.58	49.12	50.25	61.67	63.91	65.42
7	56.62	61.31	63.32	71.74	62.00	65.79	69.16	72.14	77.86
8	74.35	78.05	74.19	81.41	77.18	75.37	76.60	83.15	88.58
9	80.1	92.02	80.22	90.14	76.30	69.50	84.77	73.62	92.25

Stability studies

Thorough stability evaluations were performed to assess the physical and chemical stability of the optimized HA–BSA-coated nanostructured lipid carriers under various storage conditions. This research systematically investigated the impact of storage temperature on essential quality attributes, including particle size, zeta potential, and drug retention, to verify the formulation’s robustness and appropriateness for shelf-life, in alignment with regulatory guidelines for lipid-based nanocarriers [37].

The optimized formulations were stored under both refrigerated conditions (2–8 °C) and ambient room temperature for a predetermined period, during which they underwent periodic evaluations for alterations in particle size and polydispersity index (PDI). When stored under refrigerated conditions, the HA–BSA-coated nanostructured lipid carriers consistently maintained their nanoscale particle size and narrow size distribution throughout the duration of the study. The minimal fluctuations in particle size and PDI observed indicate robust resistance to aggregation and high physical stability at lower temperatures. In contrast, formulations stored at room temperature exhibited relatively large increases in the particle size and PDI over time. These changes are attributed to increased lipid matrix mobility, partial lipid recrystallization, and heightened particle–particle interactions at elevated temperatures. Despite these variations, they remained within acceptable limits, indicating that the HA–BSA surface coating effectively mitigated aggregation and preserved colloidal stability, even under less favorable storage conditions.

Zeta potential measurements served as a critical metric for evaluating the surface charge stability and electrostatic repulsion during storage. Under refrigerated conditions, the zeta potential values of the optimized formulations remained largely stable, indicating the preservation of surface functionalization and minimal changes in interfacial properties. This stability suggests strong adsorption of hyaluronic acid and bovine serum albumin on the nanoparticle surface, which contributes to sustained electrostatic and steric stabilization. In contrast, at room temperature, a gradual decrease in the magnitude of the zeta potential was observed, particularly during prolonged storage. This decline may be attributed to the partial rearrangement or desorption of surface-bound biomolecules. Nevertheless, the surface charge remained sufficiently high to prevent aggregation, highlighting the effectiveness of the dual HA–BSA coating strategy for maintaining colloidal stability throughout storage [38].

Drug retention studies were conducted to evaluate the effectiveness of nanostructured lipid carriers in preserving flavonoid encapsulation during storage. Under refrigerated conditions, the carriers exhibited minimal drug leakage, indicating robust drug–lipid interactions and stable entrapment of flavonoids within the nanostructured lipid matrix. These results affirm the appropriateness of the selected lipid composition and QbD-optimized formulation parameters for ensuring long-term stability. On the other hand, formulations stored at room temperature displayed a relatively higher degree of drug leakage over time, likely because of the increased lipid fluidity and enhanced diffusion of drug molecules from the carrier matrix. Nevertheless, the overall drug retention remained within acceptable limits, demonstrating that the NLC system effectively protected the encapsulated flavonoids from premature release and chemical degradation [39].

The physical and chemical stability of the optimized HA-BSA coated nanostructured lipid carrier (NLC) formulations Optimized Batch, was evaluated over a 6-month period under refrigerated (5 ± 3 °C) and room temperature (25 ± 2 °C) storage conditions. Stability parameters included particle size, zeta potential, and drug content, which are critical for ensuring colloidal stability and therapeutic efficacy (Table 4).

Table 4.

Stability data for Optimized Batch HA-BSA coated NLC formulations

Parameter	Storage Condition	Initial	1 Month	3 Months	6 Months
Particle Size (nm)	5 ± 3 °C	162.5 ± 5.0	163.0 ± 5.2	164.2 ± 5.5	165.1 ± 5.8
	25 ± 2 °C	162.5 ± 5.0	164.0 ± 5.4	166.2 ± 5.9	169.0 ± 6.3
Zeta Potential (mV)	5 ± 3 °C	-27.8 ± 1.8	-27.5 ± 1.9	-27.3 ± 2.0	-27.0 ± 2.1
	25 ± 2 °C	-27.8 ± 1.8	-27.3 ± 2.0	-26.8 ± 2.2	-26.2 ± 2.4
Drug Content (%)	5 ± 3 °C	100.0 ± 1.7	99.5 ± 1.8	99.0 ± 2.0	98.2 ± 2.1
	25 ± 2 °C	100.0 ± 1.7	99.1 ± 1.9	98.2 ± 2.2	96.8 ± 2.5

Anticancer activity

HA-BSA Coated Nanostructured Lipid Carrier Anticancer activity against A549 cell line at the different concentrations drug and formulation assessed for their anticancer activity, out of the tested samples, the formulation showed a high percentage of inhibition against the lung adenocarcinoma A549 cancer cell line as compared to the drug [40]. On the basis of percent inhibition, we can conclude that the formulation shows good anticancer activity against the lung cancer A549 cell line sand the drug has moderate anticancer activity (Fig. 9).

Fig. 9.

Cytotoxicity (percent inhibition) of HA–BSA coated nanostructured lipid carriers against A549 lung cancer cells at varying concentrations. A % Inhibition of Paclitaxel (free drug), (B) Microscopic image of cells treated with Paclitaxel, (C) % Inhibition of HA-BSA coated NLC (Paclitaxel + Flavokawain A), (D) Microscopic image of cells treated with HA-BSA coated NLC (Paclitaxel + Flavokawain A), (E) % Inhibition of HA-BSA coated NLC (Fisetin + Flavokawain A) , (F) Microscopic image of cells treated with HA-BSA coated NLC (Fisetin + Flavokawain A)

The present study demonstrates the successful development of quality-by-design (QbD)-optimized hyaluronic acid–bovine serum albumin (HA–BSA)-coated nanostructured lipid carriers as a rational nano-delivery platform for flavonoids in lung cancer. By integrating formulation engineering, surface functionalization, and systematic optimization, the developed system addresses the key limitations associated with phytochemical delivery, including poor solubility, instability, and lack of reproducibility [41]. The discussion below contextualizes the findings with respect to nanoparticle stability, phytochemical encapsulation, and the role of QbD in ensuring translation anticancer ability [42].

The Table 5 illustrates the effects of different formulations against A549 cell lines.

Table 5.

Effects of compound against A549 Cell lines

SR NO	SAMPLE CODE	Conc. (µg/ml)	OD			Mean	% Of Inhibition	% Of Viability	IC50 (µg/m l)
1	Control		1.534			-	-	-	-
2	Drug (PTX)	20	1.427	1.426	1.426	1.426	7.04	92.96	72.35
		40	1.312	1.314	1.311	1.312	14.47	85.53
		60	1.103	1.106	1.081	1.096	28.55	71.45
		80	0.841	0.843	0.847	0.844	44.98	55.02
		100	0.634	0.637	0.639	0.637	58.47	41.53
3	Formulation Pacli+Flavokawain A	20	1.461	1.461	1.462	1.465	4.64	94.37	70.45
		40	1.165	1.173	1.168	1.169	23.87	77.17
		60	0.990	0.994	0.992	0.993	36.39	65.60
		80	0.952	0.960	0.954	0.955	58.87	44.17
		100	0.851	0.868	0.8462	0.855	70.32	39.70
									72.38
4	Formulation Fisetin+Flavokawain A	20	1.462	1.464	1.461	1.463	4.62	95.38
		40	1.168	1.171	1.166	1.168	23.85	76.15
		60	0.991	0.992	0.990	0.991	35.39	64.61
		80	0.950	0.956	0.953	0.953	57.87	42.13
		100	0.850	0.866	0.8461 .	0.854	61.32	38.68

Figure 9 discusses a comparative study of the cytotoxic activity and cellular morphology of free Paclitaxel and HA-BSA coated NLC formulations. The inhibition profiles (Fig. 9. A, C, E) show a dose-dependent increase in cytotoxicity with higher inhibition for NLC formulations than that of the free drug. Based on findings in (Fig. 9B, D, F) microscopic images treated cells began to undergo various morphological changes like shrinkage, detachment and decreased cell density. It appears that the NLC formulations containing phytochemicals, especially those with a combination of phytochemicals, induce cellular damage, which indicates better drug delivery and synergistic therapeutic effect as compared to standard treatment. The cytotoxicity of free Paclitaxel and HA-BSA coated NLC formulations was evaluated through dose-dependent inhibition studies accompanied by morphological studies. The inhibition profile of free Paclitaxel (Fig. 9A) expressed an increasing trend of cytotoxicity with increasing concentration and reached about 58% inhibition with a maximum dose. The overall effect was moderate, indicating limitations in the cellular uptake and bioavailability of the free drug. This is evident from the microscopic image (Figure B), wherein the treated cells express a few morphological changes which include minor shrinkage and less density, whereas most of the cells remain unaffected. Conversely, the HA-BSA-coated NLC formulation containing Paclitaxel and Flavokawain A (Fig. 9C) exhibited a markedly enhanced cytotoxic effect, displaying a greater percentage inhibition at all tested concentrations in comparison to the free drug. The drug delivery, cellular uptake, and sustained release characteristics of the nanocarrier system could enhance this delivery. The subsequent micrographic image supports this assertion, with the notable increase in detachment, damage to the membrane, and decreased viability of cells. Moreover, the research revealed on NLC gel containing Fisetin and Flavokawain A (Fig. 9E), recorded maximum cytotoxic activity among all groups at higher concentrations. As a result, this may testify to a synergistic effect of the phytochemicals with the nanocarrier system. The microscopic image (Fig. 9F) provides strong visual evidence of this effect as there is considerable distortion and loss of normal morphology of cells along with reduction in the number of cells. The cytotoxicity results demonstrated a concentration-dependent increase in the percentage inhibition of A549 cells for all tested samples. Among the formulations, HA–BSA-coated NLCs containing Fisetin and Flavokawain A exhibited the highest percentage inhibition at higher concentrations, followed by the formulation containing Paclitaxel and Flavokawain A. Microscopic examination further supported these findings, revealing pronounced morphological alterations such as cell shrinkage, membrane damage, cellular detachment, and reduced cell density in treated groups compared with the control. These observations indicate enhanced cellular interaction and effective anticancer activity of the developed nanocarrier formulations. The improved cytotoxic response may be attributed to enhanced cellular uptake, sustained intracellular drug retention, and the combined action of the flavonoids delivered through the HA–BSA-coated nanostructured lipid carrier system. Thus, the percentage inhibition data together with the morphological changes suggest that the developed formulations possess promising anticancer potential against A549 lung cancer cells.

Overall, the finding demonstrates the NLC formulations significantly enhance cytotoxic efficacy as opposed to the free drug, Combination of nanocarrier-mediated delivery along with bioactive compounds enhances drug stability, cellular uptake, and therapeutic performance which is a potential strategy for improved anticancer activity [43].

The process of surface functionalization using bovine serum albumin (BSA) followed by hyaluronic acid (HA) coating has been instrumental in significantly enhancing the physicochemical stability of nanostructured lipid carriers. This dual HA–BSA coating delivered both steric and electrostatic stabilization, which effectively curtailed particle aggregation during storage and ensured the maintenance of the nanoscale dimensions. The slight increase in particle size observed after surface modification serves as evidence of successful coating, while retaining the size characteristics essential for applications in nanomedicine. Furthermore, zeta potential analysis highlighted the stabilizing impact of the HA–BSA coating. The retention of high surface charge values, especially under refrigerated conditions, suggests the robust adsorption of surface modifiers and the preservation of interfacial integrity. Such stabilization mechanisms have been widely documented for protein- and polysaccharide-functionalized lipid nanocarriers.

Flavonoids have been selected as model phytochemicals due to their extensive anticancer properties and favorable safety profiles. However, their clinical application is frequently constrained by challenges such as poor aqueous solubility, chemical instability, and limited bioavailability. The encapsulation of flavonoids within nanostructured lipid carriers has proven to be an effective strategy to overcome these limitations by enhancing drug solubilization, safeguarding labile molecules from degradation, and facilitating sustained drug release. Solid-state characterization demonstrated a significant reduction in flavonoid crystallinity post-encapsulation, suggesting the presence of molecular dispersion or amorphous drug states within the lipid matrix. These transformations are beneficial, as they correlate with improved dissolution behavior and enhanced therapeutic efficacy. Furthermore, sustained release from the lipid matrix may prolong drug exposure while mitigating peak-related systemic toxicity [44].

The adoption of a Quality by Design (QbD) framework was instrumental in developing a reproducible and robust nano formulation. By clearly defining the Quality Target Product Profile and systematically identifying critical quality attributes, material attributes, and process parameters, the formulation process was driven by a mechanistic understanding rather than relying on empirical trial-and-error methods. This methodology allows for precise control over the particle size, drug entrapment efficiency, and release behavior [43]. The design of experiments is essential for establishing a well-defined design space, ensuring consistent performance of the formulation. The reproducibility of the physicochemical properties and stability outcomes across the optimized formulations underscores the scalability of the developed system. Furthermore, the application of high-pressure homogenization, a widely recognized and industrially scalable technique, significantly enhances the potential of the formulation for translation.

The hyaluronic acid-functionalized surface of nanostructured lipid carriers offers a strategic approach for targeting lung cancer through CD44 receptor-mediated interactions, given that CD44 is often overexpressed in aggressive and drug-resistant lung tumors. While the current study primarily focused on formulation and stability evaluation, the established physicochemical properties provide a solid foundation for future research on receptor-mediated cellular uptake and enhanced therapeutic efficacy [38]. The integration of targeted surface modification, phytochemical loading, and QbD-guided optimization positions the developed HA–BSA-coated nanostructured lipid carriers as a promising platform for lung cancer therapy. These systems have the potential to improve therapeutic outcomes while minimizing off-target toxicity, thereby addressing the significant limitations of conventional chemotherapeutic approaches.

Summary of Key Findings: The QbD-optimized HABSA-coated nanostructured lipid carriers (NLCs) exhibited particle sizes ranging from 115 to 125 nm, with a polydispersity index (PDI) of less than 0.3, and a zeta potential of approximately − 20 mV. The drug entrapment efficiency exceeded 70%, and the carriers demonstrated a sustained release profile. Additionally, a reduction in drug crystallinity was confirmed by FTIR, DSC, and PXRD analyses. The carriers also showed excellent stability under refrigerated conditions, with minimal alterations in size, charge, or drug content [45].

The results validate the effective creation of a dual-coated (HA-BSA) NLC platform designed for the simultaneous delivery of fisetin and flavokawain A. This innovative platform offers enhanced physicochemical stability, a controlled release mechanism, and improved anticancer activity against A549 NSCLC cells when compared to paclitaxel. Comparison with Existing Literature After coating, the HABSA-NLCs experienced a modest increase in size compared to their uncoated counterparts, yet they showed improved zeta potential and stability. The drug release rate was significantly slower than that of free flavonoids, with optimized NLCs reaching about 80–90% cumulative release at 9 h, as opposed to a rapid initial release. Cytotoxicity data revealed that these NLCs inhibited cells more effectively than paclitaxel across concentrations of 20–100 µg/mL.

The high-pressure homogenization process resulted in an imperfect lipid matrix, which contributed to the high encapsulation efficiency (EE). The BSA coating plays a crucial role in providing steric and electrostatic stabilization. HA is instrumental in facilitating CD44 receptor-mediated endocytosis, specifically targeting NSCLC. The amorphization process enhanced solubility, and the Higuchi kinetics model indicated that the release was diffusion-controlled, occurring through the lipid core and the dual coating barrier. Implications of Low Particle Size and Zeta Potential Nanoscale particles (< 200 nm) are advantageous for EPR-mediated tumor accumulation and cellular uptake. A negative zeta potential (>|20 mV|) is key to preventing aggregation, extending the circulation time, and minimizing opsonization. These characteristics are vital for parenteral delivery, improved bioavailability, and effective targeting of lung cancer cells [46].

The limitations encompass studies that are exclusively in vitro, lacking in vivo pharmacokinetics/PD data, assessments limited to short-term stability, unverified industrial scalability of high-pressure homogenization, the absence of direct CD44 binding assays, and flavonoid MDR modulation that is suggested but not mechanistically validated. Future study should involve in vivo efficacy and xenograft studies Included - CD44 knockdown experiments to confirm targeting achieve long-term accelerated stability testing Scale up using microfluidics for industrial translation Test chemo-flavonoid combinations for potential synergy Validate the QbD design space with expanded DoE batches [47].

Conclusion

The therapeutic management of non–small cell lung cancer (NSCLC) remains constrained by multidrug resistance, systemic toxicity, and the limited clinical utility of bioactive phytochemicals due to poor solubility and pharmacokinetic instability. To address these challenges, the present study established a rational, quality-by-design (QbD)-guided strategy for engineering hyaluronic acid–bovine serum albumin (HA–BSA)-coated nanostructured lipid carriers for the combinational delivery of fisetin and flavokawain A. Systematic optimization enabled precise control over critical quality attributes, yielding a robust nanoscale formulation characterized by high entrapment efficiency, sustained drug release, favorable colloidal stability, and enhanced in vitro anticancer activity against A549 cells. Dual HA–BSA functionalization integrates steric stabilization with potential CD44-mediated targeting, thereby offering a mechanistically informed approach to improve intracellular drug delivery and overcome resistance-associated barriers. Despite these promising results, translational validation remains essential. The absence of in vivo pharmacokinetic profiling, tumor biodistribution assessment, and receptor-specific uptake confirmation represents a limitation that must be addressed before clinical progression. Future studies should incorporate xenograft efficacy models, mechanistic CD44-blocking assays, and scale-up validation under regulatory-compliant conditions to substantiate clinical feasibility. Overall, this work advances a reproducible and scalable nanoengineering platform that bridges phytochemical therapeutics and precision nanomedicine, offering a strategically designed framework for next-generation targeted interventions in NSCLC.

Abbreaviations

ABC

ATP-binding cassette

HA

Hyaluronic acid

BSA

bovine serum albumin

DoE

Design of experiments

EPR

Enhanced permeability and retention

ICH

International Council for Harmonization

MDR

Multidrug resistance

NLCs

Nanostructured lipid carriers

NSCLC

Non-small cell lung cancer

Authors’ contributions

A.N.H: Data curation, Wrote the manuscript, Methodology. D.R.T: Conceptualization, Reviewed the manuscript, Supervision. D.U.K: Supervision, Project administration and Reviewed the manuscript.

Funding

Open access funding provided by Datta Meghe Institute of Higher Education and Research. The authors did not receive support from any organization for the submitted work.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This research involved in vitro experiments using established cell lines only. No human participants or animals were involved; therefore, institutional ethical approval was not required.

This article is a review and does not involve any human participants.

Consent for publication

Not Applicable. This article is a review and does not contain any individual person’s data in any form.

Competing interests

The authors declare no competing interests.