Physalis polysaccharide-sericin nanocarriers for controlled alpha-lipoic acid delivery and improved human sperm cryopreservation

Abstract

The development of delivery systems that deliver ALA as a cryoprotectant to sperm in a controlled manner could provide a promising strategy to minimize the toxicity associated with direct use of alpha-lipoic acid (ALA) during cryopreservation. Therefore, the aim of this study was to design and evaluate a nanocarrier based on sericin (SER) and a polysaccharide extracted from Physalis alkekengi for ALA encapsulation in a way that minimizes the toxicity resulting from direct use of ALA by controlling the release. To find the optimal ratio of SER and polysaccharide (pro/poly), a polysaccharide-SER-based nanocarrier (Ph-SER-NC) was synthesized using different ratios of pro/poly. Then, based on the optimization results, Ph-SER-NC was synthesized by the nanoprecipitation method, and ALA was loaded into the nanocarriers by direct system of dissolution into the aqueous phase (ALA-Ph-SER-NC). Its physicochemical characteristics were determined by the dynamic light scattering (DLS) technique. Furthermore, Fourier transform infrared spectroscopy (FTIR) was used to characterize the chemical composition, molecular properties, and surface adsorption of functional groups of the ALA-Ph-SER-NC. Then, the effects of ALA-Ph-SER-NC on biological properties after cryopreservation were investigated. The results showed that the optimal ratio of pro/poly for Ph-SER-NC synthesis was 1:2, and the resulting Ph-SER-NC had a Z-average and PDI of 109 nm and 0.21, respectively. After ALA loading, the physicochemical properties of the ALA-Ph-SER-NC, including Z-average, PDI, and zeta potential of 186.9 nm, 0.456, and − 11.8 mV, were determined, respectively. The encapsulation efficiency for ALA was determined to be 93.7%. Biological evaluation showed that at concentrations of 0.02 and 0.05 mmol/mL, the ALA-Ph-SER-NC group significantly improved sperm parameters, including increases of approximately 26% in total motility, 24% in morphology, and 12% in viability, along with a 28% reduction in DNA fragmentation index (DFI). The results indicated that ALA-Ph-SER-NC, through controlled release of ALA, preserved key biological properties of sperm. Thus, the synthesized ALA-Ph-SER-NC offers a novel and effective approach to preserve sperm quality during cryopreservation in men at risk of reduced fertility.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-56524-3.

Introduction

Male infertility is considered one of the main reasons for the decline in the fertility rate. Sperm quality and male fertility are affected by various factors such as age, diseases, and environmental factors^1,2. Sperm cryopreservation, or sperm freezing under favorable conditions, is an effective strategy to reduce problems associated with male infertility in humans and to preserve biodiversity in animals¹. In recent decades, cryoprotectants (CPAs) have been commonly used for sperm freezing^1,3. The best known of the penetrating cryoprotectants are ethylene glycol (EG), glycerol (GLY)⁴, dimethyl sulfoxide (DMSO)⁵, and antioxidants such as alpha-lipoic acid (ALA)^6,7. CPAs protect sperm by inhibiting ice-crystal formation and preserving membrane integrity^3,5. Despite their widespread use, cryoprotectants present significant challenges. One of the main issues is cytotoxicity, which can cause physiological damage and metabolic changes in sperm. The use of cryoprotectants can cause damage to the plasma membrane, trigger osmotic stress, and promote excessive production of reactive oxygen species (ROS) in sperm. As a result, it affects the quality of sperm⁸. The most critical processes involved in sperm damage during cryopreservation include ice crystal formation, which affects the tissue’s chemical makeup and disrupts cell integrity, as well as oxidative stress caused by the excessive generation of ROS, leading to lipid peroxidation, DNA fragmentation, and damage to sperm function. Additionally, osmotic shock during cryoprotectant addition and removal, induces cell damage. Furthermore, the plasma membrane is highly sensitive to alterations in lipid composition and permeability during freeze–thaw cycles, leading to its damage^9–12. However, CPAs induce cell damage at high concentrations; therefore, maintaining their optimal levels could be a suitable strategy to reduce cytotoxicity¹³. Overcoming these challenges not only helps preserve sperm function but also has implications for alleviating male infertility-related issues. Consequently, developing delivery systems that can deliver CPA more safely and efficiently has become a major research priority.

Nanotechnology, especially nanoencapsulation strategies, can serve as a powerful tool for delivering chemical compounds, including CPAs, to cells in a controlled manner during sperm freezing¹⁴. Nanocarriers, including biogenic and engineered systems such as curcumin nanoparticles, metallic nanoparticles, and lipid nanoparticles, have been widely applied to improve sperm cryopreservation efficiency. Due to their high reactivity, large surface area, and antioxidant properties, these nanostructures help mitigate cryodamage and enhance post-thaw sperm quality. Their application has been associated with improved physiological parameters, including motility, viability, membrane integrity, mitochondrial activity, and DNA stability, making them an efficient strategy for decrease oxidative stress and protection sperm function during freezing and thawing process^15–17.

The synthesis of nanocapsules from natural, safe biopolymers such as polysaccharides and proteins has attracted considerable attention across infertility treatments, including sperm freezing²⁰. One well-characterized polysaccharide is derived from the fruit calyx of Physalis alkekengi. Its monomer composition includes fructose, glucose, xylose, galactose, arabinose, rhamnose, L-sorbose, and mannose, and these molecules are associated with strong antioxidant activity²¹ and anti-inflammatory and antibacterial activities²². Sericin protein (SER) is another macromolecule of interest in sperm freezing. This protein is a natural component of silkworm cocoons that contains 18-amino acids with amino, hydroxyl, and carboxyl functional groups^23,24. In addition to being degradable and biocompatible, it also has functions such as reducing immune response and maintaining moisture²⁵. SER also exhibits strong antioxidant activity by reducing lipid peroxidation, so it can have wide applications in the fields of biomedicine. Its protective role has been demonstrated in the freezing of stem cells derived from adipose tissue, buffalo sperm, and human sperm^23,24.

Protein–polysaccharide complexes enable nano-carriers for encapsulation with strong potential. Nanocapsules formed from these complexes benefit from a natural origin, absence of artificial agents, simple synthesis, biodegradability, and excellent biocompatibility in comparison to conventional nanoparticles like liposomes and micelles. Encapsulation becomes more efficient due to the protein’s strong binding to bioactives, with the polysaccharide further increasing stability by wrapping the protein in a protective shell. Recently, protein–polysaccharide nanocomplexes have been widely investigated as efficient delivery systems for various bioactive compounds such as curcumin, including casein, zein-pectin, carboxymethyl cellulose (CMC) and gum arabic, β-lactoglobulin/sodium alginate, and kafirin/carboxymethyl chitosan systems^26,27. This positive feature suggests that the development of hybrid systems can provide improved stability and controlled release, which are key performance metrics in CPA delivery.

The innovation of this work lies in the design of a hybrid nanocarrier system based on SER protein and Physalis alkekengi polysaccharide. SER was selected due to its biocompatibility, moisture-retaining ability, and antioxidant properties, while Physalis polysaccharide was chosen for its strong antioxidant, anti-inflammatory, and structural stability characteristics. The combination of these two natural macromolecules enables synergistic interactions, leading to improved encapsulation efficiency, enhanced structural stability, and controlled release behavior of the loaded cryoprotectant. Therefore, the research questions raised include how to design a biocompatible nanocarrier capable of reducing cryoprotectant-induced cytotoxicity while maintaining protective efficacy and how to achieve controlled and sustained release of ALA during sperm cryopreservation.

This study reports the optimization of a novel hybrid nanocarrier, engineered from SER protein and Physalis alkekengi polysaccharide, for the controlled delivery of ALA during sperm cryopreservation. Optimization focused on the protein/polysaccharide ratio and resultant physicochemical properties. The controlled release of ALA afforded by the proposed nanocarrier led to promising improvements in post-thaw sperm quality parameters, establishing it as a biocompatible and efficient strategy for fertility preservation applications.

Materials and methods

Materials

Sericin Bombyx mori (silkworm) powder (S5201, reagent grade, ≥ 99%) and all other chemicals, including ALA (CAS Number: 501-36-0, 99%, HPLC), polyethylene glycol (reagent grade, ≥ 98%, powder), DMSO, and non-solvent acetone (ACS reagent, ≥ 99.5%), were acquired from Sigma Aldrich (St. Louis, USA). Ultrapure water (UP-Water) was produced using Milli-Q (Millipore, Bedford, MA, USA). The fruits of Physalis alkekengi were obtained from the Department of Pharmacognosy, School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran. The plant material was authenticated and approved by the Department of Pharmacognosy and by Razi University for research purposes prior to use in this study. SER is a heterogeneous protein with a broad molecular weight distribution ranging approximately from 24 to 400 kDa²⁸.

Polysaccharide extraction protocol from Physalis alkekengi

A modified method of Wu et al. was used to extract polysaccharides²⁹. To extract polysaccharides, Physalis alkekengi fruits were first defrosted and then dried at 60 °C. The dried sample was ground into powder. After that, 3.5 kg of powder was mixed with 6 L of water, and the resulting mixture was filtered three times at 3-hour intervals. Next, it was centrifuged for 20 min at 3500 rpm, and the supernatant was separated, and the pellet was discarded. The centrifugation step was repeated three times. Then, the supernatant was mixed with three times its volume of 85% ethanol. The mixture was placed on a rotary evaporator at 150 rpm and 4 °C for 24 h to evaporate the solvent. After 24 h, the mix was centrifuged, and the supernatant was mixed with 70% ethanol. Subsequently, the solution was centrifuged for 15 min at 3500 rpm. This centrifugation step was repeated three times. Similar to the previous step, the supernatant was washed with 50% ethanol. Finally, the supernatant was concentrated using a rotary evaporator, and the residue was dried in an oven at 45 °C. Thus, a pure polysaccharide was produced. Physalis alkekengi polysaccharides typically have a molecular weight of about 16 to about 300 kDa¹⁹.

Preparation of solutions

In this study, a polysaccharide-protein-based nanocarrier (Ph-SER-NC) was prepared, which was used to load three cryoprotectants: ALA, DMSO, and PEG. The protein solution was formulated by dissolving silk SER in ultra-pure (UP) water (5% w/v) under moderate stirring (800 rpm for 4 h at 25 °C). Subsequently, the prepared solution was stored at 4 °C for 24 h. The polysaccharide solution was synthesized using the same procedure (0.25 mg/mL). These solutions had a pH of 7.4, which was subsequently adjusted to 4 and 5 by the addition of HCl.

Optimization of the protein and polysaccharide ratio

To determine the appropriate ratios, SER and polysaccharide were mixed in different proportions. To find the best protein/polysaccharide concentration for preparing nanocapsules as a Ph-SER-NC, the turbidity and clarity of the 10 different ratios at two pH levels, 4 and 5 (Table 1), were measured and expressed in NTU (nephelometric turbidity units) using a turbidometer. Based on these results, six formulations corresponding to samples 1, 3, 5, 7, 9, and 12 (Table 1) were selected for the preparation of Ph-SER-NC.

Table 1.

Optimization of the protein and polysaccharide ratio based on turbidity.

Formulation No.	Pro/Poly ratio	pH	Turbidity (NTU)	Approved for synthesis
1	1:2	4	2.38	✓
2	Blank-SER	5	1.31
3	1:4	5	1.27	✓
4	2:1	4	1.3
5	1:4	4	1.2	✓
6	Blank-Poly	5	1.28
7	1:2	5	1.23	✓
8	1:1	5	1.23
9	4:1	4	1.16	✓
10	1:1	4	0.74
11	2:1	5	0.54
12	4:1	5	0.6	✓

Preparation of polysaccharide-protein-based nanocapsules

The polysaccharide-protein-based nanocapsule was prepared via a flash-nanoprecipitation system adapted from Nelemans et al.³⁰ with a few modifications. Briefly, a protein/polysaccharide solution in various ratios (1:1, 2:1, 4:1, 1:2, and 1:4, with 2 pHs of 4 and 5) was added dropwise (10 µl/5 sec) into an organic phase of a non-solvent (acetone) under vigorous stirring (1000 rpm) at room temperature. The selected formulations corresponding to samples 1, 3, 5, 7, 9, and 12 (Table 1) were processed via solvent evaporation to remove water and acetone, yielding six optimized Ph-SER-NC variants.

Drug loading to polysaccharide-protein-based nanocapsules

The alpha lipoic acid (ALA) and ethylene glycol (EG) were loaded within Ph-SER-NC via the direct system of dissolution into the aqueous phase (protein/polysaccharide phase) at the final ratio of 2:1 in pH:4. While the DMSO, as a loaded cryoprotectant, was prepared by dissolving it in the anti-solvent phase (acetone). For the synthesis of the ALA-PH-SER-NC, the next step involved the addition of the dissolved ALA-PH-SER (aqueous phase) into the acetone organic (anti-solvent phase) under forceful stirring (800 rpm) and pH 7.45 (1 wt% ALA in blank-Ph-SER-NC). This addition was performed without specialized tools, using only a Hamilton syringe, delivering one drop every 5 s under intense stirring. Similarly, a procedure was done for the synthesis of EG-Ph-SER-NC. Also, the synthesis of the DMSO-Ph-SER-NC followed the same protocol; the only difference was the incorporation of this cryoprotectant into the acetone organic (anti-solvent) phase.

Also, for the synthesis of the DMSO-Ph-SER-NC, the synthesis protocol was the same; only this cryoprotectant was added to the acetone organic (anti-solvent phase). All solutions were further stirred at 600 rpm for one hour after preparation.

Purification

To purify the nanoparticle complex without free protein, carbohydrate, and cryoprotectant compounds, centrifugation was performed at 8000 rpm for 20 min. Centrifugation causes the free compounds to remain in the supernatant, while the nanocarrier complex is collected in the pellet. The Ph-SER-NCs were prepared in triplicate, freeze-dried, stored at − 20 °C, and protected from light. Beyond assessing physical appearance, ease of reconstitution, and storage stability, the ALA/EG/DMSO-Ph-SER-NC were evaluated for potential variations in quality characteristics upon addition to sperm-freezing media.

Examination of physical and chemical characteristics of Ph-SER-NCs

Three cryoprotectants—ALA, DMSO, and EG—were loaded into the Ph-SER-NC, and their physical and chemical properties were assessed. Following evaluation of the basic physicochemical properties, it was determined that two cryoprotectants (DMSO and EG) were not suitable for loading into Ph-SER-NC (results are provided in the supplement); consequently, they were not selected for biological testing. In contrast, the results for ALA were promising.

Properties such as particle size, polydispersity index (PDI), and particle surface charge (Zeta Potential) of Ph-SER-NC and ALA-Ph-SER-NC were determined by the dynamic light scattering (DLS) technique via the Zetasizer instrument (Zetasizer, Nano-ZS, Malvern Instruments Ltd., Worcestershire, UK) at 25 °C. Additional characterization included assessment of aggregation, size, morphology, and shape via scanning electron microscopy (SEM) (KYKY-EM3200) operated at an accelerating voltage of 25 kV. For SEM analysis, a drop of diluted nanoparticle suspension was placed on a silicon wafer and air-dried at room temperature. The dried samples were then sputter-coated with gold prior to imaging. No staining procedures were applied. DLS measurements were performed in deionized water under standard laboratory conditions at room temperature using disposable cuvettes.

Fourier transform infrared spectroscopy (FTIR)

An FTIR spectrometer (IR Prestige-21, Shimadzu, Japan) was used to characterize the chemical composition, molecular properties, and surface adsorption of functional groups on Physalis, SER, ALA, Blank-Ph-SER-NC, and ALA-Ph-SER-NC. According to FTIR protocol, 100 mg of potassium bromide (Sigma-Aldrich) was mixed with 1–2 mg of pure cisplatin, lyophilized blank Ph-SER-NC (B-Ph-SER-NC), and ALA-Ph-SER-NC. After pouring the mixture into the FTIR sample holder and compaction, the spectra were recorded in the scan range of 400–4000 cm^− 1 with a spectral resolution of 4 cm^− 1.

Investigation of the entrapment efficiency (EE%) and drug loading (DL%)

The EE% and DL% of ALA-Ph-SER-NC were determined using UV–vis spectrophotometry (Philips PU 8620, USA). First, the encapsulated drugs’ quantity was obtained as described previously³¹. Briefly, to separate the unbound free ALA from the ALA-Ph-SER-NC solution, it was ultracentrifuged for 30 min at 18,200 rpm, and after removing the supernatant, a pellet of the un-entrapped drug was obtained. Then 1 mL of this medium was assessed via UV–vis spectrophotometry for evaluating ALA concentrations at 336 nm, respectively. Next, a standard curve was drawn using different concentrations of ALA between 0.1 and 500 µg/mL (a straight line with R² = 0.98). (Fig. 9. In supplementery file).

Finally, the entrapment efficiency (EE%) and drug loading (DL%) of ALA were calculated using Eqs. 1 and 2, respectively³².

1

2

In vitro drug release

In the present study, a dialysis bag method, as described in^31,33,34, utilizing a 12 kDa molecular-weight cutoff dialysis bag (Sigma-Aldrich), was employed to examine the release of ALA from ALA-Ph-SER-NC at pH 7.4. To investigate the ALA release, a fixed volume test environment containing 80 ml of PBS with pH 7.4 was prepared, then 4 ml of ALA-Ph-SER-NC was poured into the dialysis bag and immersed in a test environment and incubated at 37 ± 0.5 °C for 55 h at 300 rpm in an orbital mixer (Benchmark Scientific). At specific times (0.5, 1, 2, 4, 24, 30, and 48 h), 1 ml of PBS dialysate was taken from the test environment and then replaced to examine the release rate of ALA. UV-vis spectrophotometry (Mini 1240, Shimadzu, Japan) at λmax corresponding to 336 nm was used to analyze the removed samples. The amount of ALA released from ALA-Ph-SER-NC was determined as the drug release percentage at specified time intervals, relative to the amount of entrapped drug. Subsequently, the kinetics release of ALA was examined using mathematical models of first-order (ln Q_t = ln Q₀k₁.t), Korsmeyer-Peppas (M_t/M_∞ = atⁿ), zero-order (Q₁ = Q₀ + k₀.t), Hixson-Crowell (W₀^1/3 − W_t^1/3 = k_st), and Higuchi (f_t = k_H.t^1/2) (2 new).

In vitro biological evaluation

Ethics statement of sperm analysis

Semen samples were obtained at the experimental station of Hospital Motazedi IVF Unit, Kermanshah, Iran. Ethical approval for this study was obtained from the Research Ethics Committee of Kermanshah University of Medical Sciences in accordance with the principles of the Declaration of Helsinki.

Informed consent statement

Written informed consent was obtained from all participants prior to their participation after they had been fully informed about the objectives and procedures of the study.

Experimental design

For this study, semen samples were collected from 25 healthy fertile men. Following initial analysis, samples were frozen using the basic freezing medium supplemented with varying concentrations of free ALA and ALA-Ph-SER-NC (0 [control], 0.01, 0.02, 0.05, 0.1, 0.5, and 1 mmol/mL) for at least 48 h. After thawing as described below, samples were centrifuged at 300 × g for 7 min and then immediately assessed for sperm motility, viability (stained with 0.5% eosin), and DNA fragmentation (assessed by the SCD assay). In this study we used ALA-Ph-SER-NC that was freshly prepared in all biology experiments.

Semen analysis and preparation

Semen samples were collected via masturbation into sterile containers after 3–5 days of sexual abstinence. Participants reported no history of drug addiction, smoking, alcohol use, chronic conditions such as varicocele, or drug/vitamin use. Samples were liquefied at 37 °C with 5% CO₂ for 20–30 min; any sample that did not liquefy within this period was excluded. Basic sperm parameters—motility, morphology, and viability —were assessed in both experiments according to World Health Organization guidelines³⁵ using light microscopy and a CASA system. Briefly, normospermic samples exhibited volume ≥ 1.5 mL, cell concentration ≥ 15 × 10⁶ cells/mL, total motility ≥ 40%, and sperm morphology ≥ 4% (Table 2). After the basic sperm analysis, the freshly analyzed semen samples for DNA fragmentation by the sperm chromatin dispersion (SCD) test were used for each of the two separate experiments.

Table 2.

Fresh semen analysis of the study subjects before cryopreservation.

Characteristics	Mean ± SD
Age (years)	33.53 ± 4.43
Volume (mL)	3.83 ± 1.54
pH	7.15 ± 0.24
Viability (%)	83 ± 13.91
Sperm concentration (10 6 /mL)	64.50 ± 22.50
Sperm with normal morphology (%)	8.96 ± 1.83
Sperm motility (%) Class A + B	77 ± 12.35

Sperm freezing and thawing process

To investigate the efficacy and protective role of ALA-Ph-SER-NC on sperm, a freezing and thawing process was performed as previously by Aghaz et al.²⁴. In this regard, semen samples were collected from healthy and fertile men. In the freezing process, 500 µl of sperm sample was gradually diluted with ALA-Ph-SER-NC at a ratio of 1:0.7 drop by drop with freezing medium (SpermFreeze, a commercial cryoprotectant consisting of 15% glycerol in HEPES buffer (Fertipro, Beernem, Belgium)). After 30 min of storage at room temperature, the cryovials were exposed to liquid nitrogen vapor on a metal surface for 15 min (slowly frozen) and then immersed in liquid nitrogen (-196 °C) for at least 48 h. In the thawing process, the cryostored were placed at 35 °C for 4 to 5 min. Next, sperm samples were resuspended for 30 min in Ham’s F-10 medium containing 10% HSA and then centrifuged at 300 × g for 7 min. Finally, they were investigated for sperm motility, viability, and DNA fragmentation. Each experiment was repeated five times. In this study we used ALA-Ph-SER-NC that was freshly prepared.

Investigation of sperm motility and viability

To examine sperm viability, staining with 0.5% eosin was used, according to the method presented by Banihani et al.³⁶, and examined under a light microscope with a magnification of ×400. In this assay, 10 µl of sperm suspension was poured onto a slide, and at least 200 sperm were examined per sample. In this regard, live sperm were colorless, while dead sperm were partially or completely stained red. The motility and morphology Index parameters were also evaluated by a light microscope with a magnification of ×400. Based on WHO criteria (2010), they were divided into three grades: progressive (Grade A + B), non-progressive (Grade C), and immobile (Grade D)³⁵. Morphology of human sperm was assessed on stained smears (eosin stain) using light microscopy at 1000× magnification with oil immersion, according to WHO criteria³⁷. The morphology index parameter comprised three levels:

• Level 1 (1–5): abnormal, defined as spermatozoa that are morphologically unfinished, immature, or malformed.

• Level 2 (5–10): head normal, but body and tail abnormal.

• Level 3 (10–15): predominantly mature, with more uniform morphology.

Examination of DNA fragmentation

DNA fragmentation was investigated using a modified version of the Sperm Chromatin Dispersion (SCD) method (Halosperm Kit; INDAS Laboratories), according to the manufacturer’s protocol³⁸. In brief, an aliquot from each semen sample was diluted to a concentration of roughly 10–15 million sperm per milliliter using phosphate-buffered saline (PBS). Separately, an Eppendorf tube containing melted agarose (prepared by heating a solid block in a water bath at 95–100 °C for 5 min, then equilibrated at 37 °C) was used for the assay. After temperature equilibration, 60 µL of the diluted semen was added to the tube and mixed gently with the molten agarose. From this mixture, 20 µL was placed onto slides already coated with agarose and covered with a 22 × 22 mm coverslip. The slides were then positioned on a cold plate at 4 °C to allow the agarose to solidify. After five minutes, the slides were removed from the fridge, and the coverslips were carefully detached. The slides were immediately immersed horizontally in an acid solution prepared by combining 80 µL of HCl with 10 mL of UP-water and incubated at room temperature (about 22 °C) for 7 min. Next, the slides were fully immersed in 10 mL of a lysing solution for 25 min, followed by a gentle wash for 5 min in a tray with ample UP-water. The slides were then dehydrated sequentially in 70%, 90%, and 100% ethanol for 2 min each and air-dried. Finally, the slides were allowed to air-dry before staining. For bright-field microscopy, the slides were treated with a 1:1 mixture of Wright’s stain solution and PBS for 5–10 min, briefly rinsed with tap water, and left to dry. Using a light microscope (Olympus BX-40, Olympus U-RFL-T, Tokyo, Japan) at 400x magnification, the samples were examined (at least 300 sperm were counted per semen sample). Sperm showing a large, striated halo around the nucleus contained intact DNA, whereas sperm with a small or no halo around the nucleus contained fragmented DNA³⁹.

Statistical analysis

Data were analyzed with GraphPad Prism 10.3.1. A two-way ANOVA was employed to assess statistically significant differences among groups. All experiments were conducted five times, and results are presented as means ± standard deviation (SD).

Results

Physicochemical characterization and encapsulation results

Our synthesized polysaccharide-protein-based nanocarrier, Ph-SER-NC, was prepared via a flash-nanoprecipitation system. The approved protein and polysaccharide concentrations were 5% w/v and 0.25 mg/mL, respectively. As shown in Tables 1 and 3, six different protein–polysaccharide (pro/poly) ratios were first evaluated by turbidometry in order to identify the ratios that were capable of forming stable dispersions. Among the ratios that produced acceptable turbidity profiles, DLS analysis was then used as the main criterion for selecting the final formulation. Based on the DLS results, the pro/poly ratio of 1:2 was selected as the optimal ratio, as it exhibited the most suitable particle size and distribution (Z-average = 109 nm; PDI = 0.21).

Table 3.

Physicochemical characteristics of formulations.

Formulation No.	Pro/Poly ratio	pH	hydrodynamic diameters (nm)	PDI
1	1:2	4	109	0.21
2	Blank-SER	5	–	–
3	1:4	5	258	0.629
4	2:1	4	1634	1
5	1:4	4	814	0.86
6	Blank-Poly	5	–	–
7	1:2	5	572	0.64
8	1:1	5	401	0.449
9	4:1	4	1787	0.88
10	1:1	4	649	0.69
11	2:1	5	359	0.292
12	4:1	5	2663	1

ALA was loaded into Ph-SER-NC through a direct dissolution system in the aqueous phase. After ALA loading, the Z-average of Ph-SER-NC size increased slightly to 186.9 nm (Fig. 1a), indicating successful drug loading. According to the PDI of 0.456 (Fig. 1a), the particle dispersion was slightly lower than that of the Blank-Ph-SER-NC, possibly due to the more uniform distribution of the Ph-SER-NC in the presence of the ALA. Zeta potential results (-11.8 mV) showed that the ALA-Ph-SER-NC had a negative surface charge and showed an approximately moderate stability (Fig. 1b). SEM morphology results indicated that the ALA-Ph-SER-NC had an average length of 160 nm and a good distribution, which confirmed the DLS result (Fig. 1c). These results confirm the nanometer size, homogeneous distribution, and non-aggregation of ALA-Ph-SER-NC. Also, the EE and DL results (93.74% and 62.75%, respectively) of the designed nanocarrier system showed that this system has high efficiency and effectiveness. Therefore, this evidence confirms the successful loading of ALA into the designed Ph-SER-NC. The DLS, Zp, and SEM results of DMSO-Ph-SER-NC and PEG-Ph-SER-NC are presented in the supplementary data file. (Fig. 1b: DLA and ZP of DMSO-Ph-SER-NC and Fig. 2b: SEM of DMSO-Ph-SER-NC Fig. 2c SEM of PEG-Ph-SER-NC ; in supplementary file)

Fig. 1.

Analysis of size distribution (a), zeta potential analysis (b), and SEM (c) of ALA-Ph-SER-NC.

Fig. 2.

FTIR Spectra for polysaccharide (Ph), serecin (SER), alpha lipoic acid (ALA), Ph-SER-NC, and ALA-Ph-SER-NC.

FTIR results

As shown in Fig. 2, the chemical-molecular structure of the precursors and the fabricated nanocarriers was analyzed using FTIR. The presence of Physalis-polysaccharide indicator groups in the structure of our final synthesis (Ph-SER-NC) was confirmed using FTIR. The results revealed 5 sharp peaks for Physalis-polysaccharide (3425, 2924, 1728, 1627, and 1431 cm^− 1). These groups are the main component of the Physalis-polysaccharide structure and indicate hydrogen bonds. The broadband at 3425 cm^− 1 indicates the stretching vibration of hydroxyl groups (OH). This band may also include contributions from hydrogen-bonded water molecules due to the highly hydrophilic nature of the polysaccharide and its aqueous extraction process. The band at 2924 cm^− 1 is related to the –NH stretching vibration. The peaks at 1728 cm^− 1 and 1431 cm^− 1 are due to C–H vibrations and indicate the carbon skeleton of the physalis-polysaccharide. Also, the peak at 1083 cm^− 1 indicates the C–N stretching vibration. Which can indicate nitrogenous groups or side bonds in this macromolecule. The FTIR spectrum of SER confirms the presence of amide bonds, which are characteristic of proteins. Six sharp peaks were reported for SER (3400, 2924, 1654, 1512, 1384, and 1076 cm^− 1). The peak at 1654 cm^− 1 is due to the carbonyl stretching vibration (C = O) in the peptide bond of SER, which represents the amide I band. The peak at 1525 cm^− 1 represents the N–H bending vibration, which is known as amide II. The band at 1244 cm^− 1 is attributed to amide III, which includes the C–N and N–H vibrations. The presence of N–H was revealed by the peak at 1652 cm^− 1. The peak at 2924 cm^− 1 represents the C–H vibration in the side chain of amino acids. In addition, the presence of the spectrum at 2248 cm^− 1 can be attributed to the C = C vibration in some side groups. Using the results obtained from FTIR, the synthesis of Ph-SER-NC from Physalis-polysaccharides and SER-proteins was confirmed so that the spectrum examination shows changes compared to the precursors. Five sharp peaks appeared in blank-nanocarriers (Ph-SER-NC) (3429, 2924, 1631, 1458, and 1083 cm^− 1). The –OH group in the 3429 cm^− 1 peak is slightly shifted compared to the Physalis-polysaccharide (3425 cm^− 1), likely due to the formation of new hydrogen bonds between the two precursors. In both SER and Physalis-polysaccharide, a 2924 cm^− 1 band is observed, which is also present in the Ph-SER-NC, which is due to the C–H and N–H groups. The spectrum at 1631 cm^− 1 in the nanocarrier is due to the C = O stretching vibration. This spectrum shifts relative to the SER bands (1654 cm^− 1), which may indicate interactions between the two components. Also, another shift relative to the Physalis-polysaccharide appeared in Ph-SER-NC, such that the band at 1458 cm^− 1 was due to -OH in the Ph-SER-NC, while in the Physalis-polysaccharide, the band at 1431 cm^− 1 was due to C-H bending, which may indicate a change in the chemical environment of both functional groups during Ph-SER-NC formation. The presence of the 1083 cm^− 1 peak (C–N group) in Physalis-polysaccharide and Ph-SER-NC confirms the presence of this macromolecule in the synthesized Ph-SER-NC. Regarding the spectra of ALA alone, the presence of bands related to functional groups of this compound was revealed. The FTIR spectra showed slight shifts in O–H and amide-related peaks compared to the individual components. Overall, these peak shifts and changes in intensity indicate that the formation of Ph-SER-NC is mainly driven by non-covalent interactions, particularly hydrogen bonding between functional groups of SER and Physalis-polysaccharide. These interactions contribute to the self-assembly and stabilization of the nanocarrier without the formation of new covalent bonds. The FTIR results showed 8 sharp peaks for ALA (2927, 1693, 1427, 1303, 1249, 1199, 937, and 675 cm^− 1). The peak at 1693 cm^− 1 is due to the carbonyl group (C = O). Bands at 1249 and 1199 cm^− 1 are attributed to C-N stretching vibration. Also, the spectra at 675, 937, and 1477 cm^− 1 are due to C–H bending vibration. In addition, the band at 2927 cm^− 1 is due to the N–H group. The band at 1427 cm^− 1 is due to the O–H bending, and at 2858 cm^− 1 is due to the stretching vibration of the hydroxyl. Five sharp peaks were revealed in ALA-Ph-SER-NC (3429, 2924, 1477, 1091, and 867 cm^− 1). After loading ALA into the Ph-SER-NC, the FTIR spectrum results showed that the bands at 1477 cm^− 1 (C–H bending) and 2858 cm^− 1 (O–H stretching) observed in ALA are also present in the nanocarrier, which indicates the detection of ALA in the Ph-SER-NC. Also, the presence of a broad band at 3429 cm^− 1 is due to the stretching vibration of the O–H group. This band could be due to hydrogen interactions between ALA and the SER-protein/Physalis-polysaccharide present in the Ph-SER-NC. In addition, slight changes in some peaks, such as shifts (e.g., 2927 → 2924 cm^− 1) and changes in the intensity of the bands (including the carbonyl band), were shown (Fig. 2). The same new peaks, 867 and 1091 cm^− 1 were observed in the ALA-Ph-SER-NC. These changes could indicate the occurrence of interactions between the ALA and the Ph-SER-NC matrix. Full spectra and further details are provided in the Supplementary Information (Figs. 4, 5, 6 and 7, and 8).

Results of in vitro release of ALA from ALA-Ph-SER-NC

The percentage of ALA release from ALA-Ph-SER-NC was investigated using a 12 kDa dialysis bag at pH 7.4 for 55 h under in vitro conditions. As shown in Fig. 3, initially, a 19% release was observed after about the first 2–3 h. This initial rapid release may be related to the ALA located near the nanocarrier surface. From about 3 to 37 h (by 37 h, approximately 29% of the ALA was released), a gradual and almost linear increase in ALA release was observed. Between 37 to about 41 h (40%), a notable increase in the rate of ALA release was observed, with cumulative release reaching about 40%. This increase is most likely associated with the continued diffusion of ALA from more deeply entrapped regions of the nanocarrier. After about 41 h, the release plateaued and reached a stable and constant level of 40%. This indicates that the ALA release has reached equilibrium.

Fig. 3.

ALA release from ALA-Ph-SER-NC using a 12 kDa dialysis bag for 55 h.

The results of the DMSO release diagram are presented in the Supplementary Data File, and together with the DL and EE values, they indicate that DMSO was not loaded into the Ph-SER-NC (Supplementary File).

As shown in Fig. 4, five different mathematical models were used to determine the in vitro kinetics of ALA release. Using these models, the release mechanism from the ALA-Ph-SER-NC was determined. After collecting the data, they were fitted to different models. The results of the data fitting showed that the Higuchi model exhibited the highest similarity (R² = 0.8619). Given this similarity, the ALA release probably occurs through a diffusion-controlled mechanism from the ALA-Ph-SER-NC system. The Higson-Crowell (R² = 0.8314), first-order kinetics (R² = 0.8438), and zero-order kinetics (R² = 0.8015) models showed reasonable agreement. However, the Korsmeyer-Peppas models (R² = 0.4921) were poorly fitted. The regression equations and corresponding fitting parameters are summarized in Table 4.

Fig. 4.

Mathematical models, first-order kinetics, Korsmeyer−Peppas, zero-order kinetics, Higuchi, and Hixson−Crowell for the kinetics of ALA release in vitro.

Table 4.

Regression equations and kinetic fitting parameters for the release of ALA from Ph-SER-NC.

Model	Equation	Slope / Constant	Intercept	R²
Zero-order	y = 0.5906x + 13.176	0.5906	13.176	0.8015
First-order	y = -0.0035x + 1.9387	-0.0035	1.9387	0.8438
Higuchi	y = 4.5081x + 8.5239	4.5081	8.5239	0.8619
Hixson–Crowell	y = 0.0112x + 0.2139	0.0112	0.2139	0.8314
Korsmeyer–Peppas	y = 0.4824x + 0.8138	0.4824	0.8138	0.4921

Results of biological evaluation

To evaluate the efficiency and protective role of the synthesized ALA-Ph-SER-NC in preserving sperm function during the freezing and thawing process, an in vitro study was performed on fresh human sperm samples. The characteristics of sperm collected from 25 fertile men are summarized in Table 2. Sperm samples were frozen using different concentrations of free ALA and ALA-Ph-SER-NC. After thawing, the main biological parameters, including motility, morphology, viability, and DNA fragmentation index (DFI), were examined.

Sperm motility and morphology evaluation

Regarding total motility (A + B) using light microscopical evaluation, a significant difference was observed between the free ALA and ALA-Ph-SER-NC groups at concentrations of 0.01-1 mmol/mL. At low concentrations (0.01, 0.02, and 0.05 mmol/mL), higher total motility with a significant difference was observed in the free ALA and ALA-Ph-SER-NC groups, and in the three concentrations, the ALA-Ph-SER-NC groups had a better result. But at the highest concentrations (0.5 and 1 mmol/mL), a decrease in total motility compared to the control group in both free ALA and ALA-Ph-SER-NC groups was observed. There is a significant difference between the free ALA and ALA-Ph-SER-NC groups. Sperm morphology preservation did not differ significantly between the two groups (free ALA and ALA-Ph-SER-NC groups), with concentrations of 0.01 mmol/mL and 0.02 mmol/mL, whereas it showed a significant difference at concentrations of 0.05-1 mmol/mL. Morphology preservation at 0.05 mmol/mL and 0.1 mmol/mL emphasizes the importance of the synthesized Ph-SER-NC during the sperm freezing process (Fig. 5b).

Fig. 5.

Total motility (a) and morphology (b) in different concentrations of ALA and ALA-Ph-SER-NC.

Sperm viability and DNA fragmentation

The results of examining sperm viability at concentrations of 0.05 mmol/mL to 1 mmol/mL revealed the importance of Ph-SER-NC in maintaining sperm, although at concentrations less than 0.02 mmol/mL there was no statistically significant difference (Fig. 6). The importance of nanocarriers at concentrations of 0.05 − 0.01 mmol/mL is particularly evident in maintaining sperm viability (Fig. 6). Also, in comparing the percentage of Viability and Total motility between different concentrations of ALA and different concentrations of ALA-Ph-SER-NC with each other, it was shown that free ALA was significant at concentrations less than 0.02 mmol/mL compared to the control, while in ALA-Ph-SER-NC this significance was determined at concentrations less than 0.05mmol/mL (Figure S1and S2). This result is reflected in the Morphology Index across varying concentrations of free ALA and across varying concentrations of ALA-Ph-SER-NC (Figure S3).

Fig. 6.

Viability curve and image of eoin stain in different concentrations of ALA and ALA-Ph-SER-NC.

The results of the sperm-DNA fragmentation using the SCD method indicated a significant decrease at concentrations of 0.01, 0.02, 0.05, and 0.1 mmol/mL compared to the control group (30%), although there was no statistically significant difference in DFI% between the two groups, free ALA and ALA-Ph-SER-NC (approximately 12–22%), in these concentration values (Fig. 7). There was a statistically significant difference between the two groups at concentrations of 0.5 mmol/mL and 1 mmol/mL. Notably, DNA integrity at low concentrations, especially 0.02 mmol/mL and 0.05 mmol/mL, showed better results, highlighting the protective role of Ph-SER-NC in preserving DNA. When comparing viability across different concentrations, free ALA showed a significant positive effect versus control at concentrations below 0.05 mmol/mL. Similarly, for ALA-Ph-SER-NC, significance occurred at concentrations below 0.05 mmol/mL (Figure S4).

Fig. 7.

Sperm DNA fragmentation and DFI% in different concentrations of ALA and ALA-Ph-SER-NC.

Overall, these findings indicate that the synthesized Ph-SER-NC could play a significant protective role on sperm DNA, morphology, motility, and viability at some concentrations, especially 0.02 mmol/mL and 0.05 mmol/mL.

Discussion

Sperm freezing using CPAs is considered a valuable strategy to preserve fertility in men who are at risk of losing their reproductive potential for various reasons^40,41. However, CPAs direct exposure can impair sperm quality by damaging the cell membrane and increasing ROS production during the freezing–thawing process⁸. Developing delivery systems that introduce CPAs to cells in a controlled manner can mitigate these adverse effects⁴². Recently, polysaccharide- or protein-based nanocarriers have attracted considerable attention in modern medicine⁴³. In this context, our synthesized polysaccharide–protein–based nanocarrier demonstrated that this class of nanocarriers can serve as an effective nanodelivery system for CPAs, such as ALA, and represents a promising strategy to minimize the toxicity associated with direct CPA use during freezing by enabling controlled release⁴⁴. Our results of the physicochemical properties of ALA-Ph-SER-NC revealed a Z-average, PDI, and zeta potential of 186.9 nm, 0.456, and − 11 mV, respectively. The Z-average of ALA-Ph-SER-NC increased compared to Blank-Ph-SER-NC (Z-average of 109 nm, PDI of 0.21), indicating successful ALA loading in our nanocarriers (Fig. 1a and b; Table 1). The selective encapsulation of ALA, compared to the negligible incorporation of DMSO and PEG, can be explained by differences in molecular interactions and partitioning behavior. ALA can establish both hydrogen bonding and hydrophobic interactions with the SER–Physalis polysaccharide matrix, facilitating its entrapment within the Ph-SER-NC. In contrast, the high hydrophilicity and aqueous solubility of DMSO and PEG likely prevent their retention during nanoprecipitation, resulting in their diffusion into the external phase rather than encapsulation. According to the SEM images, ALA-Ph-SER-NC has an average length of 160 nm, a perfectly spherical morphology, and a very good dispersion, which is consistent with the DLS results. In a previous study investigating the supplementation of semen extender with alpha-lipoic acid-loaded liposomal nanocarriers for sperm cryopreservation, the developed system exhibited a Z-average of 171.80 ± 2.60 nm and an EE of 96.46 ± 0.12%⁶. Compared with these findings, the developed Ph-SER-NC system demonstrated comparable nanoscale physicochemical properties, confirming its suitability as an efficient natural carrier for controlled bioactive delivery. These findings corroborate that the synthesized Ph-SER-NCs are effectively internalized by sperm cells. Also, FTIR results highlighted strong pro-poly hydrogen bonding and interpolymer interactions that support the core-shell architecture, improve structural stability, and facilitate efficient encapsulation of ALA (Figs. 1c and 2). In addition, given the acceptable values of EE and DL (93.74% and 62.75%, respectively) and the controlled diffusion release of ALA according to the Higuchi model, this designed nanocarrier can play a significant role in maintaining sperm function and reducing high-dose ALA-induced toxicity. These findings may be attributed to our nature-based synthesis approach, in which the macromolecules used were natural: the protein was pure silk SER, and the polysaccharide was extracted using the modified Wu et al. method²⁹, supporting the properties of the ALA-Ph-SER-NC. Our Ph-SER-NC was first synthesized using different ratios of pro/poly to determine the optimal concentration of these two macromolecules, and finally, based on the results of turbidometry and DLS, a 1:2 ratio was selected as the optimal ratio (Tables 1 and 3). Similarly, Zeng et al.⁴⁵ synthesized nanogels from ovalbumin protein and pullulan polysaccharide for curcumin delivery. DLS results showed that the nanogels in unloaded and drug-loaded states had particle diameters of 160 and 190 nm and PDIs of 0.146 and 0.227, respectively. Also, the zeta potential for both was reported to be about − 1.3, and the EE value in the nanogel was 88.38%. Also, in another study by Liu et al., a nanocarrier consisting of three compounds—polysaccharide zein, egg white-derived peptides (EWDP), and chitosan—was synthesized to encapsulate curcumin. The results showed that their nanocarriers had a particle size, PDI, and zeta potential of 139.67 nm, 0.17, and 45.93 mV, respectively. The EE for curcumin in this nanocarrier was reported to be 93.87⁴⁶. The results of these studies were consistent with our study, confirming the importance of synthesizing nanocarriers with pro/poly bases. These results confirm that natural compounds such as proteins, polysaccharides, and nucleotides for the manufacture of carriers have been considered. Polymers made from these compounds are considered a suitable option for drug delivery due to their biocompatibility, biodegradability, and the creation of biological interactions with cells and tissues during modern reproductive medicine⁴⁷.

Our biological findings regarding the protective role of ALA-Ph-SER-NC showed a significant difference in total sperm motility, as a key parameter in sperm analysis, between the free ALA and ALA-Ph-SER-NC groups at the lowest concentrations of 0.01, 0.02, and 0.05 mmol/mL. ALA-Ph-SER-NC groups indicated better performance at all five concentrations. According to our results on sperm morphology, the important use of the ALA-Ph-SER-NC compared to free ALA at concentrations of 0.05 and 0.1 mmol/mL was revealed, with more significant effects observed at 0.05 mmol/mL during sperm cryopreservation. Although ALA has been shown to improve sperm motility at low concentrations, different results may be observed at high concentrations⁴⁸. This may be because encapsulation seems to control the release of ALA and minimize its toxic effects at high concentrations. Previously published studies revealed the advantage of encapsulation; for instance, Tan et al.⁴⁹, demonstrated that testicular tissue encapsulation using gelatin methacryloyl (GelMA) can improve the morphology, mitochondrial activity, and antioxidant capacity of testicular tissue after thawing by reducing the required concentration of DMSO. Despite promising results, the study is limited by the absence of human data and the technical complexity of the encapsulation procedure. Also, in another study, Fatmi et al.,⁵⁰ demonstrated that encapsulation of vitamin E using liposomes can improve sperm motility after chilling. However, the study by Fatmi et al. has limitations, including the absence of complete freezing–thawing cycles and evaluation restricted to motility parameters; these findings therefore require validation in full cryopreservation. Ex vivo studies showed that curcumin-loaded niosomal nanoparticles (Cur-LNN) can improve total and progressive motility in horse sperm⁵¹. Similarly, Aghaz et al. reported that tretinoin-loaded solid lipid-core nanocapsules (TTN-SLN) improved sperm motility in mouse sperm⁵². However, these studies did not examine the effects of these nanoformulations on human sperm. Although further clinical trials and evaluation of fertility outcomes are necessary. In terms of sperm protective efficacy, previous studies investigating the supplementation of semen extender with ALA-loaded nanoliposomes demonstrated significant improvements in post-thaw of buffalo sperm quality compared with control groups, with progressive motility increasing from 36.0 ± 1.87 to 47.0 ± 2.00, vitality from 39.4 ± 2.75 to 50.4 ± 2.56, and membrane integrity from 40.6 ± 2.40% to 48.4 ± 2.38%⁶. In comparison, the ALA-Ph-SER-NC system in the present study further supports and extends these findings by providing a natural protein-polysaccharide-based carrier with controlled release behavior, suggesting improved modulation of ALA bioavailability and reduced cytotoxicity at higher concentrations.

Our sperm viability evaluation showed a significant difference between both groups at concentrations of 0.05-1 mmol/mL. The importance of ALA-Ph-SER-NC at concentrations of 0.05–0.1 mmol/mL was particularly evident in maintaining sperm viability (Fig. 6). The DFI% results also indicated that DNA integrity at low concentrations, especially 0.02 and 0.05 mmol/mL, showed better results (Fig. 7). Consistent with our results, Gosálvez et al. indicated that sperm microencapsulation using alginic acid after storage for 24 h at 37 °C improved cumulative sperm viability and reduced cumulative DNA fragmentation in the nanoencapsulated group compared to the control group⁵³, although their study did not investigate the kinetics and efficiency of sperm release from the microcapsules. Likewise, Jannatifar et al. showed that the use of Mito-Tempo-loaded nanoliposomes improves the viability and DFI of human sperm after freezing⁵⁴; however, their promising results were obtained in asthenoteratozoospermia samples, and no samples from normozoospermic men were examined. In previous reports highlighting the effect of nano-encapsulation methods in improving sperm cryoprotection, similar enhancing effects have been demonstrated using lipid- and liposome-based carriers that were incorporated into semen extenders. Quercetin-loaded nanostructured lipid carriers (NLCs) at 15 µM showed the protective effects on rooster sperm post-thaw quality. Specifically, sperm viability significantly increased to 72.92% compared with the control group (58.46%), as well as compared to quercetin-loaded nanoliposomes at the same concentration (64.27%) and free quercetin (71.99%). Although sperm morphology did not show significant differences among treatments (P > 0.05)⁵⁵. Compared with free antioxidants, both NLC and Ph-SER-NC systems enhance post-thaw sperm quality, likely via controlled release and oxidative stress protection, with Ph-SER-NC offering additional biocompatibility advantages. However, the natural polymer-based structure of Ph-SER-NC may provide additional advantages in terms of biocompatibility and sustained stabilization.

Overall, our findings confirmed that free ALA, as a CPA-based antioxidant, maintains the quality and reduces damage to sperm after the freezing/thawing process compared to the control group. Nevertheless, it has limitations such as poor solubility in water and instability, and the use of free ALA at high concentrations can reduce sperm quality⁵⁶. To overcome these drawbacks, Physalis/SER was used in the design of our nanocarrier. SER is a suitable candidate for nanoformulation and polymer formation with other compounds due to its structural features, including functional groups and polar amino acids, which improve compounds stability. Moreover, SER exhibits antibacterial, antioxidant, anti-aging, and anti-inflammatory properties⁵⁷. Likewise, the polysaccharide employed in the synthesis of this nanocarrier has remarkable antioxidant properties²¹. The antioxidant activity of the carrier components, by reducing ROS levels and inhibiting lipid peroxidation, contributes to the stability of the plasma membrane and provides a more suitable cellular environment for maintaining DNA integrity. By reducing oxidative damage to DNA, nanocarriers help the stability of the expression of genes related to sperm function. In addition, the controlled release of CPA reduces the damage caused by CPA-induced damage further helping to maintain nuclear integrity and prevent DNA damage. Together, these molecular mechanisms may contribute to the increased sperm quality observed in the ALA-Ph-SER-NC group. Consequently, the ALA-Ph-SER-NC system can be introduced as a bioactive and efficient solution for maintaining sperm quality during the freezing and thawing process. It not only improves post-thaw sperm quality in terms of motility, morphology, viability, and DNA integrity but also demonstrates scalability and reproducibility suitable for industrial applications. The use of natural macromolecules and the optimized nanoprecipitation method allows for efficient and safe cryoprotectant delivery. This nanocarrier provides a safe and effective option for use in infertility clinics for men at risk of reduced sperm quality and also offers significant practical benefits for the long-term, high-quality storage of animal sperm, supporting biodiversity conservation and large-scale breeding programs.

Limitation

Despite the promising results of this study, it is also accompanied by some limitations. As the molecular interactions between the poly-ser matrix and ALA are not fully characterized and require advanced analytical techniques. The fertility quality of the frozen sperm was not evaluated, which limits the direct translation of the findings to clinical applications. In addition, the long-term stability of the sperm and their freezing under different cryogenic conditions were not evaluated. Therefore, considering these limitations, further studies are needed to validate this cryopreservation system.

Future scope

It is suggested that a program to evaluate the fertilization capacity and quality of offspring derived from sperm frozen with ALA-Ph-SER-NC be included in future research. Further studies are also needed to optimize the loading efficiency for different CPAs and to investigate targeted surface modifications to improve sperm interaction. Furthermore, the use of ALA-Ph-SER-NC for sperm samples of other species or for other cryobiology applications could expand its potential use.

Conclusion

In summary, this study introduces—for the first time—a nanocarrier constructed from silk SER and Physalis alkekengi polysaccharide, optimized at a 1:2 pro/poly ratio and produced by a flash nanoprecipitation system. This formulation produced a uniform nanocarrier with a Z-average of 109 nm and a PDI of 0.21. Biological assays showed that encapsulation of ALA in Ph-SER-NC significantly improved sperm parameters after thawing. Compared to free ALA, motility increased by about 26%, morphology improved by approximately 24%, viability increased by about 12%, and DFI% decreased by approximately 28%. These improvements highlight the protective effects of the ALA-Ph-SER-NC, which modulates ALA release and reduces concentration-dependent toxicity. From a practical perspective, the synthesis method, the use of natural macromolecules, and the reproducibility of the nanoprecipitation process demonstrate favorable scalability for expansion from the laboratory to industry. Overall, the ALA-Ph-SER-NC system is proposed as a promising, biocompatible, efficient, and clinically safe strategy for maintaining human sperm quality during freeze-thaw cycles and for long-term, high-quality storage of animal sperm in biodiversity conservation programs.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

F.A., writing–review, and editing; S.A.H. and P.A., writing–original draft; P.A., E.A., R.J., K.B., and Z.R., methodology; P.A., E.A., R.J., K.B., and Z.R., investigation; F.A. and S.A.H., formal analysis; F.A. and Z.R., conceptualization; F.A. and E.A., supervision; and F.A., funding acquisition.

Funding

This study was financially supported by Kermanshah University of Medical Sciences (IR.KUMS.REC.1401.487) (ID number: 4020008). And we confirmed the study is reported in accordance with ARRIVE guidelines.

Data availability

The data that support the findings of this study are listed in the article and are available from the corresponding author upon reasonable request. Contact: **Faranak Aghaz** , Nano Drug Delivery Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran; ***Orcid.org*** /0000-0002-7311-4071; E-mail: [Faranak_aghaz@yahoo.com](mailto: Faranak_aghaz@yahoo.com).

Declarations

Competing interests

The authors declare that they have no conflicts of interest with the contents of this article.