Development of Curcumin-Loaded Hybrid Lipid-Polymeric Nanoparticles for Hypoxia-Related Applications

Abstract

Introduction

Hypoxic and ischemia-associated tissues are often characterized by acidic microenvironments, highlighting the need for drug delivery systems that enable enhanced drug release under acidic conditions. Curcumin (CUR) has antioxidant and cytoprotective properties, but its therapeutic application is limited by poor aqueous solubility and low bioavailability. This study aimed to optimize hybrid lipid-polymeric nanoparticles (HLPNs) as a CUR delivery platform and evaluate their physicochemical properties, colloidal stability, CUR release at pH 7.4 and pH 5.5, and cellular effects under normoxic and hypoxia-mimetic conditions.

Methods

In this study, hybrid lipid-polymeric nanoparticles (HLPNs) were optimized as a curcumin (CUR) delivery platform by varying the lipid-to-polymer ratio (DSPE-PEG/cholesterol: PLGA). Particle size, polydispersity index, and zeta potential were determined by dynamic light scattering (DLS) and laser Doppler electrophoresis. The formulations were further characterized in terms of colloidal stability, encapsulation efficiency, morphology, and in vitro CUR release at pH 7.4 and pH 5.5. The effect on cell viability of CUR-loaded HLPNs and free CUR was evaluated in human foreskin fibroblasts (HF) using the MTT assay under normoxic and CoCl₂-induced hypoxia-mimetic conditions.

Results

Among the tested formulations, HLPNs with lipid-to-polymer ratios of 1:2 and 1:3 showed the most favourable physicochemical profile, with initial particle sizes of ~127 and ~144 nm, respectively, low polydispersity index (~0.12), and negative zeta potentials around −42 mV. After CUR loading, particle size increased to 160 nm for HLPN-CUR 1:2 and 173 nm for HLPN-CUR 1:3. In contrast, encapsulation efficiency reached 38.6% and 40.0%, respectively. Both formulations exhibited pH-dependent CUR release, with minimal release during the first 6 h (<2.0%) and markedly higher cumulative release at pH 5.5 than at pH 7.4 after 72 h. In HF, both CUR-loaded formulations reduced cell viability in a concentration-dependent manner. Free CUR was generally better tolerated than the nanoparticle-loaded formulations. HLPN-CUR 1:3 produced a higher MTT signal under hypoxia-mimetic conditions than under normoxia at 10 µM. However, higher concentrations were associated with marked morphological changes, including cell rounding, aggregation, and reduced cell density.

Conclusion

Among the tested formulations, HLPN-CUR 1:3 demonstrated suitable physicochemical properties, colloidal stability, CUR encapsulation, and enhanced CUR release under acidic conditions at later time points. However, the concentration-dependent effects on cell viability and morphology indicate that further studies are needed to confirm its biological activity and potential relevance for hypoxia-related applications.

Introduction

Nanotechnology has brought significant progress to the field of drug delivery. By exploiting the unique properties of nanomaterials, researchers have developed innovative strategies to deliver drugs and other therapeutic agents, thereby overcoming numerous limitations associated with traditional therapeutic approaches.

Traditional drug delivery systems often face challenges such as poor solubility, low bioavailability, and nonspecific distribution, which can lead to adverse side effects. Nanotechnology enables the development of nanocarriers that protect drugs and allow for their controlled and targeted release.^1,2 Moreover, nanoparticles (NPs) can enhance the solubility of poorly water-soluble drugs, improve their stability, and prolong their circulation time in the body.³ One of the key advantages of NPs is the ability to engineer particles with targeting ligands that enable them to selectively accumulate in specific tissues, thereby increasing the drug concentration at the desired site and minimizing off-target effects.⁴ Ligand-coated NPs can target specific cell receptors,⁵ enzymes,⁶ proteins,⁷ or other biological structures, further enhancing their therapeutic effectiveness. Another significant advantage of nanosystems is their ability to serve not only as drug carriers but also as diagnostic tools, allowing therapy and disease monitoring to be integrated into a single system.⁸ Nanomaterials have enabled the development of versatile platforms in which a single nanoparticle can perform diverse tasks, including drug delivery and disease marker detection-an integrated approach, known as theranostics.

All NPs share a common characteristic: their dimensions are in the nanometer range, typically between 80 and 200 nm.^9,10 The size of these particles can vary based on the concentration and type of lipids used in their production, with higher lipid concentrations resulting in larger NPs.¹¹

NPs can be categorized based on the materials used in their synthesis into three types: organic, inorganic, or hybrid. Organic nanocarriers comprise polymer-, lipid-, protein-, and carbohydrate-based nanoparticles and are widely investigated for improving drug solubility, stability, and controlled release.¹² Within this group, polymeric micelles represent a promising class of nanocarriers that can be engineered for stimuli-responsive drug delivery. For example, spermine-modified polymeric micelles have been shown to enhance cellular uptake through interactions with the polyamine transport system and to promote pH-responsive drug release under acidic conditions.¹³ Inorganic nanoparticles include carbon-based materials and metal particles.¹² For example, copper carbonate nanoparticles can deliver multiple therapeutic agents and support multimodal cancer therapy by combining starvation therapy, chemodynamic therapy, and gene silencing.¹⁴ Recently, hybrid nanoparticles, which combine organic and inorganic components, have been gaining attention for their potential to improve drug selectivity and effectiveness. In our study, we developed hybrid lipid-polymeric nanoparticles (HLPNs), advanced drug delivery systems combining the advantages of lipid- and polymer-based nanoparticles. These nanoparticles possess a dual-phase structure. For the lipid phase, scientists often use phospholipids,¹⁵ solid lipids,¹⁶ cholesterol¹⁷ and other compounds, which contribute to membrane stability and improved NPs absorption. In addition, pegylated lipids, such as DSPE-PEG, are frequently incorporated to provide stabilization and reduce aggregation and protein adsorption. The polymer phase is typically composed of natural polymers,¹⁸ synthetic polymers¹⁹ or copolymers.²⁰ These polymers are often used to enable controlled and sustained drug release.²¹ A hybrid nanoparticle approach combining lipid and polymer phases produces NPs that are stable and effective for delivering therapeutic agents. The lipids form a biocompatible membrane, while the polymers regulate the kinetics of drug release. These hybrid systems are continuously explored for applications such as cardiovascular disease treatment,²² cancer therapy,²³ gene delivery²⁴ and other applications where precise control of drug release and biological interactions is essential.

Since pathological tissues may present both extracellular and intracellular delivery barriers, nanocarriers should be designed to release their cargo in biologically relevant compartments. For therapeutic molecules that act inside cells, efficient cytosolic delivery is required, as cargo internalized by endocytosis can remain trapped in endosomes or be degraded in lysosomes.²⁵ Therefore, recent delivery systems are being developed to promote endosomal escape and release therapeutic molecules into the cytosol, where they can remain active and exert their biological effects.²⁶ In ischemic and hypoxic tissues, reduced oxygen availability alters cellular metabolism and promotes glycolysis, which may lead to lactate accumulation and extracellular acidification.²⁷ These changes create a pathological microenvironment that may influence the behaviour of nanoparticle-based drug delivery systems. At the same time, the lipid-to-polymer ratio is an important formulation parameter that can affect particle size, dispersity, colloidal stability, and drug release. However, the influence of this ratio on the overall performance of HLPNs remains insufficiently characterized. In this study, we aimed to optimize HLPNs by varying the ratios (1:10, 1:5, 1:3, 2:5, 1:2, 3:5, 2:3, 3:4, 4:5, 1:1) of lipids and polymers (DSPE-PEG/cholesterol: PLGA). Following synthesis, we evaluated the formulations and selected the most effective ones based on their physicochemical properties. Cholesterol was chosen for the lipid phase as it is a naturally occurring sterol found in cellular membranes and is essential for maintaining membrane integrity and permeability.²⁸ In addition, DSPE-PEG was incorporated to provide steric stabilization and improve colloidal stability in biological media by reducing NPs aggregation and nonspecific interactions. Together, these properties enhance the stability and functionality of these NPs, which are widely used as drug delivery systems.

Formulations exhibiting the most suitable physicochemical properties and colloidal stability were loaded with CUR, which is a bright yellow polyphenolic compound derived from the root of Curcuma longa.²⁹ It has been widely studied for its antioxidant, anti-inflammatory, and anticancer properties.^30,31 These properties make CUR a promising candidate for treating various chronic diseases, including cardiovascular diseases and cancer. Despite its significant therapeutic potential, clinical application of CUR is hindered by challenges such as poor bioavailability due to low water solubility, rapid metabolism, and limited absorption.³² NPs can enhance CUR bioavailability and overall effectiveness, offering a promising strategy for clinical applications. The selected CUR-loaded formulations were further evaluated for CUR release at pH 7.4 and pH 5.5 and for their effects on HF viability under normoxic and CoCl₂-induced hypoxia-mimetic conditions.

Materials and Methods

Preparation of Hybrid Lipid-Polymeric Nanoparticles

To prepare the aqueous phase, 3.2 mg of cholesterol (Sigma-Aldrich, USA) and 4.8 mg of DSPE-PEG(2000) (Avanti Polar Lipids, USA) were weighed and dissolved in 0.8 mL of ethanol and 3 mL of water at 65 °C. This solution was then diluted with warm water (65 °C) to a total volume of 20 mL. 4.5 mL of the previously prepared aqueous solution was transferred to a separate vial. For the organic phase, 40 mg of PLGA (Sigma-Aldrich, USA) was dissolved in 2 mL of acetonitrile (Sigma-Aldrich, USA). Then, 0.5 mL of this organic solution was added to the aqueous phase while maintaining 65 °C and stirring at 600 rpm. The resulting mixture was sonicated in an ultrasonic bath for 10 minutes and subsequently stirred at room temperature for 2 hours. The final mixture was transferred to filters and centrifuged at 5580×g for 10 minutes. After centrifugation, the nanoparticles were collected and resuspended in 1 mL of water.

HLPNs with different lipid-to-polymer ratios were synthesized using the previously described method. To prepare CUR-loaded HLPNs (HLPN-CUR), 0.5 mg of CUR was added to the polymer-containing organic phase before combining it with the aqueous phase.

Characterization of Hybrid Lipid-Polymeric Nanoparticles

The physicochemical properties of the hybrid lipid–polymer nanoparticles (HLPNs) were assessed using dynamic light scattering and laser Doppler electrophoresis with a Zetasizer Ultra device (Malvern Panalytical, UK). For size and polydispersity index (PdI) measurements, 20 µL of the nanoparticle suspension was diluted in 980 µL of Milli-Q water. The surface charge (zeta potential) was measured in a capillary cell (DTS 1070). All measurements were performed at room temperature.

HLPNs Stability Studies

The in vitro stability of HLPNs was evaluated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, UK) containing 10% fetal bovine serum (FBS) (Gibco, UK). A volume of 200 μL of HLPNs was incubated with 1 mL of medium at 37 °C with continuous stirring at 110 rpm for 72 hours. At each time point (15 and 30 minutes, 1, 2, 4, 6, 8, 24, 48, and 72 hours), 20 μL samples were collected, and the average particle size and PdI were measured using DLS. Additional stability studies were performed for selected formulations (1:2 and 1:3) in DMEM without FBS and in phosphate-buffered saline (PBS) (Gibco, UK) under the same conditions.

Evaluation of Curcumin Entrapment Efficiency and Drug Loading

To determine the entrapment efficiency (EE) of CUR (Thermo Scientific, USA), 20 μL of HLPN-CUR was centrifuged at 10,000 rpm for 5 minutes at room temperature. The supernatant was removed, and 500 μL of DMSO (Sigma-Aldrich, USA) was added and mixed for 1 minute on a shaker. The nanoparticles were completely disrupted by 2 minutes of ultrasonic treatment in a water bath. Subsequently, 100 μL of the DMSO solution was transferred to microplate wells for fluorescence measurement. Fluorescence was measured using a fluorimeter with an excitation wavelength of 485 nm and an emission wavelength of 538 nm. CUR concentration was calculated from a standard calibration curve prepared in DMSO. EE (%) was determined as the amount of encapsulated CUR relative to the initial CUR added during formulation. Drug loading (DL, %) was calculated as the amount of encapsulated drug relative to the total weight of drug-loaded nanoparticles using the following equation: DL (%) = (amount of encapsulated drug / total weight of drug-loaded nanoparticles) × 100.

Storage Stability Assessment

The physical stability of the empty (HLPN 1:2 and HLPN 1:3) and CUR-loaded (HLPN-CUR 1:2 and HLPN-CUR 1:3) nanoparticles was evaluated during storage at 4 °C in a refrigerator for four weeks. Samples were kept in sealed Eppendorf tubes and analyzed weekly. At weeks 0, 1, 2, 3, and 4, particle size, PdI, and zeta potential were determined as described previously. All measurements were performed in triplicate.

Morphology of the HLPN-CUR

HLPN-CUR 1:3 and HLPN-CUR 1:2 were examined using a Hitachi S-3400N scanning electron microscope (Tokyo, Japan). To preserve their native morphology, the specimens were imaged without conductive coating to avoid topographical distortions caused by sputter-coated layers. Imaging was performed at a low accelerating voltage of 5 keV to maximize surface sensitivity and reduce beam-induced charging, thereby minimizing structural damage in these non-conductive biological samples.

Release Profile in Nanoparticles

The in vitro CUR release assay was performed using a dialysis method. A volume of 220 μL of freshly prepared HLPN-CUR 1:2 and HLPN-CUR 1:3 was placed into dialysis filters with a molecular weight cut-off (MWCO) of 15 kDa. The filters were immersed in 150 mL of acceptor medium consisting of PBS supplemented with 0.5% Tween 80 (Sigma-Aldrich, USA). The assay was conducted under physiological (pH 7.4) and acidic (pH 5.5) conditions, with continuous magnetic stirring at 37 °C and 120 rpm. All samples were protected from light throughout the experiment. At each sampling point, 100 μL of the acceptor medium containing released CUR was collected and immediately replaced with an equal volume of fresh medium. The release study was carried out over 72 hours, with samples collected at 15, 30, and 60 minutes, and at 2, 4, 6, 24, 48 and 72 hours.

Cytotoxicity in Fibroblasts

The cytotoxicity of empty HLPNs, HLPN-CUR and free CUR was evaluated using the MTT assay under normoxic and CoCl₂-induced hypoxia-mimicking conditions. Human foreskin fibroblasts CRL-4001 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HF were seeded in 96-well plates at 3×10⁴ cells per well in a volume of 100 μL and incubated for 24 hours. Cells were then treated with 100 μL of different concentrations of empty HLPN, HLPN-CUR and CUR (undiluted). Normoxic conditions corresponded to standard cell culture conditions, whereas hypoxia-mimicking conditions were induced by adding 200 μM cobalt (II) chloride (anhydrous, 97%, Carl Roth GmbH, Germany) to the cell culture medium. After 72 hours, cells were incubated with 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL in medium) (Life Technologies, Oregon, USA) for 1 hour at 37°C. Following incubation, the supernatant was removed, and the resulting formazan crystals were dissolved in 100 µL of 2-propanol (Sigma-Aldrich, USA) with gentle shaking to ensure complete dissolution. Absorbance was measured at 570 and 630 nm wavelengths. Cells were imaged using an inverted microscope before the MTT assay. Phase-contrast images of cells were captured using an Olympus IX73 inverted microscope (Olympus Europe Holding GmbH). Random fields of the wells with cells were photographed with a 20× lens.

Statistical Analysis

Statistical analysis was performed using Microsoft Office Excel 2007 (Microsoft Corporation, Redmond, WA, USA) and IBM SPSS Statistics (Version 20). Data are presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical significance was assessed using analysis of variance (ANOVA), followed by post hoc multiple comparisons testing. Differences were considered statistically significant at p < 0.05.

Results

Nanoparticle Size, PdI and Zeta Potential

HPLNs were initially synthesized without drug loading using various lipid-to-polymer ratios. The physicochemical characteristics, including particle size, PdI and zeta potential, are summarized in Table 1. The average HLPN size ranged from approximately 116 nm to 205 nm, depending on the formulation. Among all tested ratios, the 1:10 formulation, with the highest polymer content, resulted in the largest nanoparticle size (205.7 ± 0.6). PdI values ranged from 0.113 to 0.176, indicating a narrow size distribution. All HLPNs had a negative surface charge, with zeta potential ranging from −40.9 mV to −54.9 mV. The particles with the highest polymer content (lipid-to-polymer ratio of 1:10) exhibited the most negative charge (−54.9 ± 1.8 mV).

Table 1.

Particle Size, Polydispersity Index (PdI), and Zeta Potential of Empty Hybrid Lipid-Polymeric Nanoparticles (HLPNs) Prepared Using Different Lipid-to-Polymer Ratios. Results Represent the Mean ± Standard Deviation (SD), n=3

HLPNs Formulation	Size (nm)	PdI	Zeta Potential (mV)
1:10	205.7 ± 0.6	0.149 ± 0.01	−54.9 ± 1.8
1:5	182.3 ± 2.3	0.117 ± 0.03	−44.8 ± 2.5
1:3	143.7 ± 2.5	0.123 ± 0.02	−42.4 ± 3.5
2:5	175.3 ± 1.2	0.113 ± 0.01	−46.6 ± 2.0
1:2	127.0 ± 5.0	0.119 ± 0.03	−42.0 ± 2.8
3:5	173.7 ± 2.1	0.126 ± 0.01	−53.7 ± 3.2
2:3	147.7 ± 9.3	0.136 ± 0.01	−40.9 ± 4.7
3:4	175.3 ± 1.5	0.114 ± 0.01	−47.2 ± 2.0
4:5	191.0 ± 11.5	0.168 ± 0.01	−40.9 ± 2.0
1:1	116.0 ± 13.1	0.176 ± 0.03	−41.5 ± 1.7

Following synthesis, HLPNs with particle sizes up to 150 nm were selected. Four formulations, corresponding to lipid-to-polymer ratios of 1:3, 1:2, 2:3, and 1:1, were chosen for further experiments (Figure 1).

Figure 1.

Physicochemical characteristics of empty hybrid lipid-polymeric nanoparticles (HLPNs) prepared using different lipid-to-polymer ratios. Particle size, polydispersity index (PdI), and zeta potential are shown. The dashed line indicates the 150 nm particle size threshold used for formulation selection. Light-colored bars indicate formulations selected for further studies. Results represent the mean ± standard deviation (SD), n=3.

HLPNs Stability

Stability studies were performed with selected HLPN formulations (1:3, 1:2, 2:3, 1:1). The results indicated that the 1:1 formulation exhibited a significant increase in PdI from 0.168 to 0.330 (Figure 2A, p < 0.05). The 2:3 formulation also showed a significant increase in particle size over time, from approximately 151 to 179 nm (Figure 2B, p < 0.05). Among all formulations, 1:2 and 1:3 (Figure 2C and D) demonstrated the highest stability, with no significant changes in particle size or PdI. These stable formulations were selected for further experiments.

Figure 2.

Colloidal stability of selected empty HLPN formulations in DMEM supplemented with 10% fetal bovine serum over 72 h. Particle size and polydispersity index (PdI) were monitored for HLPN 1:1 (A) HLPN 2:3 (B) HLPN 1:2 (C) and HLPN 1:3 (D). Results represent the mean ± standard deviation (SD), n=3.

An additional stability study was conducted for the 1:2 and 1:3 formulations in DMEM without FBS to evaluate the effect of serum on HLPNs’ stability. The stability of these formulations was also assessed in PBS, which is used for subsequent HLPN characterization. As shown in Figure 3, both the 1:2 and 1:3 formulations remained stable in DMEM without serum and in PBS.

Figure 3.

Colloidal stability of selected empty HLPN formulations in DMEM without fetal bovine serum and in phosphate-buffered saline (PBS) over 72 h. Particle size and PdI were monitored for HLPN 1:2 in DMEM (A) HLPN 1:2 in PBS (B) HLPN 1:3 in DMEM (C) and HLPN 1:3 in PBS (D). Results represent the mean ± standard deviation (SD), n=3.

Encapsulation of Curcumin Within HLPNs

HLPNs 1:2 and 1:3 lipid-to-polymer ratios were loaded with CUR at concentrations of 2, 1, and 0.5 mg/mL. Due to the statistically significant change in the characteristics of HLPNs using 2 mg/mL and 1 mg/mL of CUR, further studies were conducted using only the 0.5 mg/mL concentration. The particle size, PdI, and zeta potential of HPLNs after loading 0.5 mg/mL CUR are shown in Figure 4. CUR incorporation increased particle size, from 127 to 160 nm for the 1:2 formulation and from 143 to 173 nm for the 1:3 formulation. PdI increased from 0.119 to 0.300 for the 1:2 formulation and from 0.123 to 0.250 for the 1:3 formulation. Upon CUR loading, the zeta potential became slightly less negative, changing from −42.0 to −39.0 mV for the 1:2 formulation and from −42.4 to −40.0 mV for the 1:3 formulation.

Figure 4.

Physicochemical characterization of CUR-loaded HLPN formulations prepared with 0.5 mg/mL CUR. Particle size, polydispersity index (PdI), and zeta potential are shown for HLPN-CUR 1:2 and HLPN-CUR 1:3. Results represent the mean ± standard deviation (SD), n=3.

Table 2 shows the EE and DL of CUR in the 1:2 and 1:3 formulations. Analysis of the results revealed that increasing the polymer content slightly increased EE from 38.6% (1:2) to 40.0% (1:3). This trend may be attributed to higher polymer content providing more favourable conditions for CUR encapsulation, although the observed increase is minor. Although HLPN-CUR 1:3 showed slightly higher EE, the calculated DL was higher for HLPN-CUR 1:2, likely due to the lower total polymer content in this formulation.

Table 2.

Entrapment Efficiency (EE) and Drug Loading (DL) of CUR in HLPN-CUR 1:2 and HLPN-CUR 1:3 Formulations. Results Represent the Mean ± Standard Deviation (SD), n=3

HLPNs Formulation	EE (%)	DL (%)
1:2	38.6 ± 1.6	1.64 ± 0.07
1:3	40 ± 2.6	1.27 ± 0.08

Morphology of HLPNs

SEM images of HLPN-CUR 1:2 and HLPN-CUR 1:3 revealed that both formulations exhibited a similar spherical morphology with slightly rough surfaces (Figure 5). The HLPN-CUR 1:2 formulation had an average diameter of approximately 170 nm, whereas 1:3 formulation was slightly larger, about 180 nm. These results indicate that varying polymer content and incorporating CUR primarily influence particle size rather than morphology or surface characteristics.

Figure 5.

Scanning electron microscopy images of CUR-loaded HLPNs. Representative images show HLPN-CUR 1:2 (A) and HLPN-CUR 1:3 (B). White arrows indicate nanoparticles. Scale bars represent 1 µm.

Nanoparticle Storage Stability

For the empty nanoparticles (HLPN 1:2 and HLPN 1:3), storage at 4 °C for 4 weeks resulted in only a slight increase in particle size, from 134 to 143 nm for HLPN 1:2 and from 146 to 155 nm for HLPN 1:3 (Figure S1). The PdI values remained low, changing only from 0.119 to 0.113 for HLPN 1:2 and from 0.123 to 0.091 for HLPN 1:3. The zeta potential also remained highly negative, varying from −40.2 to −35.3 mV for HLPN 1:2 and from −42.4 to −34.4 mV for HLPN 1:3. Statistical analysis indicated no significant changes in particle size, PdI, or zeta potential (p > 0.05), confirming the physical stability of the empty NPs.

CUR-loaded nanoparticles (HLPN-CUR 1:2 and HLPN-CUR 1:3) showed comparable stability profiles (Figure S2). The initial particle size after CUR loading was higher than that of the empty NPs, increasing from 163 to 170 nm for HLPN-CUR 1:2 and from 171 to 178 nm for HLPN-CUR 1:3. The PdI of HLPN-CUR 1:2 changed slightly from 0.269 to 0.258, while its zeta potential increased from −39.2 to −33.9 mV. Similarly, for HLPN-CUR 1:3, the PdI decreased from 0.252 to 0.213, and the zeta potential changed from −39.8 to −33.9 mV. No statistically significant changes (p > 0.05) in particle size, PdI, or zeta potential were observed for the loaded formulations, confirming that their physical stability remained consistent throughout 4 weeks of storage.

In addition, the CUR encapsulation efficiency remained stable throughout storage at 4 °C for 4 weeks, in both HLPN-CUR 1:2 and HLPN-CUR 1:3 formulations.

The in vitro Release

The in vitro release of CUR from HLPN-CUR formulations 1:2 and 1:3 was investigated in PBS containing 0.5% of Tween 80 under acidic (pH 5.5) and physiological (pH 7.4) conditions (Figure 6). The release profiles were both pH- and formulation-dependent. For both formulations, minimal CUR release (< 2.0%) was observed during the first 6 h at both pH values.

Figure 6.

In vitro CUR release from HLPN-CUR 1:2 (A) and HLPN-CUR 1:3 (B) at pH 5.5 and pH 7.4 over 72 h. CUR release was evaluated in PBS supplemented with 0.5% Tween 80. Results represent the mean ± standard deviation (SD), n=3.

For the 1:2 formulation, at pH 7.4, CUR release gradually increased to 25.2% at 24 h, 35.5% at 48 h, and 54.7% at 72 h. Under acidic conditions (pH 5.5), CUR release reached 27.8% at 24 h, 65.0% at 48 h, and 90.0% at 72 h. Although release was comparable between pH 7.4 and pH 5.5 at 24 h, it became significantly higher under acidic conditions at 48 h and 72 h (p < 0.05). The 1:3 formulation showed a time-dependent release pattern. At pH 7.4, CUR release increased from 30.7% at 24 h to 43.4% at 48 h and 60.4% at 72 h. Under acidic conditions (pH 5.5), CUR release reached 25.5% at 24 h, 53.0% at 48 h, and 88.3% at 72 h. At 24 h, CUR release was lower at pH 5.5 than at pH 7.4, whereas at 48 h and 72 h, release became significantly higher under acidic conditions (p < 0.05).

The results demonstrated a pH-dependent release of CUR. For both formulations, CUR release was greater in the acidic medium (pH 5.5) than in the physiological medium (pH 7.4) at later time points. Moreover, the 1:3 formulation exhibited a slightly higher cumulative release than the 1:2 formulation at physiological pH. This observation suggests that the lipid-to-polymer ratio may influence the release rate of CUR.

Nanoparticle Effect on Cell Viability and Morphology

MTT assay results showed that after 72 h of exposure, both HLPN-CUR 1:2 and HLPN-CUR 1:3 formulations induced a concentration-dependent decrease in cell viability under both normoxic and hypoxia-mimetic conditions.

Following exposure to HLPN-CUR 1:2 under normoxia (Figure 7A), HF viability remained high at the lowest concentration (90.9 ± 7.9% at 0.625 µM) and progressively decreased to 69.2 ± 4.4%, 56.7 ± 4.1% and 49.1 ± 10.9% at 1.25, 2.5, and 5 µM, reaching 25.9 ± 12.0% at 10 µM. A similar dose-response pattern was observed under hypoxia-mimetic conditions, with viabilities of 85.4 ± 19.8%, 76.0 ± 17.5%, 62.1 ± 7.5% and 51.6 ± 12.7% at 0.625–5 µM. Although these differences were not statistically significant (p > 0.05), HF viability was consistently more reduced under normoxia than hypoxia-mimetic conditions within the 1.25–5 µM concentration range. At 10 µM, hypoxia-mimetic conditions led to a more pronounced loss of viability than normoxia, with cell viability decreasing to 9.4 ± 6.5%.

Figure 7.

Viability of human fibroblasts treated with HLPN-CUR 1:2 (A) and HLPN-CUR 1:3 (B) under normoxic and hypoxia-like conditions. Cell viability was assessed after 72 h using the MTT assay. Results represent the mean ± standard deviation (SD), n=3. Statistical significance was assessed using ANOVA followed by post hoc multiple comparisons testing; *p < 0.05 compared with the corresponding normoxic group.

Under exposure to HLPN-CUR 1:3 in normoxia (Figure 7B), HF viability also remained high at the lowest concentrations (91.1 ± 10% and 85.6 ± 13.4% at 0.625 and 1.25 µM, respectively). Cell viability decreased to 70.1 ± 1% at 2.5 µM, 47.7 ± 14.6% at 5 µM and 28.8 ± 7.2% at 10 µM. Under hypoxia-mimetic conditions, a similar reduction in viability was observed up to 2.5 µM (91.0 ± 8.7%, 80.0 ± 8.9%, and 66.7 ± 9.0% at 0.625, 1.25 and 2.5 µM, respectively). At higher concentrations, hypoxia-mimetic conditions resulted in a less pronounced loss of cell viability compared with normoxia. At 5 µM, HF viability reached 60.1 ± 10.0% under hypoxia compared with 47.7 ± 14.6% under normoxia (approximately 1.3-fold higher). At 10 µM, viability under hypoxia-mimetic conditions was 52.9 ± 11.3% compared with 28.8 ± 7.2% in normoxia (about 1.8-fold higher). Although the difference at 5 µM was not statistically significant, a significant effect was observed at 10 µM (p < 0.05), with HF viability higher under hypoxia-mimetic conditions following HLPN-CUR 1:3 exposure.

Morphological analysis revealed that, under normoxia, treatment with HLPN-CUR 1:2 and HLPN-CUR 1:3 at low concentrations (0.625–2.5 µM) did not markedly alter cell appearance (Figure S3A). As the concentration increased, both formulations induced gradual morphological changes, including cell rounding, detachment, and localized clustering. Under hypoxia-mimetic conditions, nanoparticles triggered more rapid and pronounced morphological alterations (Figure S3B). Even at low concentrations, early signs of cell rounding and small aggregate formation were evident. At 10 µM, extensive aggregation was observed for both HLPN-CUR 1:2 and HLPN-CUR 1:3, resulting in localized regions of low cell density.

The effect of free CUR on HF viability was also assessed under normoxic and hypoxia-mimetic conditions (Figure 8). Compared with nanoparticle formulations, free CUR caused a less pronounced decrease in HF viability, indicating greater cellular tolerance than that observed for CUR delivered via nanoparticles. Free CUR showed higher MTT-derived viability than HLPN-CUR 1:2 at selected concentrations, reaching statistical significance at 2.5 and 10 µM under normoxia and at 10 µM under hypoxia-mimetic conditions (p < 0.05). No significant differences were observed between free CUR and HLPN-CUR 1:3. For free CUR, cell viability at 5 and 10 µM tended to be higher under hypoxia-mimetic conditions than under normoxia. Although these differences were not statistically significant, the results suggest higher cell viability under a hypoxia-mimetic environment. Exposure to free CUR induced noticeable changes in HF appearance compared with untreated controls under both normoxic and hypoxia-mimetic conditions (Figure S3).

Figure 8.

Viability of human fibroblasts treated with free CUR under normoxic and hypoxia-mimetic conditions. Cell viability was assessed after 72 h using the MTT assay. Results represent the mean ± standard deviation (SD), n=3. No statistically significant differences were observed between normoxic and hypoxia-mimetic conditions.

Discussion

In this study, HLPNs were successfully developed and characterized as potential carriers for CUR delivery. Our study results demonstrate that the lipid-to-polymer ratio significantly influences the physicochemical properties of the nanoparticles, and formulations with intermediate polymer content (1:2 and 1:3) exhibited the most favourable profiles for further biological evaluation. Formulations 1:2 and 1:3 showed particle sizes of approximately 127 and 144 nm, respectively, low PdI values below 0.130, and zeta potential values of −42.0 and −42.4 mV, supporting their selection for further CUR loading.

The mean particle size of all synthesized HPLN formulations ranged from ~116 nm to ~205 nm. The PdI values were below 0.180, confirming a relatively narrow size distribution and indicating a monodisperse system.³³ All formulations exhibited negative zeta potentials (from −40.9 to −54.9 mV), suggesting appropriate colloidal stability mediated by electrostatic repulsion, which reduces the risk of aggregation. Variations in lipid-to-polymer ratio influenced both size and surface charge of HLPNs, reflecting the interaction between the polymeric core and the stabilizing lipid monolayer. Similar observations have been reported for other polymer–lipid hybrid systems.¹¹

Particle size plays a crucial role in NPs biodistribution, cellular uptake and therapeutic efficiency. NPs smaller than 10 nm are rapidly cleared by renal filtration, whereas larger particles (> 200 nm) may activate the complement system and be quickly removed from the bloodstream, accumulating in the liver and spleen. NPs smaller than 200 nm exhibit slower clearance, allowing prolonged circulation compared with larger particles.^33,34 Therefore, in this study, particles in the intermediate range (< 150 nm) were selected, as this size is widely recognized as optimal for efficient endocytosis, extended circulation, and enhanced accumulation at pathological sites. Moghimi et al proposed that spherical nanoparticles should not exceed ~150 nm to minimize splenic filtration and subsequent clearance, thereby prolonging systemic circulation.³⁵

Stability studies showed that the 1:2 and 1:3 formulations better maintained their size and PdI compared with the 1:1 and 2:3 formulations. Additional testing in DMEM without serum and in PBS confirmed that the selected (1:2 and 1:3) formulations were stable under these conditions, supporting their suitability for further evaluation.

Upon exposure to biological media, nanoparticles readily adsorb biomolecules, primarily proteins, leading to the formation of a protein corona on the particle surface.³⁶ Adsorbed proteins can alter surface charge, promote aggregation, or otherwise destabilize nanoparticles, highlighting the need for stability studies under conditions that closely mimic the in vivo serum environment.

Incorporation of 0.5 mg/mL CUR led to an increase in nanoparticle size and PdI. CUR loading also altered the surface charge, suggesting drug-induced surface modification. PdI values increased most notably in the 1:2 formulation, indicating greater particle heterogeneity upon drug encapsulation. Encapsulation efficiency was moderate, with 38.6% for the 1:2 formulation and a slightly higher 40% for the 1:3 formulation. The EE values obtained in this study were lower compared with other reports, in which CUR-loaded hybrid, lipid, or polymeric nanoparticles achieved higher EE, depending on formulation parameters and synthesis techniques. Literature data show that EE of CUR-loaded nanoparticles varies widely, often exceeding 90%.^37,38 In contrast, some studies have reported that comparable hybrid nanoparticles exhibit only about 12% CUR entrapment.³⁹ Other types of NPs, such as CUR-loaded solid lipid NPs synthesized using a microfluidic device, achieved EE above 60%, highlighting the importance of the synthesis technique in maximizing drug encapsulation.⁴⁰ Rabanel et al observed EE of approximately 50% for polymeric NPs, with EE decreasing as CUR concentration increased, indicating saturation effects.⁴¹ Previous studies suggest that CUR EE can be enhanced by increasing the lipid fraction during synthesis. The use of appropriately selected surfactants can improve the solubilization of hydrophobic compounds, such as CUR, and stabilize interfaces to reduce drug leakage.^37,42,43 Surfactants may also be employed during sample washing to minimize post-formation drug loss. In another study, β-cyclodextrin, a cyclic oligosaccharide with a hydrophobic cavity, was used to encapsulate CUR into hybrid nanoparticles, significantly improving entrapment efficiency.⁴⁴

SEM images showed that both formulations exhibited spherical morphology with rough surfaces, consistent with previous reports on lipid–polymer hybrid nanoparticles, which demonstrated stability, monodispersity, and suitability for drug delivery applications.^11,39,45

CUR release from HLPNs depended on both environmental pH and NPs composition. Release was enhanced under acidic conditions, likely due to accelerated polymer degradation in acidic environments, thereby facilitating drug diffusion.⁴⁶ These results align with previous studies on pH-responsive hybrid carriers for hydrophobic drug delivery, highlighting their efficacy for controlled drug release in acidic pathological environments.^47,48 At neutral pH, HLPN-CUR 1:3 released slightly more CUR than HLPN-CUR 1:2, suggesting that the lipid-to-polymer ratio may influence release kinetics. The effect of acidic pH was time-dependent, particularly for HLPN-CUR 1:3, where CUR release at pH 5.5 was slightly lower than at pH 7.4 after 24 h but became greater after 48 and 72 h. At 72 h, CUR release from HLPN-CUR 1:2 reached 90.0% at pH 5.5 compared with 54.7% at pH 7.4, while HLPN-CUR 1:3 released 88.3% and 60.4%, respectively.

Morphological analysis of HF indicated that cellular responses to CUR-loaded HLPNs were strongly influenced by treatment concentration and experimental conditions. Under normoxia, cells maintained normal morphology at low nanoparticle concentrations, whereas higher doses led to cell detachment and the formation of small cell clusters. These results also suggest that the lipid-polymer ratio or CUR release profile may influence particle-cell interactions. In contrast, hypoxia-mimetic conditions increased cellular susceptibility. Even moderate nanoparticle concentrations under hypoxia-mimetic conditions caused reduced cell monolayer integrity and lower overall cell density. Previous studies have shown that hypoxia modulates HF behavior, including altered proliferation,⁴⁹ changes in protein expression⁵⁰ and differentiation toward different phenotypes.⁵¹ These alterations may render cells more vulnerable to additional environmental factors.

In this work, CUR was encapsulated into HLPNs and used as a model cargo. CUR demonstrates clinically relevant anti-inflammatory and antioxidant activities⁵² and exhibits intrinsic fluorescence, allowing assessment of both cytoprotective effects and intracellular uptake and distribution. The maximum CUR concentration of 10 µM was selected for cell viability assays based on published dose–response data. Cianfruglia et al reported that human dermal fibroblasts begin to undergo CUR-induced cell death at concentrations above 10 µM, whereas in MCF-7 breast cancer cells, viability decreased by more than 50% at concentrations exceeding 20 µM.⁵³ Similarly, in H9C2 cardiomyoblasts, CUR inhibited cell proliferation at concentrations exceeding 10 μM.⁵⁴

Pharmacokinetic studies further support the choice of 10 µM. Oral CUR exhibits low peak serum concentrations. In a Phase I clinical trial, mean serum levels of 0.51–1.77 µM CUR were reported following daily doses of 4–8 g.⁵⁵ Other studies reported low nanomolar or undetectable plasma levels after administration of doses up to 12 g/day.⁵⁶ Taken together, these data justify the use of 10 µM CUR as the highest concentration in this study, as it is close to the in vitro toxicity threshold while remaining within physiologically relevant ranges.

In this study, the MTT assay was employed to characterize the effects of CUR-loaded nanoparticles on cell viability under normoxic and hypoxia-mimetic conditions. The 72-hour MTT assay demonstrated that exposure to CUR-loaded nanoparticles (HLPN-CUR 1:2 and HLPN-CUR 1:3) induced a dose-dependent reduction in HF viability under both normoxic and hypoxia-mimetic conditions. For the HLPN-CUR 1:2 formulation, a gradual decline in cell viability was observed, and this pattern remained largely consistent between normoxia and hypoxia-mimetic conditions, as no statistically significant differences were detected. In contrast, the HLPN-CUR 1:3 formulation exhibited a less pronounced reduction in viability at lower concentrations under normoxic conditions. When comparing the effect of HLPN-CUR 1:3 under normoxic and hypoxia-mimetic conditions, HF viability was higher under hypoxia-mimetic conditions at 5 and 10 µM. This difference reached statistical significance (p < 0.05) at 10 µM, with higher MTT-derived viability observed under hypoxia-mimetic conditions than under normoxia. These findings indicate that the response to HLPN-CUR 1:3 may differ between normoxic and hypoxia-mimetic conditions and warrant further investigation in ischemia-relevant models. Under the tested conditions, HF showed greater tolerance to free CUR than to the CUR-loaded HLPN formulations. This suggests that the cellular response was influenced by the formulation type and may be related to carrier-associated effects, particle-cell interactions, or differences in CUR availability. The possible contribution of the empty carrier should also be considered. Empty HLPNs showed limited effects at lower concentrations, while changes in MTT-based cell viability became more evident at higher concentrations, particularly under hypoxia-mimetic conditions. Therefore, carrier-associated effects cannot be excluded, especially at higher HLPN-CUR concentrations. However, a limitation of this study is that cell viability was assessed only using the MTT assay, which reflects cellular metabolic activity rather than directly measuring cell survival. Because CoCl₂ may alter mitochondrial metabolism, changes in the MTT signal may not fully correspond to changes in viable cell number. Although the CUR and nanoparticle concentrations used in this study were relatively low, minor interference with the final MTT absorbance values cannot be fully excluded due to the optical properties of CUR and CUR-loaded nanoparticles. Therefore, these findings should be considered preliminary and confirmed in future studies using additional methods, such as ATP measurement, membrane integrity testing, direct cell counting, and others.

CUR is increasingly recognized for its ability to support cell viability under ischemia-like conditions through activation of multiple cytoprotective pathways, including antioxidant and anti-inflammatory mechanisms. These findings are consistent with broader antioxidant-based strategies reported in animal models. For example, sugarcane extract has been shown to reduce ROS accumulation and improve stress resistance and longevity in Caenorhabditis elegans, highlighting that modulation of oxidative stress is an important therapeutic direction.⁵⁷ Previous studies have shown that CUR enhances the survival of H9C2 cardiomyocytes subjected to hypoxia/reoxygenation injury by inhibiting apoptosis and excessive autophagy.⁵⁸ In a simulated ischemia/reperfusion model, CUR also improved H9C2 viability, reduced lactate dehydrogenase release and apoptosis, and partially preserved mitochondrial membrane potential.⁵⁴ The cytoprotective properties of CUR have been investigated not only in its free form but also in NP-based delivery systems designed to overcome its poor aqueous stability and limited bioavailability. For example, CUR-loaded polymeric nanoparticles have been reported to preserve the viability of H9C2 cardiomyocytes and reduce stress-induced cell apoptosis when applied at higher CUR concentrations (up to 100 µM) over a 24 or 48-hour period.⁵⁹ In contrast, the present study evaluated CUR-loaded HLPNs over a longer incubation period (72 h) and within a limited concentration range (≤10 µM) to reflect physiologically relevant exposure levels. Although such methodological differences limit direct comparisons between studies, our findings demonstrate that formulation composition and experimental conditions influenced cellular responses even at lower concentrations. Future studies using different concentration ranges, exposure times, disease-relevant cell models, and additional biological evaluation are needed to clarify the differences between free and nanoparticle-loaded CUR under hypoxia-mimetic conditions.

The findings obtained for HLPN-CUR 1:3 provide a basis for further evaluation in ischemia-relevant experimental models. The greater CUR release observed at pH 5.5 than at pH 7.4 at later time points suggests that this formulation may be relevant to acidic pathological microenvironments associated with ischemia and hypoxia. However, further studies in disease-relevant cellular and in vivo models are required to evaluate its biological effects, tissue distribution, and therapeutic potential.

Our study has several limitations. The biological evaluation was limited to HF, which does not fully represent cardiac ischemia-related conditions. In addition, CoCl₂ was used as a chemical hypoxia-mimicking agent, and the model was not validated using HIF-1α or other hypoxia-related biomarkers. Cellular uptake, oxidative stress, apoptosis-related markers, and hypoxia-associated signalling were also not assessed. Therefore, further studies using controlled hypoxia conditions, more relevant cardiac cell models, and additional biological endpoints are needed to evaluate better the cellular effects and mechanisms of the selected formulations.

Conclusions

In this study, optimized hybrid lipid-polymeric nanoparticles were developed by varying the lipid-to-polymer ratio. Among the tested formulations, the 1:2 and 1:3 ratios exhibited favourable physicochemical characteristics and optimal stability in physiologically relevant media. CUR-loaded nanoparticles (HLPN-CUR 1:2 and HLPN-CUR 1:3) exhibited pH-dependent release, with greater cumulative CUR release at pH 5.5 than at pH 7.4 at later time points. This release profile supports further investigation of these formulations in acidic pathological microenvironments associated with ischemia and hypoxia.

In vitro experiments with human fibroblasts showed that nanoparticle composition and experimental conditions influenced cellular morphology and MTT-derived viability. HLPN-CUR 1:3 was associated with higher MTT-derived viability under hypoxia-mimetic conditions than under normoxia at selected higher CUR concentrations. However, this observation alone does not confirm a cytoprotective effect.

Overall, the results support the selection of HLPN-CUR 1:3 for further development based on its physicochemical properties, colloidal stability, and release behaviour. Further studies using disease-relevant cell models, validated hypoxia-related experimental models, cellular uptake analysis, and other biological assays are required to determine its biological effects and potential relevance to hypoxia-associated pathological conditions.

Funding Statement

This research was supported by the Science Foundation of Lithuania University of Health Sciences project “Development of hybrid lipid-polymeric nanosystems targeting ischemic myocardium”, 2025.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.