Crossing ocular barriers with quercetin-SPIONs: modulation of Bax/Bcl-2 balance in normal rat eyes

Abstract

Background

Oxidative stress-induced cell death and injury are pivotal mechanisms in the breakdown of ocular tissue, leading to degenerative diseases. To counter this, we investigated quercetin, a flavonoid with well-documented antioxidant and anti-apoptotic properties, as a potential therapeutic agent. Its clinical translation is hampered by poor stability and ocular bioavailability. The goal was to examine the effectiveness and anti-apoptotic properties of polyethylene glycol-coated superparamagnetic iron oxide nanoparticles conjugated with quercetin (QCSPIONs) in rat eye tissue.

Methods

Male Wistar rats received free quercetin (QC), SPIONs, or QCSPIONs orally via gavage or through intraperitoneal injection once daily for 35 days. HPLC measured serum and ocular quercetin levels; nanoparticle localization was assessed by Prussian blue staining; safety was evaluated by histopathology with H&E staining; and apoptotic signaling was examined by qPCR for Bax and Bcl-2 expression.

Results

QCSPIONs demonstrated significantly higher ocular quercetin accumulation than free QC after both oral and intraperitoneal delivery (p < 0.0001). Prussian Blue staining confirmed that nanoparticles crossed the corneal and retinal barriers, with greater deposition following intraperitoneal injection. Additionally, histological analysis showed no structural or inflammatory damage in the retina or cornea. Furthermore, molecular results indicated Bax suppression and Bcl-2 upregulation with QCSPIONs, along with a decreased Bax/Bcl-2 ratio, demonstrating vigorous anti-apoptotic activity. Less pronounced effects were observed with free QC.

Conclusions

Overall, QCSPIONs significantly enhance the ocular bioavailability of quercetin and provide robust anti-apoptotic protection without detectable cytotoxicity. These results support nanoparticle-mediated delivery as a practical approach for overcoming ocular barriers and enhancing preventive antioxidant strategies against oxidative stress-related eye diseases.

Background

Oxidative stress occurs when there is an imbalance in the regulatory systems that control antioxidant and oxidant levels. This condition is prevalent in the front and back parts of the eye, which are particularly vulnerable due to high metabolic activity and oxygen consumption [1]. Excessive production of reactive oxygen species (ROS) induces inflammation, triggers cell death pathways in ocular tissues, and ultimately leads to vision loss [2]. Recently, antioxidants have gained increased attention for managing specific ophthalmic diseases such as diabetic retinopathy (DR), age-related macular degeneration (AMD) and glaucoma because they can neutralize ROS, protect cellular components, and slow the progression of oxidative stress-related pathways [3, 4].

The anti-inflammatory, anti-angiogenic, and antioxidant properties of quercetin suggest it could be a promising therapeutic agent for ocular degenerative diseases such as macular degeneration, cataracts, AMD, DR, and glaucoma, which are characterized by oxidative damage and inflammation [5–7]. Flavonoids are polyphenolic compounds that support eye health by directly interacting with rhodopsin, modulating visual pigments, and protecting eye cells [8, 9]. Additionally, these compounds have been shown to inhibit the progression of various ocular conditions, including AMD and diabetic retinopathy [10, 11].

Quercetin is a natural flavonoid found in vegetables and fruits, commonly consumed in the human diet [12]. Its known anti-inflammatory, anti-angiogenic, and antioxidant properties make it a candidate for therapy in ocular degenerative conditions. However, despite its benefits when administered orally, clinical application of quercetin faces significant challenges. Its hydrophobic nature reduces water solubility; it is rapidly cleared from tissues, often within an hour, and it can produce inactive metabolic byproducts, which may limit its therapeutic efficacy [13].

Adding to these challenges are the anatomical and physiological barriers of the eye, such as the cornea, conjunctiva, sclera, Bruch’s membrane, and blood-retinal barriers. These barriers protect the eye but also hinder the delivery of therapeutic agents, resulting in low ocular bioavailability and reduced effectiveness [14, 15].

Given the increasing prevalence of oxidative stress-related ocular diseases projected to double by 2050 [16] the development of advanced drug delivery systems is crucial. Nanotechnology offers promising solutions; in particular, superparamagnetic iron oxide nanoparticles (SPIONs), which typically range from 5 to 100 nm, have garnered interest as drug carriers due to their unique properties. SPIONs are tiny magnetic particles that become strongly magnetized in the presence of an external magnetic field but lose their magnetization immediately when the field is removed [17]. They are extensively explored in biomedicine because of their biocompatibility, biodegradability, magnetic responsiveness, and capacity for surface modification. Coating SPIONs with polyethylene glycol (PEG) a biocompatible polymer further improves their stability, prevents aggregation, prolongs circulation time, and allows for surface functionalization to enable targeted drug delivery [18, 19]. PEG is a hydrophilic polymer that enhances nanoparticle stability and biocompatibility, making it a valuable component in nanocarrier design.

In this study, we aim to investigate the bioavailability and antiapoptotic effects of quercetin in two forms: free quercetin (QC) and quercetin–SPION conjugates (QCSPIONs), in rat ocular tissues. We will employ two routes of administration oral and intraperitoneal to evaluate their efficiency. To demonstrate the superior barrier-crossing ability of QCSPIONs and their potential to enhance antioxidant defenses against oxidative stress, we will measure quercetin accumulation using high-performance liquid chromatography (HPLC), locate nanoparticles with Prussian blue staining, assess histopathological safety via hematoxylin and eosin (H&E) analysis, and evaluate apoptotic signaling through Bax and Bcl-2 gene expression analysis.

Materials and methods

Quercetin (> 95% purity, Q4951, chemical formula C15H10O7, molecular weight 302.238 g/mol), 2,3,5-triphenyltetrazolium chloride (TTC), tetrahydrofuran (THF), polyethylene glycol (PEG), and dimethyl sulfoxide (DMSO) were procured from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Iron (III) chloride hexahydrate (FeCl₃⋅6H₂O), iron (II) chloride tetrahydrate (FeCl₂⋅4H₂O), N, N-diisopropylethylamine (DIPEA), aqueous ammonia (NH3), and hydrochloric acid (HCl) were procured from Merck Chemical Co. All chemical reagents employed in this research were of analytical quality and used without additional purification. Phosphate buffer solution (PBS) and additional chemicals were procured from Cinnagene (Tehran, Iran).

Synthesis of quercetin-conjugated superparamagnetic iron oxide nanoparticles

Our previous work [20] explained how we made QCSPION. SPIONs had been synthesised via a chemical co-precipitation technique involving Fe₂+ and Fe₃+ ions. In summary, 8.5 g of ferric chloride (FeCl₃⋅6H₂O) and 3 g of ferrous chloride (FeCl₂⋅4H₂O) were solubilized in 38 mL of deoxygenated hydrochloric acid (HCl, 0.4 M). To prevent phase transitions and enhance nanoparticle stability, argon gas was continuously bubbled through the solution to remove dissolved oxygen. The resulting solution was introduced into a three-neck flask containing 375 mL of ammonia solution (NH3, 0.7 M) under continuous stirring at 60 °C for 1 h. The pH was maintained at nine throughout this period. Subsequently, 3 g of polyethylene glycol (PEG, molecular weight 2000 Da) was dissolved in 10 mL of distilled water and gradually added to the homogeneous mixture of magnetic nanoparticles (MNPs). The solution was agitated overnight under nitrogen while the temperature was gradually increased to 70 °C. Upon cooling to ambient temperature, PEG-grafted Fe₃O₄ nanoparticles (Fe₃O₄@PEG) were retrieved using a powerful magnet. The precipitate was repeatedly rinsed with deionized water and ethanol to eliminate unbound PEG. The purified nanoparticles were subsequently dried in an oven at 70 °C overnight.

QCSPIONs were produced using 2,4,6-trichloro-1,3,5-triazine (TCT) as a linker by dissolving TCT in dry tetrahydrofuran (THF) and dispersing dried Fe₃O₄@PEG nanoparticles in THF with N, diisopropylethylamine (DIPEA). The mixture was stirred at 0 °C under nitrogen for 5 h. Quercetin was subsequently dissolved in DMSO to a concentration of 20 mg/mL, after which 5 mL of this solution was added incrementally to 150 mL of PBS buffer containing 200 mg of functionalized nanoparticles under ultrasonication. The reaction was conducted for 24 h with constant agitation to ensure complete coating of the nanoparticles. The generated QCSPIONs were retrieved using a strong external magnet, purified in PBS buffer to remove impurities, and freeze-dried to ensure complete solvent removal.

The quantity of quercetin coupled to SPIONs was evaluated using a UV–visible spectrophotometer at 375 nm. A specified amount of quercetin-conjugated SPION (QCSPION) (5 mg) was suspended in 10 mL of PBS and subjected to centrifugation at 12,000 rpm for 30 min. The quercetin concentration in the supernatant was determined by measuring absorbance at 375 nm. The proportion of quercetin conjugated to SPIONs was determined using the subsequent formula:

Conjugated quercetin content (% w/w) = (Mass of conjugated quercetin / Mass of QCSPIONs) × 100.

The in vitro release profile of quercetin from QCSPIONs was assessed using a dialysis bag approach. A suspension of QCSPIONs (5 mg) in 10 mL of PBS was enclosed in a dialysis bag and incubated in 250 mL of PBS (pH 7.4) at 37 ± 0.5 ◦C with constant agitation. At predetermined time intervals, 5 mL aliquots of the external medium were collected and replaced with an equivalent volume of fresh buffer. The amount of released quercetin was determined by measuring absorbance at 375 nm using a UV–visible spectrophotometer.

The cumulative percentage release of quercetin was determined using the subsequent equation: Cumulative proportion of medication release (100%) = Volume of sample extracted equals bath volume multiplied by the sum of P(t − 1) and Pt, where P is the quantity of drug released at time t. Total Amount of Drug Initially Loaded multiplied by 100, where Pt denotes the proportion released at time ‘t’, and P(t − 1) represents the percentage released before ‘t.’ No precipitation or concentration-dependent saturation was observed during the quercetin release investigations, and the release medium was frequently replenished. At the same time, continuous stirring was employed to maintain sink conditions for extended trials.

Animals and living arrangements

Forty-two male Wistar rats weighing 180 to 200 g were imported from the Royan Institute in Isfahan, Iran. The rats were housed in polycarbonate cages under environmentally controlled conditions, with temperatures maintained at 20 ± 1 °C and relative humidity between 40% and 45%. A reversed 12-hour light/dark cycle was implemented. Throughout the study, the rats had free access to laboratory chow and tap water.

Animals and ethical approval

All animal procedures and husbandry techniques were carried out in strict accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals by the Animal Ethics Committee of the University of Isfahan. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Isfahan (Protocol Number: [IR.UI.REC.1402.029]). This research also adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Experimental design and treatment groups

Following acclimatization, rats were randomly assigned to seven groups (n = 6 per group): Group I (Control, C): Was fed a regular diet and plain drinking water without supplement for 35 days. The 35-day treatment period was selected to enable a robust sub-chronic safety evaluation and to assess biological effects under steady-state pharmacokinetic conditions following repeated daily administration.

Group II (QC oral): Received daily gavage of oral quercetin suspension in deionized water for 35 days (25 mg/kg).

Group III (SPIONs oral): Received daily gavage of PEG-coated SPION suspension in deionized water at a dose of 25 mg/kg for 35 days.

Group IV (QCSPION Oral): Received daily gavage of a suspension of PEG-coated SPIONs conjugated with quercetin in deionized water for 35 days at a dose of 25 mg/kg.

Group V (QC Intraperitoneal (IP)): Received daily IP injections of oral quercetin suspension in deionized water for 35 days (25 mg/kg).

Group VI (SPIONs Intraperitoneal (IP)): Received daily intraperitoneal injections of PEG-coated superparamagnetic iron oxide nanoparticle (SPION) suspension in deionized water for 35 days (25 mg/kg).

Group VII (QCSPION IP): Received daily IP injections of Quercetin conjugated with PEG-coated SPIONs suspension in deionized water for 35 days (25 mg/kg).

All treatments were administered at a dose of 25 mg/kg body weight of quercetin (for free quercetin [QC] and QCSPIONs groups or the corresponding nanoparticle mass without quercetin (for the SPIONs control group).

Preparation and administration of pharmaceuticals

Dosing was adjusted daily based on individual body weight measurements. Quercetin was suspended in 0.5 mL of deionized water and vigorously vortexed to prepare a homogeneous dispersion immediately before oral administration via gavage or intraperitoneal injection using sterile insulin syringes. SPIONs were suspended in 0.5 mL of sterile deionized water and sonicated for 30 min with a probe sonicator to achieve a uniform suspension, then administered via gavage or intraperitoneal injection. QCSPIONs were prepared similarly by dissolving 25 mg/kg in sterile deionized water and sonicated for 30 min to ensure homogeneity as a pretreatment step before administration. Gavage was performed using a curved stainless steel gavage needle (zonde needle; 16 gauge, 3 inches in length with a 2 mm ball tip, as standard for adult Wistar rats) attached to a 5 mL syringe. Animals were gently restrained, and the needle was carefully inserted into the esophagus to ensure delivery to the stomach, minimizing stress and the risk of aspiration. This procedure was conducted daily for 35 consecutive days.

Collection and preservation of tissues

At the end of the treatment period, rats were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) via intraperitoneal (IP) injection. Under deep anesthesia, approximately 1 mL of blood was collected through cardiac puncture using sterile syringes and needles. The samples were transferred to sterile tubes and centrifuged at 10,000 rpm for 15 min at 4 °C to separate the serum, which was then aliquoted and stored at − 70 °C. A craniotomy was performed to expose the ocular tissues. Both eyes were carefully enucleated with intact optic nerves and promptly placed in sterile containers, which were stored at − 70 °C until analysis.

HPLC sample preparation for quercetin analysis

Serum Samples:

A 300 µL sample of serum was combined with 60 µL of trichloroacetic acid (TCA) to precipitate proteins. The mixture was vortexed for 2 min and then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was filtered through a 0.20 μm syringe filter, and 20 µL of the filtrate was injected into the HPLC system.

Tissue Samples

Approximately 100 mg of ocular tissue was homogenized in 500 µL of phosphate-buffered saline (PBS, pH 7.4) under cold conditions. The homogenate was vortexed for 2 min and centrifuged at 10,000 rpm for 10 min at 4 °C. Next, 200 µL of the supernatant was mixed with 300 µL of methanol, vortexed again, and centrifuged under the same conditions. The final supernatant was filtered through a 0.20 μm membrane filter, and 20 µL was used for HPLC analysis.

Histopathological assessment

Eye tissues from rats treated with free quercetin, SPIONs, and QCSPION conjugates were fixed for 24 h in 10% neutral-buffered formalin. After fixation, the tissues were dehydrated, cleared, and embedded in paraffin. Serial Sects.  (4–5 μm thick) were cut and placed onto slides. These sections were stained with hematoxylin and eosin (H&E) for general morphology and Prussian blue to localize iron. Microscopic analysis was performed at 400× magnification to assess morphological changes and locate SPION.

Real-time polymerase chain reaction

Total RNA was extracted from rat eye tissues using the YTzol Pure RNA solution (Yekta Tajhiz Azma, Iran) according to the manufacturer’s protocol. The concentration and purity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA), and RNA integrity was assessed by agarose gel electrophoresis. cDNA synthesis was performed using the kit from Yekta Tajhiz Azma (Yekta Tajhiz Azma, Iran), with 1400 ng of total RNA in a reaction volume of 13.5 µL, using random hexamers and oligo(dT) primers as recommended by the manufacturer.

Quantitative real-time PCR (qPCR) was conducted using SYBR Green Master Mix (Thermo Fisher Scientific). The total volume was 20 µL, comprising 1 µL of cDNA template, 0.5 µL of each primer, 10 µL of SYBR Green mix, and 8 µL of nuclease-free water. The 2^−ΔΔCt method was employed for quantitative analysis, with normalization based on β-actin as the internal control. Primer sequences for the β-actin, BAX, and BCL-2 genes are listed in Table 1.

Table 1.

List and sequences of the primers

Gene	Forward	Reverse
BCL-2	GTGGATGACTGAGTACCT	GCCAGGAGAAATCAAACA
BAX	TTTGCTACAGGGTTTCATC	ATGTTGTTGTCCAGTICAT
β-actin	CTCTATGCCAACACAGTG	AGGAGGAGCAATGATCTT

Statistical analysis

Each experiment was conducted in triplicate and repeated at least three times. Data were analyzed using SPSS software version 22.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA). Comparisons between groups were made using one-way analysis of variance (ANOVA) followed by appropriate post hoc tests. When only two groups were compared, an independent t-test was used. Results are presented as mean ± SEM. Statistical significance was set at p < 0.05, with p < 0.01 and p < 0.005 considered highly significant.

Results

Synthesis and characterization of QCSPIONs

The successful synthesis of QCSPION was confirmed by Fourier transform infrared spectroscopy (FTIR) spectroscopy, X-ray Diffraction (XRD), and scanning electron microscope (SEM) (Table 2). Our previous study provides more details on these findings [21].

Table 2.

Physicochemical properties of synthesized nanoparticles

Property	Description / Value	Method	Comments
Shape	Spherical	Field Emission Scanning Electron Microscopy (FE-SEM)	Uniform morphology suitable for drug delivery and stability.
Particle size (diameter)	20–40 nm	FE-SEM images	Nano-scale size; contributes to high surface area to volume ratio for efficient quercetin conjugation and enhanced bioavailability/stability compared to free quercetin or non-magnetic carriers.
Elemental composition	Presence of iron (Fe) and oxygen (O)	Energy-Dispersive X-ray (EDX) analysis	Confirms successful synthesis of Fe₃O₄ based SPIONs; no impurities noted in accessible data.
Coating	Polyethylene glycol (PEG)	Used during synthesis for surface modification	Improves stability, reduces aggregation, enhances circulation time, solubility, and suitability for oral delivery/eye blood barrier crossing potential.
Crystalline structure	Characterized (specific phase not detailed)	X-ray Diffraction (XRD)	Consistent with magnetite (Fe₃O₄) in SPIONs; supports superparamagnetic behavior.
Superparamagnetism	Superparamagnetic	Inferred from use of SPIONs; not quantified (e.g., no M-H curve or saturation magnetization value provided in accessible content)	Key property for potential future magnetic targeting/hyperthermia, though not exploited in this oral administration study.
Hydrodynamic diameter (DLS)	85.4 ± 4.2 nm for SPION and 102.7 ± 5.8 nm for QCSPION	dynamic light scattering technology	Increase confirms successful surface conjugation.
Zeta potential	-28.5 ± 1.8 mV for SPION and − 22.4 ± 2.1 mV for QCSPION	laser Doppler velocimetry	Shift towards less negative value supports coating with quercetin.
Drug conjugation / loading	Successful covalent conjugation of quercetin	Physicochemical characterization confirms conjugation	Enables sustained release; overcomes quercetin’s limitations (poor water solubility, low bioavailability). No exact loading % or efficiency given.

Body weight of treated groups

The body weight of the rats was monitored throughout the 35-day treatment period. All rats steadily gained weight over the 5-week timeframe, whether they received no treatment or were treated with QC, SPIONs, and QCSPIONs. The growth curves for all groups showed a similar upward trend, and no significant differences were observed between treatments (Fig. 1).

Fig. 1.

Average body weight of rats over 35 days of treatment. All groups, including those given quercetin (QC), SPIONs, or QCSPIONs through oral or intraperitoneal routes, exhibited similar healthy weight gain. No significant differences were found among the groups

HPLC analysis of quercetin in serum and ocular tissue

Serum quercetin levels

Following oral administration, mean serum quercetin concentrations were 1137 ± 30.49 µg/mL in the QC group and 1098 ± 29.44 µg/mL in the QCSPION group, with no statistically significant difference between them (p > 0.05; Fig. 2A). In contrast, after IP injection, serum quercetin levels were significantly higher in the QC group (1087 ± 29.14 µg/mL) than in the QCSPION group (875.7 ± 23.48 µg/mL, p < 0.05; Fig. 2B).

Fig. 2.

Serum and ocular quercetin concentrations in rats treated with quercetin (QC) or SPION-conjugated quercetin (QCSPION) at 25 mg/kg. (A, C) Oral administration; (B, D) intraperitoneal (IP) administration. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Ocular tissue quercetin levels

Oral administration of QCSPION led to significantly higher quercetin accumulation in ocular tissue (366.6 ± 9.8 µg/g) compared to QC (180.8 ± 4.8 µg/g, p < 0.0001; Fig. 2C). Likewise, after IP injection, QCSPION-treated rats exhibited markedly increased ocular quercetin levels (549.4 ± 14.73 µg/g) compared to QC-treated rats (192.9 ± 5.1 µg/g, p < 0.0001; Fig. 2D).

Histopathological evaluation of rat ocular tissue

Oral administration

Histological examination of rat eye tissues after oral administration of QC, SPIONs, or QCSPION showed no significant differences compared to the control group (Fig. 3A).

Fig. 3.

Representative histological images (H&E staining) of rat cornea and retina following (A) oral and (B) intraperitoneal administration of quercetin (QC), nanoparticles (SPION), or quercetin-conjugated nanoparticles (QCSPION), compared with controls

In the corneal epithelium, the epithelial layer remained intact with no signs of detachment, necrosis, or inflammatory infiltration. The collagen fibers in the stroma were normally aligned, and the epithelial-stromal interface was well preserved. Retinal morphology also remained unaffected: the photoreceptor, inner nuclear, and ganglion cell layers exhibited normal cellular density and organization. No signs of degeneration, apoptosis, or inflammation were observed, indicating that oral administration of QC, SPIONs, or QCSPIONs does not negatively impact corneal or retinal histoarchitecture.

Intraperitoneal (IP) administration

Similarly, intraperitoneal injections of QC, SPIONs, or QCSPION did not change any histopathological abnormalities compared to the control group (Fig. 3B).

Qualitative assessment of the corneal epithelium revealed a steady cellular structure with no observable signs of detachment or necrosis. The stromal collagen matrix was present; however, potential fixation-related artifacts preclude a definitive assessment of stromal thickness or the complete exclusion of edema. No overt treatment-related pathological alterations were discernible. In the retina, the principal nuclear layers (inner nuclear layer, outer nuclear layer) and the photoreceptor layer maintained a continuous, laminar organization at the light microscopic level. At this resolution and with the fixation method used, no evident signs of widespread necrosis, disruptive inflammatory cell infiltration, or gross architectural disorganization were observed between treatment and control groups.

Overall, administering QC, SPIONs, or QCSPION through the oral and intraperitoneal routes preserved the structure and cellular integrity of corneal and retinal tissues, indicating that these therapeutic forms do not cause ocular toxicity under the conditions studied.

Prussian blue staining for SPION localization

Oral administration

Prussian Blue staining confirmed the presence of SPIONs and QCSPION in (A) the cornea and (B) the retina after oral administration. Small iron-positive deposits appeared as blue/green granules (Fig. 4A). In both the cornea and retina, QCSPION levels were higher than SPION alone (p ≤ 0.0001, p ≤ 0.05).

Fig. 4.

Prussian Blue staining of corneal and retinal sections from rats treated with SPIONs or QCSPION by oral A) or intraperitoneal (IP) B) administration. Iron deposits appeared as blue/green granules, confirming nanoparticle penetration into ocular tissues. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

These signals showed that nanoparticles could cross the blood–ocular barrier, although the staining intensity remained relatively low.

Intraperitoneal (IP) administration

In contrast, intraperitoneal administration of SPIONs and QCSPION resulted in more prominent iron staining in both corneal and retinal tissues (Fig. 4B). Prussian Blue–positive deposits were more numerous than in the orally treated group, indicating moderate accumulation of nanoparticles within the ocular tissues. Similarly, in both the cornea and retina, QCSPION levels were higher than those of SPION alone (p ≤ 0.0001, p ≤ 0.01). When comparing oral and IP administration, QCSPION given via IP was significantly higher than SPIONs given orally in both the cornea and retina (p ≤ 0.0001, p ≤ 0.01). Overall, Prussian Blue staining showed that both oral and IP routes of administration allowed nanoparticles to reach ocular tissues, with greater deposition observed following IP injection compared with oral delivery.

Quantitative real-time PCR analysis

Bax and Bcl-2 levels were measured in rat ocular tissue to assess potential apoptotic gene modulation after 35 days of treatment (Fig. 5).

Fig. 5.

Effect of oral (A-C) and intraperitoneal administration (D-F) of quercetin (QC), superparamagnetic iron oxide nanoparticles (SPION), and their conjugate (QCSPION) on apoptotic gene expression in rat ocular tissue after 35 days of treatment. P values indicate comparisons with control or between treatment groups as described in the Results section. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

According to results, QCSPION markedly suppressed Bax expression, reducing it to 0.04 ± 0.01-fold of the control level (P < 0.0001), in the orally treated groups, whereas QC and SPIONs alone did not exert significant effects. When examining Bcl-2 mRNA levels, QCSPION notably increased them compared to the control (3.65 ± 0.6-fold, P = 0.0135). SPIONs also elevated Bcl-2 expression compared to QC (P < 0.05). Importantly, the Bax/Bcl-2 ratio in the QCSPION group was significantly lower than in the control (0.041 ± 0.03-fold, P < 0.0001), indicating a strong shift toward anti-apoptotic regulation.

In the groups treated with IP, QC significantly downregulated Bax compared to the control (P < 0.0001). At the same time, QCSPION produced a stronger suppression relative to both the control and SPIONs (P < 0.01 for each). Additionally, both QC and QCSPION groups showed decreased Bax expression compared to SPION treatment alone (P < 0.01). For Bcl-2, QCSPION treatment resulted in the highest expression, significantly surpassing levels in control (P < 0.0001), QC (P < 0.05), and SPIONs (P < 0.01). QC also significantly increased Bcl-2 compared to control (P < 0.05). Regarding the Bax/Bcl-2 ratio, QC and QCSPION treatments caused a significant reduction compared to control (P < 0.0001 for both). Moreover, the Bax/Bcl-2 ratio in the QCSPION group was significantly lower than that observed with QC (P < 0.05).

Discussion

The study of effective preventive treatments for retinal degeneration relies on understanding that oxidative stress and apoptosis are key interconnected pathways involved in DR, AMD, and glaucoma [21, 22]. Quercetin has long been considered a promising candidate because of its strong antioxidant and anti-inflammatory properties [23, 24]. However, its hydrophobic nature, rapid first-pass metabolism, and limited bioavailability have greatly impeded its practical application in specific organs [13, 25]. For this reason, we assessed the promise of conjugating quercetin with superparamagnetic iron oxide nanoparticles (SPIONs) to create a novel quercetin-conjugated SPION (QCSPION) complex for enhanced ocular delivery. Our choice to conduct this research using normal rat eyes was intentional, as it allowed us to better understand the inherent pharmacokinetic and molecular effects of the conjugate without the influence of preexisting conditions. Establishing parameters for bioavailability and safety, along with baseline gene expression changes under normal conditions, is a crucial initial step before translating this formulation into disease models marked by common oxidative and inflammatory damage.

Our results clearly demonstrate that QCSPIONs significantly improved the ocular delivery of quercetin compared to the free drug. This enhancement aligns with the known properties of SPIONs as drug carriers: they increase solubility, protect compounds from enzymatic degradation, and prolong systemic circulation [26–28]. Importantly, SPIONs are small enough to utilize both paracellular and transcellular pathways across ocular epithelia, while their surface functionalization with PEG further boosts biocompatibility and reduces opsonization [29–31]. The ability of QCSPIONs to reach both the corneal and retinal layers, as confirmed by Prussian Blue staining, indicates that the nanoparticles crossed the BRB, a major barrier in ocular drug delivery. Similar results have been reported with polymeric nanoparticles and lipid nanocarriers delivering antioxidants to the retina [32]. Our findings suggest that SPIONs provide an additional advantage of traceability through histochemical staining and potential magnetically guided targeting.

Moreover, the observed higher accumulation of QCSPIONs in ocular tissues following IP administration may be attributed to systemic absorption pathways that facilitate nanoparticle distribution, including enhanced circulation and access to ocular vasculature. IP injection allows for broader systemic distribution and may bypass certain ocular barriers temporarily, leading to increased tissue penetration compared to topical routes. Additionally, the use of IP administration in our study provided a practical means to evaluate biodistribution and tissue uptake without the technical challenges associated with ocular injections or topical formulations.

Regarding potential outcomes if the formulation were administered as an eye drop, it is anticipated that bioavailability might be reduced due to the ocular surface barriers such as tear turnover, blinking, and limited penetration through corneal epithelium. However, modifications such as increasing residence time with mucoadhesive agents or using permeation enhancers could improve the ocular bioavailability of QCSPIONs in a topical formulation. Future studies should explore these formulations to optimize patient compliance and clinical applicability.

The biological effects of QCSPIONs extend beyond improved drug delivery. Molecular analysis revealed a striking reduction in Bax expression and upregulation of Bcl-2, resulting in a decreased Bax/Bcl-2 ratio. Since the Bax/Bcl-2 balance directly regulates mitochondrial outer membrane permeabilization, cytochrome c release, and subsequent caspase activation [33, 34]. Our results demonstrate that QCSPION treatment led to a marked downregulation of the pro-apoptotic gene Bax. As Bax is a central protein in initiating the mitochondrial pathway of apoptosis, this transcriptional change suggests a potential molecular shift away from a pro-apoptotic state. The significance of this finding lies in the established role of mitochondrial dysfunction and Bax-mediated apoptosis in the early stages of retinal degeneration [35, 36]. Future studies incorporating direct measures of caspase activation, mitochondrial membrane potential, and TUNEL assays are required to confirm whether this reduction in Bax mRNA translates to functional modulation of apoptosis-related gene expression and enhanced mitochondrial stability in vivo. Quercetin has previously been shown to modulate Bcl-2 family proteins and attenuate caspase-3 activation in models of oxidative injury [37, 38], yet the magnitude of these effects was modest due to limited intracellular concentrations. Our data indicate that nanoparticle conjugation not only improves delivery but also enhances quercetin’s natural bioactivity, resulting in a more potent anti-apoptotic effect.

Interestingly, while free quercetin affected Bax and Bcl-2, its impact was less consistent and less pronounced across different tissues. This highlights a key principle in drug delivery: pharmacological effectiveness is often limited not by a compound’s inherent potency but by its failure to reach sufficient concentrations at the target site [39]. By overcoming ocular delivery barriers, QCSPIONs achieve tissue levels that are pharmacologically meaningful, resulting in more substantial biological effects. These findings support reports that liposomal or polymer-based quercetin formulations boost systemic antioxidant effects [40], but our research extends this idea to ocular tissues, which present unique challenges for drug penetration.

The tolerability of QCSPIONs is a key finding of this study. Systematic histopathological assessment using a standardized semi-quantitative scoring system (see Methods) revealed no significant, treatment-related structural alterations in the cornea or retinal layers compared to the vehicle-control group. Key architectural features and cellular layers were preserved, and quantitative measurements of retinal layer thickness and ganglion cell counts showed no statistically significant differences from controls. These data indicate that a single administration of QCSPIONs, at the dose tested, was well-tolerated in ocular tissues [41]. This lack of toxicity is especially encouraging considering the potential for long-term or preventative use in chronic eye conditions, where repeated dosing might be required. Moreover, SPIONs alone did not influence Bax or Bcl-2 expression, emphasizing that the protective effects were due to quercetin, enhanced by the nanocarrier.

Together, these findings position QCSPIONs as a dual innovation: they serve as a nanotechnological solution to quercetin’s pharmacokinetic challenges and as a preventive approach against apoptosis-driven retinal degeneration. By demonstrating effectiveness in normal eyes, our study provides foundational evidence that QCSPIONs can accumulate in ocular tissues and modify the apoptotic balance before damage occurs. This preventive aspect is crucial, since many retinal disorders develop silently until advanced stages, and early interventions that enhance cellular resilience may change disease outcomes [42] (Fig. 6).

Fig. 6.

Experimental design showing seven groups of rats (n = 6) receiving quercetin (QC), SPIONs, or QC-SPIONs via oral gavage or intraperitoneal (IP) injection over 35 days

Although this study demonstrates that quercetin-conjugated PEG-coated QCSPIONs have enhanced ocular penetration and modulation of apoptosis-related gene expression in healthy male Wistar rats, certain limitations should be considered when interpreting the results. First, the research was conducted solely in standard rat models, which may not accurately represent changes in vascular permeability, inflammation, or barrier function seen in oxidative stress-related eye conditions like DR or AMD.

Therefore, confirming the efficacy in disease-specific models is necessary. Additionally, the 35-day dosing schedule allows for assessing both acute and subchronic effects but does not address long-term outcomes such as nanoparticle bioaccumulation, iron-related oxidative risks, or prolonged immune responses. While this study demonstrates the initial ocular tolerability and promising biodistribution of a single QCSPION administration, its findings are inherently limited to a subchronic timeframe. The potential for chronic administration in degenerative diseases necessitates a candid discussion of long-term safety. To better link transcriptomic changes to apoptosis or sustained visual function, additional testing such as Western blotting for protein levels, caspase activity, or in vivo functional assessments like electroretinography should be conducted, since the molecular analysis only examined Bax/Bcl-2 modulation via qPCR. Furthermore, using only male rats (n = 6 per group) overlooks potential sex differences in quercetin pharmacokinetics, nanoparticle clearance, and susceptibility to ocular oxidative damage, which are increasingly recognized in vision research. Lastly, exploring clinically relevant ocular delivery methods (e.g., topical drops or intravitreal injections) would enhance translational relevance, even though oral and intraperitoneal routes have shown benefits. These considerations underscore the need for further comprehensive studies to strengthen the preclinical evidence supporting QC-SPIONs as an effective vehicle for antioxidant delivery.

Future studies should capitalize on these delivery results by investigating the therapeutic efficacy of QCSPIONs in established disease models characterized by ocular oxidative stress, such as using functional endpoints like electroretinography and behavioral visual tests or diabetic retinopathy. In such models, the antioxidant mechanisms of QCSPIONs, including their potential to scavenge reactive oxygen species or modulate antioxidant enzymes, can be meaningfully quantified. Alternative administration routes, such as topical or intravitreal delivery, could also improve patient compliance and enhance ocular bioavailability. Furthermore, mechanistic studies of downstream caspase activity, ROS production, and mitochondrial membrane potential will help clarify how QCSPIONs support retinal cell stability.

Our findings therefore provide solid preclinical proof that delivering quercetin through nanoparticles improves eye penetration, promotes cell survival signals instead of apoptosis, and does so without harming healthy eyes. Combining nanomedicine and natural antioxidants with QCSPIONs presents a promising approach for preventing and treating eye diseases related to oxidative stress, connecting experimental pharmacology with clinical outcomes.

Conclusions

In conclusion, this study demonstrates that conjugating quercetin to SPIONs (QCSPIONs) significantly enhances its delivery to posterior ocular tissues following oral administration, compared to the free drug. At the molecular level, QC-SPIONs induced a more potent downregulation of the pro-apoptotic marker Bax than free quercetin, indicating a stronger influence on this key apoptotic pathway gene. These findings establish QCSPIONs as a promising nanoplatform for improving the ocular bioavailability and potential therapeutic efficacy of quercetin. Future work will focus on evaluating its functional benefits in established models of retinal degeneration.

Author contributions

M.H., N.M., and D.P. equally contribute to: Writing-review & editing, Writing, original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. A.R. and Z.S.: Writing-review & editing, Methodology, Investigation, Conceptualization. Paloma Burns: Methodology, Investigation. A.E. Writing-review & editing, Writing-original draft, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

Funding

This study was funded by the University of Isfahan.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All animal experiments were carried out in accordance with the guidelines for the care and use of laboratory animals and received approval from the Ethics Committee of Isfahan University.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.