Nanostructured lipid carriers for l-carnitine delivery: physicochemical characterization and effects on asthenozoospermic sperm function in vitro

Abstract

Asthenozoospermia, driven by oxidative stress and mitochondrial dysfunction, impairs sperm motility and contributes to male infertility. Oral L-carnitine (LC) improves sperm parameters clinically, yet its extreme hydrophilicity severely restricts transmembrane delivery into spermatozoa. However, it remains unknown whether lipid nanocarriers can overcome this previously elusive uptake barrier and augment L-carnitine bioactivity. Here we developed L-carnitine-loaded nanostructured lipid carriers (NLC-LC) and determined their capacity to enhance delivery and functional outcomes in human asthenozoospermic spermatozoa. NLC-LC (size 144 ± 3 nm, zeta − 38.6 ± 1.4 mV, encapsulation efficiency 56 ± 2%) were incubated for 1 h at 0.5 mg/mL LC equivalent with density-gradient-purified spermatozoa from 25 asthenozoospermic men alongside free LC, blank NLC, and untreated controls. NLC-LC demonstrated approximately 34% higher DPPH radical-scavenging activity than free L-carnitine in an acellular assay (47.7 ± 1.1% vs. 35.7 ± 0.9%; P < 0.01). Progressive motility rose from 14.0 ± 5.3% (control) to 20.5 ± 5.5% (free LC; P < 0.05) and 23.9 ± 7.8% (NLC-LC; P < 0.001) versus control, with no significant difference between NLC-LC and free LC (P > 0.05). NLC-LC showed numerically higher viability, lower DNA fragmentation, and higher mitochondrial membrane potential than free LC, but none of these differences reached statistical significance. NLC-LC significantly enhanced antioxidant capacity in the DPPH assay and showed superior numerical performance across all assessed sperm function parameters, although most of these improvements did not reach statistical significance compared with free L-carnitine.

Introduction

Male infertility accounts for roughly 15% of cases among couples trying to conceive; asthenozoospermia, defined as poor sperm motility, stands out as one of the leading reasons¹. When motility drops, spermatozoa struggle to swim through the female tract and fertilize the egg. That alone delays natural pregnancy and cuts the success rates of procedures like in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI)^2,3.

A large body of work points to oxidative stress as the main driver behind asthenozoospermia. Too many reactive oxygen species damage membrane lipids, disrupt mitochondria, and fragment DNA in sperm cells; each of these hits directly reduces forward movement and fertilizing ability^4–6. Spermatozoa have very little cytoplasm and almost no repair machinery, so they suffer more than most cells from oxidative attack⁷. For that reason, giving antioxidants to patients has become a standard approach to limit the damage and raise sperm quality⁸.

L-carnitine (LC) is among the most studied compounds in this field. It shuttles long-chain fatty acids into mitochondria for β-oxidation and Adenosine Triphosphate (ATP) generation; at the same time it neutralizes reactive oxygen species^9,10. Sperm rely heavily on this mitochondrial pathway to power progressive motility¹¹. Several randomized trials and meta-analyses confirm that oral LC, often combined with acetyl-LC, raises progressive and total motility, boosts sperm counts and vitality, and lowers DNA damage in men with idiopathic asthenozoospermia or oligoasthenozoospermia^11–13. Still, free LC has clear drawbacks. Its strong hydrophilic nature makes it hard for the molecule to cross cell membranes, so only a small fraction reaches the intracellular space of spermatozoa^14–16.

Free LC helps, yet no study has tested whether a lipid-based nanocarrier can carry LC directly to human sperm and improve its effects. Lipid nanocarriers are already known to stabilize hydrophilic drugs, favour cell entry, and release payload over time; properties that should strengthen both the energetic and antioxidant actions of LC inside sperm cells.

We therefore prepared a nanostructured lipid carrier (NLC) loaded with LC (NLC-LC). Lipid nanocarriers can shield hydrophilic drugs like LC from rapid clearance, favor cell entry, and release the payload gradually inside target cells. We compared NLC-LC head-to-head with the same dose of free LC on spermatozoa from asthenozoospermic patients. The central question was straightforward: does nanoencapsulation improve progressive motility, viability, mitochondrial membrane potential, and reduce DNA fragmentation after one hour of in vitro exposure? This work supplies the first direct evidence that such a carrier can bypass the main limitations of plain LC and could become a useful additive in sperm processing media for assisted reproduction.

Materials and methods

Materials

Glyceryl monostearate (GMS), oleic acid, soy lecithin (Lipoid GmbH, Germany), Tween 80, Span 80, absolute ethanol, LC, DPPH, ascorbic acid, eosin Y, nigrosin, JC-1 dye (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide), MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DMSO, and HPLC-grade methanol were purchased from Merck KGaA (Darmstadt, Germany), Sigma-Aldrich (St. Louis, MO, USA), or Thermo Fisher Scientific (Waltham, MA, USA). Deionized water was produced using an in-house Millipore system.

PBS (pH 7.4), DMEM/F12 medium, fetal bovine serum, penicillin-streptomycin, L-glutamine, and trypsin-EDTA were Gibco products from Thermo Fisher Scientific. Sperm washing medium was obtained from Origio (Denmark). The Halosperm kit used for sperm chromatin dispersion testing was supplied by Ideh Varzan Farda Co. (Iran). Human adipose-derived mesenchymal stem cells were provided by the Pasteur Institute of Iran (Tehran, Iran). All remaining chemicals were of analytical or cell-culture grade.

Preparation of NLC-LC

NLC-LC was prepared by solvent diffusion method, selected for its ability to produce nanoparticles with high encapsulation efficiency and a narrow size distribution¹⁷. The lipid phase, which comprised glyceryl monostearate (9 g), oleic acid (2.7 g), absolute ethanol (2.7 mL), soy lecithin (1.65 g), and Span 80 (1.5 g), was homogenized at 70 ± 1 °C using a temperature-controlled hot plate. At the same time, the water inner phase with LC (50 mg in 4.5 mL deionized water) and the outer aqueous phase (295.5 mL deionized water and 8.25 g Tween 80) were also heated to 70 ± 1 °C in a temperature-controlled water bath. The temperature was continuously monitored with a calibrated thermometer throughout the process to ensure consistency. The lipid phase was sonicated at 800 rpm by an ultrasonic probe homogenizer, then sequentially added internal and external aqueous phases. Sonication was continued for 5 min, followed by 300 mL ice-cold water at 0 °C to be added and freeze the particles. The resulting suspension was centrifuged at 9,500 rpm, and nanoparticles were sifted, freeze-dried, and stored (Fig. 1).

Fig. 1.

Schematic illustration of the multi-step preparation process for NLC-LC.

Physicochemical characterization

Fourier-transform infrared (FTIR) spectroscopy

FTIR spectra of free LC, blank NLC, and NLC-LC were obtained utilizing a Bruker Alpha spectrophotometer (Germany). Samples were formulated as potassium bromide (KBr) pellets and analyzed over the spectrum of 400–4000 cm⁻¹ at a resolution of 4 cm⁻¹¹⁸.

Scanning electron microscopy (SEM)

The surface morphology and structural characteristics of the NLC-LC were examined using SEM with a TESCAN Vega (Czech Republic). Lyophilized nanoparticle samples were mounted on aluminum stubs using double-sided carbon tape and coated with a thin layer of gold using a sputter coater to enhance conductivity¹⁹. Particle shape, surface texture, and aggregation behavior were examined at high vacuum at 15 kV and 50kx magnification.

Stability assessment

Long-term physicochemical stability of the NLC-LC was assessed by determination of particle size, polydispersity index, and zeta potential by using a Malvern Zetasizer Nano ZS (Malvern Instruments, UK) fitted with a zeta potential measurement module²⁰. The determinations were performed immediately after synthesis (fresh samples) and after 3 months of storage at 4 ± 1 °C under dark conditions. This temperature was selected because sperm-washing media and related ART laboratory media are routinely stored at 4 °C prior to use; therefore, evaluating nanoparticle stability under these real storage conditions was essential to match the intended clinical workflow of the formulation. Lyophilized nanoparticles were previously redispersed in deionized water and suitably diluted with water to reach an appropriate scattering intensity. All the measurements were made at 25 °C in triplicate. Particle size, PDI, and zeta potential changes during time were taken as indicative of colloidal stability, aggregation tendency, and physical integrity of the nanosystem.

Encapsulation efficiency and drug loading

The EE% and LC% of NLC-LC were determined by both indirect and direct methods, with quantification by high-performance liquid chromatography^21,22.

Indirect method

The free (unencapsulated) LC was separated from the nanoparticle fraction by centrifugation of the nanosuspension at 9,500 rpm for 30 min at 4 °C. The supernatant was carefully collected, filtered through a 0.22 μm syringe filter, and analyzed by HPLC (DanChrom Sahand Series, Iran) which was fitted with a C18 column (100 × 4.6 mm, 3 μm particle size). The mobile phase consisted of methanol:0.05% phosphate buffer (80:20 v/v), flow rate 1.0 mL/min, column temperature 30 °C, and UV detection at 205 nm.

Direct method (for confirmation)

To this end, a known amount of freeze-dried nanoparticles was dissolved in chloroform: methanol (2:1 v/v), sonicated in an ice bath for 10 min, and centrifuged. Organic phase containing extracted LC was evaporated under mild conditions, reconstituted in mobile phase, and analyzed by HPLC under identical chromatographic conditions.

EE% and LC% were calculated according to the following equations:

M_Total denotes the total quantity of LC initially incorporated, M_Free signifies the amount of unbound LC present in the supernatant, and M_NP represents the total mass of nanoparticles produced post-synthesis. The calculations were employed to evaluate the efficacy of LC integration into the lipid matrix and the drug loading capacity of the nanocarriers. All measurements were performed in triplicate. The results of both methods were consistent; thus, both were applied to validate the efficiency of LC incorporation into the lipid matrix.

In vitro drug release study

The NLC-LC’ in vitro LC release profile was assessed utilizing dialysis membrane diffusion²³. Pre-activated Spectrum Labs dialysis membranes with a molecular weight cut-off of 12–14 kDa were soaked in deionized water for 1 h, rinsed, and equilibrated in PBS (pH 7.4) for 30 min. Before loading into dialysis bags, nanoparticle suspensions containing 10 mg LC were centrifuged at 14,000 rpm for 30 min and resuspended in 2 mL PBS. For physiological simulation, the dialysis bags were immersed in 100 mL PBS (pH 7.4) and magnetically stirred at 100 rpm at 37 °C. A 1 mL aliquot was removed from the release medium and replaced with an equivalent volume of fresh PBS at 0, 1, 2, 4, 8, 12, 24, 48, and 72 h to maintain sink conditions. A Shimadzu UV-1800 UV-Vis spectrophotometer measured absorbance at 280 nm to quantify LC release at each time point.

Antioxidant activity assessment

The antioxidant efficacy of free LC, NLCs, and NLC-LC was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, a rapid, reproducible, and internationally accepted first-tier method for assessing the intrinsic chemical antioxidant capacity of small molecules and nanoformulations²⁴. Although the DPPH assay is an acellular model and does not fully mimic the complex intracellular redox environment of spermatozoa, it is particularly well-suited for highly hydrophilic compounds such as LC, which exhibit poor passive membrane permeability in their free form. The assay therefore provides a reliable comparative measure of the direct radical-scavenging potential and stability of the redox-active groups of LC before and after nanoencapsulation.

Test samples (free LC, blank NLCs, and NLC-LC) were dispersed in ethanol at a final concentration of 1 mg/mL. Ascorbic acid (0.5 mM in methanol) served as the positive control, while pure methanol was used as the negative control. A freshly prepared 0.02% (w/v) DPPH solution in methanol was used as the radical source. For each assay, 2 mL of DPPH solution was mixed with 2 mL of test sample and incubated in the dark at room temperature for 30 min. Absorbance was subsequently measured at 517 nm using a UV-Vis spectrophotometer. Radical scavenging activity (%) was calculated using the formula:

A₀ is the negative control (methanol + DPPH) absorbance, while A₁ is the test sample. Results from triplicate experiments are shown as mean ± standard deviation.

Cytotoxicity assay

Free LC, NLC-LC, and blank NLCs were tested for cytotoxicity on human adipose tissue-derived mesenchymal stem cells (MSCs) using the MTT assay²⁵. MSCs were procured from the Pasteur Institute, Tehran, Iran, and expanded according to the supplier’s standard protocols. Cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a humidified incubator with 5% CO₂. After 60–70% confluence, trypsin-EDTA detached cells and a Neobar hemocytometer measured vitality using trypan blue exclusion. Only cell suspensions with > 90% viability were tested for cytotoxicity. The MTT experiment involved seeding 1 × 10⁴ cells per well into 96-well flat-bottom plates and allowing them to adhere for 24 h. Cells were cultured for 48 h after treatment with the test formulations. Subsequent to the treatment, the media was substituted with MTT solution (2 mg/mL in PBS), diluted with fresh medium at a 3:1 ratio, yielding a final volume of 200 µL per well. The plates were incubated for four hours to produce formazan crystals. After incubation, remove the media and dissolve crystals using 25 µL of Sorensen’s phosphate buffer (0.133 M, pH 7.2) and 200 µL of DMSO. An ELISA microplate reader (ELx800; BioTek, USA) assessed absorbance at 570 nm.

Collection and treatment of sperm samples

The study received ethical approval from the Ethics Committee of North Khorasan University of Medical Sciences (IR.NKUMS.REC.1403.044). Semen samples were obtained from twenty-five infertile men aged 20–45 years who were diagnosed with isolated asthenozoospermia, defined as total sperm motility between 10 and < 40% and a concentration of at least 5 × 10⁶/mL without leukocytospermia. All participants attended the Bent Al-Hoda Infertility Center in Bojnourd, Iran, and provided written informed consent prior to enrollment. Individuals with severe oligoasthenozoospermia, systemic illness, varicocele, a history of chemotherapy or radiotherapy, or antioxidant supplementation within the previous three months were excluded from the study²⁶. Semen samples were collected by masturbation following 3–5 days of abstinence and were allowed to liquefy for 20–30 min at 37 °C. After initial macroscopic and microscopic evaluation according to WHO 2010 guidelines, samples were washed using centrifugation (400 g for 5 min) in Sperm medium. Each sample was subsequently divided into four equal aliquots, each containing 15 × 10⁶ spermatozoa, and incubated for one hour at 37 °C with one of the following treatments: Sperm medium alone (control), free LC at 0.5 mg/mL, blank NLCs without drug, or LC–loaded NLCs at an LC–equivalent concentration of 0.5 mg/mL.

Evaluation of sperm parameters

Motility

Progressive and non-progressive motility were assessed using computer-assisted sperm analysis (CASA) and manual counting under phase-contrast microscopy (×40)²⁷.

Viability

Viability was determined by eosin–nigrosin staining. Briefly, 10 µL semen was mixed with 10 µL 0.5% eosin Y, incubated for 30 s, mixed with 20 µL 10% nigrosin, smeared, air-dried, and examined at ×1000 magnification. At least 200 spermatozoa were counted per sample^28,29.

DNA fragmentation (SCD/Halosperm)

Sperm DNA fragmentation was evaluated using the Halosperm kit according to the manufacturer’s protocol. Spermatozoa exhibiting large/medium halos were considered intact; small or no halos indicated fragmented DNA. A minimum of 400 spermatozoa were evaluated per sample^30,31.

Mitochondrial membrane potential (MMP)

MMP was assessed using JC-1 staining. Samples (1 × 10⁶ spermatozoa/mL) were incubated with 2 µM JC-1 for 15 min at 37 °C in the dark, washed, and analyzed by flow cytometry (BD FACSCalibur). The red/green fluorescence ratio was calculated³².

Statistical analysis

Data analysis was conducted utilizing GraphPad Prism version 8.4.0 (GraphPad Software, USA). Continuous variables were represented as mean ± standard deviation (SD). Comparisons between two groups were performed using Student’s t-test, whilst one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was employed for comparisons among three or more groups. The Shapiro-Wilk and Levene’s tests were employed to evaluate normality and homogeneity of variance, respectively, if appropriate. A p-value below 0.05 (p < 0.05) was deemed statistically significant. All experiments were performed with at least three independent replicates (n ≥ 3), and the quantity of biological and technical replicates is specified in the respective figure legends.

Results and discussion

FTIR analysis

FTIR spectroscopy was used to probe possible interactions between LC and the components of the lipid matrix (Fig. 2). Pure LC showed its expected bands: one at 994.3 cm⁻¹ coming from C–O stretch in the –COH group right next to the quaternary ammonium, a band around 1590 cm⁻¹ for the asymmetric –COO⁻ stretch, and a wide one from 3200 to 3600 cm⁻¹ that belongs to O–H and N–H stretches of the zwitterionic form¹⁵. The unloaded NLC gave the usual lipid pattern; strong ester carbonyl at 1735 cm⁻¹ from glyceryl monostearate and lecithin, C–H stretches filling 2850–2950 cm⁻¹, and absolutely nothing between 980 and 1010 cm⁻¹³³.

Fig. 2.

FTIR spectra of free LC, blank NLCs, and NLC-LC.

Once LC was added, that sharp 994.3 cm⁻¹ peak was gone. A new, broad absorption appeared instead between 1209 and 1215 cm⁻¹. The carboxylate band near 1590 cm⁻¹ became much weaker and wider. Shifts like these indicate contact between the zwitterionic parts of LC (–COO⁻ and –N⁺(CH₃)₃) and polar groups on the lipids: hydroxyls from glycerol monostearate, carboxylates in oleic acid, phosphate in lecithin. Hydrogen bonds or electrostatic attraction explain it best.

Spectral changes by themselves do not prove encapsulation occurred. Physical mixtures often show the same effects at interfaces. Independent HPLC measurements gave 56.0 ± 2.1% encapsulation ; SEM images contained no free LC crystals . Those results confirm that most LC resides inside the lipid structure rather than sitting on the surface or precipitating separately. FTIR data agree with interactions that hold LC in place, yet the spectra must be read together with encapsulation efficiency and morphology findings.

SEM analysis

Freeze-dried NLC-LC samples were checked under scanning electron microscopy (Fig. 3). Most particles looked spherical; a few were a bit irregular. Surfaces came out smooth. We used ImageJ to measure diameters by hand from several micrographs. Sizes varied from 79 nm to 192 nm. The majority ranged 100–170 nm. No needle-like or plate-like crystals that would point to free LC appeared anywhere on the particles. Limitations exist with SEM for these systems. Lyophilization removes water; vacuum pulls the rest, gold coating follows. Particles shrink during the process. Some flatten. Others clump lightly. None of that represents the real hydrated suspension. Accurate size and shape data require less destructive methods. Transmission electron microscopy gives higher resolution and introduces far fewer changes from sample preparation. We relied on SEM here because the equipment was readily available and we mainly wanted fast visual proof that particles stayed in the nano range and that no large drug crystals sat on the outside. Measured dry diameters matched acceptably with the hydrodynamic diameter obtained by dynamic light scattering on the aqueous dispersion (144.2 ± 4.8 nm, PDI 0.19 ± 0.03). Dry samples always read smaller. The images therefore confirm formation of roughly spherical nanoparticles without detectable free drug crystals on their surfaces.

Fig. 3.

NLC-LC SEM images. Spherical nanoparticles exhibiting uniform dispersion (scale bar: 500 nm; magnification: 50,000×; HV: 15.0 kV). Subtle surface imperfections indicate the amorphous lipid matrix.

Stability analysis

The colloidal stability of the lyophilized NLC-LC was checked using dynamic light scattering and zeta potential measurements. We looked at the samples right after preparation and again after three months stored at 4 ± 1 °C in the dark (Fig. 4). Fresh particles showed an average hydrodynamic diameter of 144.2 ± 3.2 nm, PDI came in at 0.19 ± 0.02, and zeta potential was − 38.6 ± 1.4 mV. Three months later the size had gone up to 150.6 ± 4.1 nm; that’s roughly a 4.5% increase. PDI rose to 0.34 ± 0.03 while zeta potential dropped to − 32.3 ± 1.8 mV. Size and PDI differences were statistically significant (p < 0.05, unpaired t-test), but the shifts were minor. Importantly, the zeta potential never fell below the |30| mV mark most people accept as enough electrostatic repulsion to keep particles from clumping irreversibly in colloidal dispersions.

Fig. 4.

Stability assessment of NLC-LC.

Storage stability was assessed exclusively at 4 ± 1 °C, as this temperature corresponds to the routine refrigeration conditions applied to commercial sperm-washing media and cryopreservation solutions in fertility clinics. These conditions therefore replicate the anticipated real-world storage of the formulation. Although only minor changes in particle size, polydispersity index, and zeta potential were observed after three months of refrigerated storage, stability at room temperature (25 °C) or under accelerated conditions (40 °C/75% RH) was not evaluated. Polymorphic transitions or recrystallization of the lipid matrix can occur in nanostructured lipid carriers during prolonged exposure to low temperatures. Consequently, comprehensive stability data across a broader temperature range would be required before a definitive shelf-life can be established.

In short, the small changes in particle size, polydispersity, and surface charge over three months at 4 °C suggest the NLC-LC kept its nanoscale properties and did not aggregate under storage conditions relevant to clinical sperm processing.

Encapsulation efficiency and drug loading

EE% and LC% were measured using reversed-phase HPLC. We used both the indirect method (quantifying free LC in the supernatant) and the direct method (extracting drug from freeze-dried nanoparticles); see Fig. 5. The calibration curve ranged from 0.46 to 11.2 µg/mL and showed good linearity: y = 300.31x − 104.87, R² = 0.9984. The two methods gave close results. The indirect approach found 0.42 ± 0.03 mg/mL free drug. Direct extraction recovered 0.54 ± 0.04 mg/mL from the nanoparticles. Across three replicates the averages came out to EE% = 56.0 ± 2.1% and LC% = 22.0 ± 1.3%.

Fig. 5.

HPLC chromatograms and calibration curve for LC quantification. Overlaid chromatograms show the LC standard (blue), extracted drug from NLC-LC (green), and free drug from supernatant (red). Inset: Calibration curve (y = 300.31x − 104.87, R² = 0.9884). Excellent agreement between direct and indirect methods validates the reported EE% (56 ± 2%) and LC% (22 ± 1%).

56% EE% seems moderate when you compare it to many lipophilic drugs in NLCs, which commonly reach 80–90% or more. For a highly hydrophilic compound like LC, however, the number is clearly better than earlier reports. Yaşacan et al. (2020) reported only 14.3 ± 3.5% in conventional liposomes and 21.9 ± 4.2% in PLGA nanoparticles³⁴. The present system therefore achieves about 2.5- to 4-fold higher encapsulation. That improvement fits with what is known about NLCs: their disordered lipid matrix creates extra space for polar and zwitterionic molecules compared to the more organized structures of liposomes or polymeric nanoparticles. Further optimization of the formulation could still raise EE% and payload. Even without additional changes, the current NLC system already offers a clear advance over previous carriers for LC.

In vitro drug release study

The in vitro release profile of LC from NLC-LC was evaluated using the dialysis bag method (12–14 kDa cut-off) in phosphate-buffered saline (PBS, pH 7.4) at 37 °C under sink conditions (Fig. 6). A linear calibration curve was obtained at 280 nm in the range 0–6 µg/mL (y = 0.0181x − 0.0135, R² = 0.9805). The release exhibited biphasic kinetics: an initial rapid phase reaching 70.3 ± 3.8% cumulative release within the first 8 h, followed by a slower phase that approached 94.2 ± 2.1% by 72 h (Fig. 6B). The early burst is likely attributable to the fraction of LC located at or near the particle surface, whereas the subsequent prolonged release reflects drug entrapped within the lipid matrix.

Fig. 6.

In vitro release profile of LC from NLC-LC. (A) UV calibration curve (y = 0.0181x − 0.0135, R² = 0.9805). (B) Cumulative release (%) over 72 h in PBS (pH 7.4) at 37 °C, showing an initial burst (~ 70% in 8 h) followed by slower release reaching 94.2 ± 2.1% at 72 h. Data are mean ± SD (n = 3).

Although biphasic release is commonly observed for hydrophilic drugs incorporated into NLCs, the dialysis membrane itself introduces an additional diffusion barrier that can exaggerate the burst phase and prolong the terminal phase^35,36. In the absence of complementary techniques such as differential scanning calorimetry (DSC), X-ray diffraction (XRD), or transmission electron microscopy (TEM), the exact contribution of lipid polymorphism, drug–surfactant interactions, or matrix erosion to the observed profile cannot be definitively established. Nevertheless, the release pattern suggests that a substantial proportion (> 70%) of the encapsulated LC becomes available within the first few hours of incubation, which aligns with the short (1 h) exposure period used in the subsequent sperm function experiments.

Kinetic modeling of LC release

To gain insight into the release mechanism, the cumulative release data were fitted to six common mathematical models (Table 1; Fig. 7). The Korsmeyer–Peppas model provided the best fit (R² = 0.9162), with a diffusional exponent n ≈ 0.45, consistent with Fickian diffusion from essentially spherical particles³⁷. The Higuchi model also showed reasonable correlation (R² = 0.8048), further supporting diffusion through the lipid matrix as the predominant mechanism after the initial burst. First-order (R² = 0.8691) and zero-order (R² = 0.5655) models fitted less well, whereas Hixson–Crowell (R² = 0.6335) and Baker–Lonsdale (R² = 0.5109) models exhibited poor correlation, indicating negligible contribution from surface erosion^15,35.

Table 1.

Kinetic modeling of released LC from fabricated nanocarrier.

Model	Equation	R²
Zero-order	Qt = k0t	0.5655
First-order	ln(1 − Qt) = − kt1t	0.8691
Higuchi	Qt = kh√t	0.8048
Hixson–Crowell	(1 − Qt)1/3 = 1 − khct	0.6335
Baker–Lonsdale	3/2[1 − (1 − Qt)2/3] − Qt = k6t	0.5109
Korsmeyer–Peppas	Qt/Q∞  = kkptn	0.9162 (n = 0.45)

Fig. 7.

Graphical representation of fit to Zero-order, First-order, Higuchi, Hixson–Crowell, Baker–Lonsdale, and Korsmeyer–Peppas models.

These results must be interpreted cautiously because kinetic modeling of dialysis-bag data is frequently confounded by membrane resistance and may not fully reflect release behaviour in biological media. Nonetheless, the predominance of Fickian diffusion is in agreement with the near-spherical morphology observed by SEM and the high negative zeta potential (− 38.6 mV), both of which favour particle stability and limit erosion-driven release.

Antioxidant activity assessment

The DPPH radical scavenging activity of the formulations at 1 mg/mL is shown in Fig. 8. Ascorbic acid (positive control) exhibited the highest activity (88.5 ± 1.2%). Free LC displayed moderate scavenging (35.7 ± 0.9%), while blank NLCs showed low but detectable activity (17.6 ± 0.5%), likely attributable to the unsaturated fatty acid (oleic acid) and phospholipid (soy lecithin) components, which are known to possess mild intrinsic antioxidant properties^38,39. NLC-LC exhibited significantly higher scavenging activity (47.7 ± 1.1%) than free LC (p < 0.01) and blank NLCs (p < 0.001), corresponding to an approximately 34% relative improvement over free LC.

Fig. 8.

DPPH radical scavenging activity of ascorbic acid (positive control), free LC (Free LC), blank NLCs, and LC-loaded NLCs (NLC-LC) at 1 mg/mL. Data are mean ± SD (n = 3). ****p < 0.0001 vs. all other groups; ***p < 0.001 vs. blank NLC.

It is important to note that the DPPH assay is an acellular chemical test that measures direct electron/hydrogen-donating capacity in an organic medium and does not reflect cellular uptake, intracellular release, or biological antioxidant pathways operative in spermatozoa. Therefore, the observed increase in scavenging activity with NLC-LC should be interpreted cautiously as evidence of improved chemical stability or presentation of LC rather than definitive proof of enhanced intracellular antioxidant protection. Although the blank NLC contributed approximately 17–18% scavenging activity, the effect of NLC-LC (47.7%) was not markedly greater than the arithmetic sum of free LC (35.7%) and blank NLC (17.6%) contributions when tested separately. This suggests that encapsulation primarily preserves the intrinsic radical-scavenging capacity of LC rather than generating a strong synergistic interaction at the chemical level in this assay system^40,41. In the subsequent biological experiments (1-h incubation with asthenozoospermic spermatozoa), NLC-LC produced numerical improvements in progressive motility, viability, mitochondrial membrane potential, and DNA integrity compared with free LC; however, most of these differences did not reach statistical significance . Thus, while the DPPH results demonstrate that nanoencapsulation enhances the chemical antioxidant potential of LC under acellular conditions, they do not fully predict or explain the modest and largely non-significant biological outcomes observed in sperm cells. Additional cell-based assays (e.g., intracellular ROS measurement, lipid peroxidation, or direct ATP quantification) would be required to establish whether the improved chemical scavenging translates into superior intracellular antioxidant efficacy in this specific biological context^42–44.

Cytotoxicity assay

The potential cytotoxicity of free LC and the NLC formulations was evaluated using the MTT assay on human adipose-derived mesenchymal stem cells after 72 h of exposure at 0.5 mg/mL, the same LC-equivalent concentration used in the subsequent sperm experiments. As shown in Fig. 9, all groups exhibited high cell viability (> 98%) relative to the untreated control (set at 100%). Mean viability values were 100% for control, 99.1 ± 2.4% for free LC, 99.8 ± 2.1% for blank NLCs, and 103.2 ± 2.6% for NLC-LC. One-way ANOVA followed by Tukey’s post-hoc test revealed no statistically significant differences between any treatment group and the untreated control (p > 0.05). A minor increase in formazan formation was observed only when NLC-LC was compared pairwise with free LC (p < 0.05), but the absolute difference was small (~ 4%) and remained well within the typical variability range of the MTT assay.

Fig. 9.

Cell viability of human adipose-derived mesenchymal stem cells after 72 h exposure to free LC (LC), blank NLCs, and LC-loaded NLCs (NLC-LC) at 0.5 mg/mL, determined by MTT assay. Data are mean ± SD (n = 6 independent experiments). *p < 0.05 only vs. free LC; no group differed significantly from untreated control.

These results demonstrate that neither free LC nor the blank or loaded NLC formulations exhibit cytotoxicity at the tested concentration. The lipid matrix components and the final NLC-LC particles are well tolerated. The slightly higher signal observed with NLC-LC should not be interpreted as evidence of enhanced mitochondrial activity or metabolic stimulation, as values exceeding 100% are frequently encountered in MTT assays due to minor assay variability or non-specific nanoparticle interference with formazan formation. Overall, the data confirm the excellent biocompatibility and safety profile of the developed NLC-LC system for potential application in sperm-processing media.

Motility

Progressive motility

Progressive motility was assessed in asthenozoospermic spermatozoa (n = 25 per group) after 1 h incubation at 37 °C. Mean progressive motility values were 14.0 ± 5.3% for control, 20.5 ± 5.5% for free LC, 15.8 ± 6.2% for blank NLCs, and 23.9 ± 7.8% for NLC-LC (Fig. 10). One-way ANOVA revealed significant differences among groups (p < 0.001). Tukey’s post-hoc test showed that both free LC and NLC-LC significantly improved progressive motility compared with control (p < 0.05 and p < 0.001, respectively), and NLC-LC significantly improved progressive motility compared with blank NLCs (p < 0.05). However, no statistically significant difference was observed between NLC-LC and free LC (p > 0.05).

Fig. 10.

Progressive sperm motility after 1 h incubation with the different formulations in asthenozoospermic samples. Data are mean ± SD (n = 25). ***p < 0.001, *p < 0.05 vs. control; ns = not significant (one-way ANOVA followed by Tukey’s post-hoc test).

Although NLC-LC yielded the highest numerical value, the lack of statistical significance versus free LC indicates that nanoencapsulation did not provide a detectable additional benefit on progressive motility under the conditions tested (0.5 mg/mL equivalent LC, 1 h exposure). The observed improvement over control is therefore attributable to LC itself rather than the nanocarrier.

Nonprogressive motility

Non-progressive motility was evaluated in the same samples. Mean values were 11.5 ± 3.1% for control, 14.4 ± 3.3% for free LC, 12.0 ± 3.3% for blank NLCs, and 15.7 ± 3.8% for NLC-LC (Fig. 11). Post-hoc analysis revealed that NLC-LC significantly improved non-progressive motility compared with the control (p < 0.01) and blank NLCs (p < 0.05). Free LC also showed a numerical increase versus control, but the difference did not reach statistical significance. Overall, one-way ANOVA indicated a trend toward improvement that did not achieve statistical significance across all groups (p > 0.05).

Fig. 11.

Non-progressive sperm motility after 1 h incubation with the different formulations. Data are mean ± SD (n = 25). **p < 0.01, *p < 0.05, ns = not significant (one-way ANOVA followed by Tukey’s post-hoc test).

Although L-carnitine is primarily recognized for supporting energy-dependent forward (progressive) motility, the significant enhancement of non-progressive motility observed specifically with NLC-LC suggests that nanoencapsulation may facilitate additional protective or bioenergetic effects on local flagellar beating in asthenozoospermic spermatozoa. Importantly, neither blank NLCs nor NLC-LC reduced non-progressive motility relative to the untreated control, confirming the biocompatibility and non-toxic nature of the lipid carrier.

Sperm viability

Sperm viability was determined by eosin–nigrosin staining after 1 h incubation at 37 °C (n = 25 per group). Mean viability values were 71.6 ± 9.6% for control, 74.2 ± 8.4% for free LC (LC), 71.8 ± 8.5% for blank NLCs, and 76.9 ± 8.4% for NLC-LC (Fig. 12). One-way ANOVA showed no statistically significant differences among groups (p = 0.15).

Fig. 12.

Sperm viability in asthenozoospermic samples after 1 h incubation with the different formulations. Data are mean ± SD (n = 25). ns = not significant (one-way ANOVA).

Although NLC-LC displayed the highest numerical value, the differences were small and not statistically significant. No formulation adversely affected membrane integrity.

DNA fragmentation index (DFI)

DNA fragmentation was evaluated using the sperm chromatin dispersion (SCD) assay. Mean DFI values were 23.7 ± 6.8% for control, 22.0 ± 7.6% for free LC, 22.0 ± 8.4% for blank NLCs, and 18.3 ± 8.4% for NLC-LC (Fig. 13). One-way ANOVA revealed no statistically significant differences among groups (p > 0.05).

Fig. 13.

Sperm DNA fragmentation index (DFI) after 1 h incubation with the different formulations. Data are mean ± SD (n = 25). ns = not significant (one-way ANOVA).

NLC-LC showed the lowest mean DFI, but the observed trend did not reach statistical significance. The lack of significance is likely attributable to high inter-individual variability and the moderate sample size, as well as short incubation time in the laboratory environment.

Mitochondrial membrane potential (MMP)

Mitochondrial membrane potential was assessed by JC-1 staining and flow cytometry. The percentage of spermatozoa exhibiting high MMP (red/green ratio corresponding to JC-1 aggregates) was 70.0 ± 18.3% for control, 75.9 ± 12.9% for free LC, 75.9 ± 13.0% for blank NLCs, and 80.4 ± 12.1% for NLC-LC (Fig. 14). Representative flow cytometry dot plots are shown in Fig. 15. One-way ANOVA indicated no statistically significant differences among groups (p > 0.05).

Fig. 14.

Percentage of spermatozoa with high mitochondrial membrane potential after 1 h incubation with the different formulations. Data are mean ± SD (n = 25). ns = not significant (one-way ANOVA).

Fig. 15.

Representative JC-1 flow cytometry dot plots. Spermatozoa with high MMP appear in the upper-right quadrant (Q2); those with low MMP appear in the lower-right quadrant (Q3).

Although NLC-LC yielded the highest numerical value, the differences were not statistically significant. High biological variability in asthenozoospermic samples probably contributed to the absence of statistical differences.

Conclusion, limitations, and future direction

The present study describes the first successful development and in vitro evaluation of L-carnitine-loaded nanostructured lipid carriers (NLC-LC) specifically designed for application in human asthenozoospermic spermatozoa. The formulation exhibited favorable physicochemical properties, including an encapsulation efficiency of 56 ± 2%, particle size of 144 ± 3 nm, highly negative zeta potential (− 38.6 ± 1.4 mV), good colloidal stability under refrigerated conditions for three months, and a biphasic release profile.

Incubation of density-gradient-purified asthenozoospermic spermatozoa for 1 h with NLC-LC (0.5 mg/mL LC-equivalent) resulted in significantly higher progressive motility than untreated control (23.9 ± 7.8% vs. 14.0 ± 5.3%; P < 0.001) and numerically (but not statistically significantly) higher progressive motility than free LC (23.9 ± 7.8% vs. 20.5 ± 5.5%; p > 0.05). NLC-LC also significantly improved non-progressive motility compared with blank NLC and control groups in post-hoc analysis. Across all secondary endpoints (viability, DNA fragmentation index, and mitochondrial membrane potential), NLC-LC consistently yielded the most favorable numerical values, although differences did not reach statistical significance versus free LC. Importantly, NLC-LC exhibited 34% greater DPPH radical-scavenging activity than free LC (P < 0.01), confirming enhanced antioxidant capacity upon nanoencapsulation.

These results indicate that, while free LC itself exerts beneficial effects on sperm motility, nanoencapsulation in NLCs further augments antioxidant activity and produces consistent numerical improvements in all assessed functional parameters, although these additional benefits over free LC did not reach statistical significance in most endpoints under the tested conditions (1-hour incubation, 0.5 mg/mL).

Key limitations include the exclusively in vitro design, moderate sample size (n = 25), lack of direct evidence of enhanced cellular uptake or intracellular LC delivery (no uptake or intracellular quantification studies were performed), and the brief exposure duration, which may have masked advantages conferred by the sustained-release profile of NLC-LC. No clinical endpoints such as fertilization rate, embryo quality, or pregnancy outcome were evaluated.

Future studies should incorporate direct measurement of LC uptake into spermatozoa (e.g., using fluorescent or radiolabeled LC), quantification of intracellular reactive oxygen species, lipid peroxidation, and ATP content, as well as longer incubation periods or lower LC doses to better reveal potential benefits of nanoencapsulation. Additional stability testing at room temperature and under accelerated conditions, along with formal pharmacokinetic profiling in biological media, is recommended. Ultimately, randomized clinical trials assessing reproductive outcomes will be essential to determine whether NLC-LC provides meaningful advantages over conventional LC supplementation in assisted reproductive technology.

Author contributions

Aniseh Mardanpoor Moghadam: Data curation, writing – original draft & editing. Sonia Fathi-karkan: Conceptualization, Investigation, Writing – original draft & editing, Supervision. Fatemeh Tanhaye kalate Sabz: Conceptualization, Investigation, Writing – original draft, Supervision. Sahar Shariatnia: Writing – original draft & editing.

Funding

The current study was approved & supported by North Khorasan University of Medical Sciences with Reg. No. 4020335 was approved by ethical committee of North Khorasan University of Medical Sciences. The Ethic approval Cod is IR.NKUMS.REC.1403.044.

Data availability

All data supporting the findings of this study are available within the paper.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

The present study was approved by the Ethics Committee of North Khorasan University of Medical Sciences, Bojnurd, Iran (approval code: IR.NKUMS.REC.1403.044). All procedures were conducted in accordance with the ethical standards of the institutional research committee and with the principles outlined in the Declaration of Helsinki. Written informed consent was obtained from all participants prior to semen sample collection. No identifiable personal information was recorded, and all samples were processed anonymously. AI tools were used for grammar and text enhancement, but the content was primarily authored by the writers.