Microneedle-based Rabies Vaccination: A Promising Approach Toward the WHO “Zero by 30” Target

1. Introduction
Rabies is a life[-threatening zoonotic encephalitis caused by the rabies virus (RABV) from the genus Lyssavirus of the family Rhabdoviridae [1]. The virus infects a broad spectrum of mammalian species worldwide and is transmitted most frequently to humans through bites, scratches, or direct contact with infected saliva on mucous membranes. Although the disease is vaccine-preventable, it still kills tens of thousands of people annually, primarily among children under 15 years of age in resource-limited settings [2].
In 2015, to address this ongoing global health threat, the World Health Organization (WHO), in collaborattion with the Global Alliance for Rabies Control (GARC), the Food and Agriculture Organization of the United Nations (FAO), and the World Organisation for Animal Health (WOAH), endorsed a global goal to achieve zero human deaths from dog-mediated rabies by 2030. Reaching this goal requires improved rabies awareness and education, increased access to high-quality, affordable human post-exposure prophylaxis (PEP) for populations at risk, and mass dog vaccination [3]. 
Currently, most licensed rabies vaccines are administered intramuscularly, which requires relatively high antigen quantities—typically several micrograms per dose. The high cost and cold chain requirements associated with these formulations have severly restricted rabies vaccination coverage in many rabies-endemic countries, especially in low- and middle-income countries (LMICs) [4]. Intradermal (ID) immunization has proven to be a cost-effective and antigen-sparing alternative, achieving comparable immunogenicity with considerably lower antigen quantities. This increased immunological efficiency is attributed to the high density of antigen-presenting cells (APCs) in the dermis, which facilitates improved antigen uptake and leads to strong immune stimulation [5]. However, its reliance on skilled personnel and cold-chain infrastructure restricts use in resource-poor settings.
To overcome these limitations and enhance global immunization efforts, recent developments in transdermal vaccine delivery systems, i.e. microneedle (MN) patch technology, have presented promising alternatives. MNs are micron-scale projections that can painlessly penetrate the stratum corneum (SC) —the main barrier to transdermal delivery— and deliver vaccine antigens directly into the epidermis and dermis [6]. Among various MN platforms, dissolving MN patches (dMNPs) fabricated from biodegradable and biocompatible polymers offer key advantages: they dissolve completely in the skin, eliminating sharps waste and the necessity for trained personnel. Moreover, their typical room-temperature stability improves logistical flexibility and minimizes dependence on cold-chain systems, which are critical in LMICs [7].
Preclinical and early-phase clinical studies have shown the safety, immunogenicity, and dose-sparing capacity of dMNPs for the administration of various vaccines, such as influenza, SARS-CoV-2, rotavirus, and poliovirus vaccines [8-10]. These findings demonstrate that dMNPs provide immunological outcomes comparable to conventional IM/ID administration, while offering a streamlined and operationally efficient vaccine delivery route. Therefore, dMNPs technology represents an attractive next-generation approach for vaccine delivery that addresses the operational, logistical, and accessibility limitations of conventional injection-based strategies. Based on these advancements, our study aimed to develop a dMNP loaded with rabies virus vaccine, formulated using hyaluronic acid (HA) and polyvinylpyrrolidone (PVP) as biocompatible and biodegradable polymers. We evaluated MN morphology, mechanical strength, skin insertion efficiency, and viral particle integrity, followed by comparative immunogenicity testing in BALB/c mice compared with standard IM vaccination. Virus-neutralizing antibody (VNA) titers were measured using the WHO-recommended rapid fluorescent focus inhibition test (RFFIT).

2. Materials and Methods

2.1. Materials
The inactivated rabies vaccine (Chirorab®, Bharat Biotech, India; lyophilized, reconstituted with 0.5 mL of sterile distilled water, containing 100 IU of rabies antigen) was obtained from the West Health Center, Tehran, Iran. HA with medium (MW≈500–750 kDa) and high (MW≈1300 kDa) molecular weights, PVP K30, and polydimethylsiloxane (PDMS; SYLGARD® 184 Silicone Elastomer Kit) were purchased from Huakang Biotech Inc. (China), Ningbo Yiho Import and Export Co. (China), and Dow Corning (Midland, MI, USA), respectively. All other solvents and reagents were of analytical grade.

2.2 Machining of MN mold
The patch design was created using SolidWorks Premium 2021 SP5.1 (SolidWorks, USA), and the aluminum master mold was fabricated via computer numerical control (CNC) machining, as described previously [11]. Precision wet cutting was performed using a Johnford VMC-550 machining center (Johnford Machinery Industries Co., Ltd., Taiwan) equipped with a diamond cutting tool (DO.6, 50L, D6, 400, China). A synthetic coolant (Syncool, Iran) was continuously applied during the machining process to minimize thermal deformation and tool wear. The workpiece was a cuboid aluminum alloy block with dimensions of 3 cm on each side. Optimized machining parameters included a spindle speed of 80,000 rpm, a cutting width of 0.6 mm, and a cutting depth of 1 mm, maintaining a dimensional tolerance of ±10 µm to ensure high-fidelity replication of the MN geometry.

2.3. Fabrication of rabies vaccine-loaded dissolving MN patches 
PDMS female molds were created from the MN master template following the manufacturer’s instructions. The PDMS base and curing agent were mixed at a 10:1 weight ratio and thoroughly de-aired under vacuum to remove trapped air bubbles. The mixture was then poured over the master mold and cured in a laboratory oven at 105 °C for 1 h, yielding a negative replica of the MN structure. Rabies vaccine-loaded dMNP were prepared by a two-step centrifugal casting process (Figure 1). The first-layer formulation was achieved by mixing medium molecular weight HA (1.5% w/v) with PVP (10% w/v) in sterile distilled water in a 1:1 volume ratio. The mixture was then subjected to magnetic stirring at room temperature for 2 hours. 100 IU of lyophilized rabies vaccine was reconstituted in 0.5 mL of this solution under gentle stirring. Next, 150 μL of the vaccine-polymer mixture was pipetted onto each PDMS mold and centrifuged twice at 3500 rpm for 20 minutes to fill all MN cavities. After centrifugation, the excess solution was removed carefully. Approximately 20 μL of the casting solution remained within each mold, corresponding to 4 IU of rabies vaccine.

Considering the volume proportion of the pyramidal part of the MN to its base (1:3), the amount of vaccine loaded in the MN tips was about 1 IU. After the initial 1-hour drying at 37 °C, 1.8 mL of the second casting solution containing high-molecular-weight HA (2.5% w/v) was applied to each mold and re-centrifuged under the same conditions to form the backing layer, followed by final drying at room temperature for 36 hours. The skin patches were carefully demolded using adhesive tape and stored in foil pouches with desiccant until use. Blank dMNPs without rabies vaccine were prepared in the same manner as negative controls.

2.4. Morphological characterization
The morphology of rabies vaccine-loaded dMNPs was examined using a stereo microscope (Stemi 305, ZEISS, Germany) to evaluate the uniformity of needles. For high-resolution morphological analysis, scanning electron microscopy (SEM; JSM-6400, JEOL Ltd., Japan) was used after gold sputter-coating (SC 7610; United Kingdom). Dimensional parameters, such as MN height and base width, were calculated from SEM images with ImageJ software, version 1.52. 

2.5. Mechanical strength testing
The mechanical strength of blank and rabies vaccine-loaded dMNPs was determined by a texture analyzer (Z050, ZwickRoell, Germany) equipped with a 500 N load cell. A 20×20 MN array was compressed against a polished stainless-steel plate at 0.5 mm/min. The patches were firmly affixed to ensure full contact during testing. Force–displacement plots were recorded to determine the failure force, defined as the point of sudden force drop indicating MN fracture, and deformation. Post-compression morphology was observed using SEM.

2.6. Skin penetration and histological evaluation
Ex vivo skin penetration experiments were conducted on dorsal cadaver skin excised from a Wistar rat, provided by the Experimental Study and Research Center of Iran University of Medical Sciences (IUMS). Following hair removal and drying, dMNPs were applied manually with consistent thumb pressure for 2 minutes. A 0.4% trypan blue solution was then applied for 10 minutes to visualize micropore formation, after which excess dye was removed and the skin rinsed. Micropuncture sites were photographed, and the number of blue-stained pores was counted to calculate insertion efficiency as the proportion of stained sites relative to the total number of MNs on the patch. Subsequently, the skin sample was fixed in 10% formalin, embedded in paraffin, sectioned to 5-10 µm thickness, and stained with hematoxylin and eosin (H&E). Histological assessment was carried out by a slide scanner (CELLNAMA LS5, Iran) to identify the depth of penetration of the MNs, with measurements taken from the SC to the deepest point of penetration.

2.7. Electron microscopy
Two weeks after fabrication, the rabies vaccine-loaded dMNPs were dissolved in 0.5 mL of distilled water to assess the size and morphology of rabies virus particles. The resulting sample was applied onto Formvar-coated copper grids. After 1–2 minutes of adsorption, excess liquid was removed, and the grids were negatively stained with 2% phosphotungstic acid (PTA, pH 5–6) for 1 minute. Grids were then air-dried and examined using transmission electron microscopy (TEM; LEO 906E, Germany) operating at an accelerating voltage of 100 kV. For comparison, the inactivated rabies vaccine solution with an equivalent concentration was similarly prepared and analyzed as a control.

2.8. Animals
The BALB/c mice (female, 6−8 weeks old, 17−20 g) were purchased from the Laboratory Animal Science Core Facility of Royan Institute, Tehran, Iran. Animal studies were conducted at the Experimental Study and Research Center of IUMS under the institution’s Laboratory Animal Welfare Guidelines.

2.9. Immunization protocol and sample collection
After one week of acclimatization, BALB/c mice were randomly assigned to three experimental groups (n=5 each): group 1 received rabies vaccine-loaded dMNPs (1 IU of rabies vaccine, equivalent to 1/100 of the antigen content of the human licensed rabies vaccine), group 2 received an intramuscular (IM) injection into the biceps femoris muscle of the posterior hind limb with 50 µL of the rabies vaccine (10 IU of rabies vaccine, equivalent to 1/10 of the antigen content of the human licensed rabies vaccine), and group 3 (negative control) received blank dMNPs containing no antigen. 
Immunizations were administered on days 0 and 7, according to the schedule of rabies pre-exposure prophylaxis. Dorsal hair was clipped and removed with a depilatory cream 4–6 hours prior to patch application. Mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) before administration. The patches were manually applied to the dorsal skin using consistent finger pressure and remained in place for 10 minutes before removal. 
Baseline blood samples were collected from the submandibular vein of mice before immunization and pooled within each experimental group for analysis. To monitor early antibody responses, additional blood samples were obtained two weeks after the primary immunization. Four weeks following the secondary immunization, mice were euthanized under deep anesthesia, and terminal blood collection was performed via cardiac puncture. Blood was allowed to clot at 37 °C for 30–60 minutes, centrifuged at 2000×g for 10 minutes at 4 °C, and serum was stored at −20 °C until analysis.

2.10. Rapid fluorescent focus inhibition test 
The RFFIT was conducted following the WHO-recommended protocol at the Rabies Reference Laboratory, Pasteur Institute of Iran. Briefly, heat-inactivated serum samples and a calibrated in-house reference serum were serially diluted in Dulbecco's modified eagle medium (DMEM) (Gibco) and mixed with the baby hamster kidney (BHK-21) subclone rapid growth selection (BSR)–adapted CVS-11 strain of rabies virus in 96-well plates. After 1 h incubation at 37 °C, BSR cells in DMEM supplemented with 8-10% FBS were added, and the plates were incubated for 20–24 h at 37 °C in a CO2 incubator. The cells were then fixed with cold acetone, stained with FITC-labeled anti-rabies conjugate, and examined under a fluorescence microscope. Titers were calculated by the Reed and Muench method based on 50% inhibition of fluorescence and expressed in IU/mL relative to the reference serum [12]. Samples with titers ≥0.5 IU/mL were considered seropositive in accordance with WHO guidelines [2].

 
2.11. Statistical analysis
All statistical analyses and graph generation were performed in SPSS software version 26. Data are presented as Mean±SD, and geometric mean titers (GMTs) with 95% confidence intervals (CI) were calculated and reported. To compare groups, one-way ANOVA was conducted (significance level set at P<0.05) and followed by Tukey’s honestly significant difference (HSD) test for pairwise comparisons.

3. Results 

3.1. Characterization of MNs
The CNC-machined MN master mold was fabricated and characterized, as illustrated in Figures 2A and 2B, comprising 400 cubical-pyramidal MNs arranged in a 20×20 array. Each MN measured 800 µm in height, with a 300×300 µm base and 800 µm tip-to-tip spacing, precisely matching the original CAD design.
Morphological analysis via stereo microscopy demonstrated that the fabricated cubical-pyramidal MNs exhibited sharp tips and uniform geometry (Figure 2C). Furthermore, SEM imaging confirmed the structural integrity and homogeneity of the MNs, revealing no evidence of deformation or fracture during demolding or drying (Figure 2D).
Subsequent post-drying dimensional analysis indicated an average needle height of 651.23±4.30 μm, representing an ~18.6% reduction relative to the master mold height of 800 μm. Despite this, the structural integrity and base width (~288.3±5.5 μm) remained stable.

3.2. Mechanical performance
To assess mechanical properties, the strength of blank and rabies vaccine-loaded dMNPs was evaluated quantitatively using a universal testing machine. The resulting force–displacement profiles (Figures 3A and 3B) showed that the blank dMNPs had an average failure force of 0.61±0.01 N per needle, while the rabies vaccine-loaded dMNPs demonstrated a slightly reduced failure force of 0.403±0.006 N per needle. Post-compression SEM images (Figure 3C) verified that, although mild tip blunting occurred, no complete structural failure was observed.

3.3. Skin insertion and histological analysis
In ex vivo experiments, the insertion test revealed that the 20×20 MN arrays achieved an insertion efficiency of over 95%, calculated as the ratio of successfully formed micropores to the total number of MNs (400), following the application of consistent thumb pressure for 2 minutes (Figure 4A). To further evaluate penetration and disruption of the SC, a 0.4% (w/v) trypan blue solution was applied to the MN-treated skin. Following 10 minutes of staining, rinsing, and visual inspection, over 80% of the MNs were found to have formed visible blue-stained micropores (Figure 4B).

For precise determination of penetration depth, histological evaluation of H&E-stained skin sections was performed. The analysis showed that MN-induced micropores extended from the SC to a depth of approximately 300 μm, reaching into the viable epidermis or upper dermis (Figure 4C).

3.4. TEM analysis
To investigate the preservation of rabies virus particles, their size and morphology within dMNPs two weeks post-fabrication were compared to those of the native inactivated rabies virus solution using TEM analysis. As depicted in Figure 5, the virions appeared as bullet-shaped particles measuring approximately 180×80 nm. Notably, no significant differences in particle size or morphology were observed between the polymer-incorporated virus and the control group.

3.5. Immunogenicity evaluation
The immunogenicity of the rabies vaccine-loaded dMNPs was evaluated in BALB/c mice using the RFFIT following a two-dose immunization schedule. As illustrated in Figure 6, baseline (day 0) VNA titers were undetectable across all experimental groups. At 14 days post-primary immunization, significant differences in antibody titers emerged among groups (one-way ANOVA, P<0.001), with the rabies vaccine-loaded dMNP group displaying the highest titers (GMT: 0.39 IU/mL, 95% CI, 0.38%, 0.4%), followed by the IM group (GMT: 0.34 IU/mL, 95% CI, 0.31%, 0.38%), while the blank dMNP group showed no increase. Tukey’s HSD test showed significant differences between both the dMNP- and IM-vaccinated groups versus the blank control (P<0.001).

Four weeks after the secondary immunization, antibody titers in the rabies vaccine-loaded dMNPs and IM groups were significantly elevated compared to the blank dMNP group (P<0.001). Specifically, the rabies vaccine-loaded dMNP group exhibited a mean titer of 7.72±0.94 IU/mL (GMT: 7.67 IU/mL, 95% CI, 6.8%, 8.64%), while the IM group had a mean titer of 7±0.79 IU/mL (GMT: 6.95 IU/mL, 95% CI, 6.2%, 7.8%). Tukey’s HSD test revealed no significant difference between the dMNP-vaccine and IM groups (P>0.05). In contrast, the blank dMNP group maintained consistently low titers (GMT: 0.10 IU/mL), significantly lower than both experimental groups (P<0.001).

4. Discussion
The present study successfully developed a dMNP loaded with an inactivated rabies vaccine using an HA/PVP matrix, achieving protective immunogenicity equivalent to conventional IM vaccination while requiring only one-tenth of the antigen dose. The CNC machining process enabled the precise fabrication of a durable MN master mold, overcoming limitations associated with other microfabrication methods. While ultra-hard metals like stainless steel offer high stability, aluminum was chosen for its ductility and lower tool deformation, allowing accurate micro-scale structuring. Optimized machining parameters—including spindle speed, cutting width, depth, and symmetrical strategies—minimized thermal and mechanical stresses, ensuring high-dimensional fidelity of the MNs.
PDMS molds derived from the master were used to fabricate rabies vaccine-loaded dMNPs via two-step centrifugal casting, localizing the antigen in the needle tips and reinforcing the patch with a backing layer. The HA/PVP matrix, previously validated for molding fidelity, mechanical robustness, and rapid dissolution [13], provided a suitable polymer base. In the referenced study, in vitro release experiments conducted at 37 °C demonstrated that more than 80% of the antigen payload was released at early time points in a molecular-weight-dependent manner, confirming the fast dissolution behavior of the HA/PVP-based MN system and efficient antigen release following application. 
The observed post-drying shrinkage, resulting in an average needle height reduction of ~18.6%, can be attributed to water evaporation and matrix densification during the drying process—a phenomenon commonly reported in polymer-based MNs [11, 14]. Base dimensions and tip sharpness remained unaffected, consistent with reports that moderate shrinkage does not impair mechanical performance or skin penetration [15]. Importantly, maintaining tip sharpness and geometrical uniformity is critical for reliable skin insertion and successful antigen delivery to the viable epidermis and upper dermis, both of which were successfully achieved in this study. 
In evaluating mechanical performance, the slight reduction in failure force for vaccine-loaded dMNPs compared to blank patches may stem from alterations in the polymer matrix structure upon vaccine incorporation, potentially involving changes in HA/PVP distribution or enhanced hygroscopic behavior. Nevertheless, the failure force of the loaded dMNPs remained substantially above the minimum insertion force (~0.05–0.1 N per needle) required to penetrate the SC and reach the viable epidermis, as previously reported [16].
For ex vivo skin insertion and histological analyses, rat skin was used because its greater thickness and surface area allow more reliable assessment of MN penetration. Skin penetration experiments confirmed the practical applicability of the dMNPs, demonstrating high insertion efficiency and clear micropore formation verified by trypan blue staining. Because trypan blue cannot cross intact skin, its presence in the micropores indicates successful transdermal penetration. These findings are consistent with previous reports showing that pyramidal HA/PVP-based dMNPs effectively traverse the SC under minimal mechanical pressure [17]. Histological analysis further confirmed that the MN-created micropores penetrated to a depth of ~300 μm, effectively breaching the skin barrier and reaching the viable epidermis or upper dermis. This penetration depth is particularly relevant from an immunological perspective, as it enables direct access to skin-resident APCs, including Langerhans cells and dermal dendritic cells. Targeting these cells facilitates efficient antigen uptake and presentation, thereby promoting robust immune activation and contributing to the enhanced immunogenicity and dose-sparing effect commonly associated with dissolving MN-based vaccination strategies [18].
Regarding the preservation of vaccine integrity, TEM analysis revealed no significant differences in the size or morphology of rabies virus particles within the dMNPs compared to the native inactivated virus, consistent with the ultrastructural morphology of lyssaviruses [19]. These results indicate that the fabrication process did not cause aggregation, deformation, or degradation of the viral particles, which is consistent with reports that polymer-based MNs preserve viral morphology during processing and storage [18, 20]. Overall, the HA/PVP-based dMNP system effectively maintained the ultrastructural integrity of rabies virions, supporting its potential as a stable and immunogenically reliable platform for intradermal vaccine delivery.

Immunogenicity evaluation in BALB/c mice showed undetectable baseline antibody titers, confirming the absence of pre-existing immunity. By day 14, the rabies vaccine–loaded dMNP group displayed higher titers than controls, indicating a strong early response. Four weeks after the booster, both dMNP and IM groups exhibited significantly increased and comparable titers, while the blank group remained low, confirming response specificity. All vaccinated mice exceeded the WHO protective threshold of 0.5 IU/mL, demonstrating successful seroconversion and robust protective immunity. These results demonstrate that the dMNP platform elicits a robust humoral immune response comparable to conventional IM injection, even though it delivers only one-tenth of the rabies vaccine antigen (1 mg via dMNP vs. 10 mg via IM). Notably, the 1 mg dose administered through dMNP corresponds to only 1/100 of the antigen content of the licensed human rabies vaccine, yet it still achieves an immune response equivalent to the higher-dose IM administration. This efficacy is attributable to the targeted delivery of antigen into the immunocompetent epidermis and upper dermis, which are rich in APCs. This targeted approach boosts antigen presentation and helps achieve a dose-sparing effect. 
These findings align with previous studies supporting dMNP-based immunization. For instance, Arya et al. demonstrated that a two-dose rabies DNA vaccine regimen (with the first dose on day 0 and a booster on day 28) induced durable neutralizing antibody titers above the protective threshold in beagle dogs [21]. Similarly, Arshad et al. first demonstrated that rabies vaccine–loaded polymeric MNPs combined with iontophoresis elicited significantly higher IgG and rabies virus neutralizing antibody (RVNA) titers compared to MNPs alone, and subsequently reported complete seroconversion in Wistar albino rats following transcutaneous delivery of rabies, BCG, and tetanus toxoid vaccines using polymer-based MNPs [22, 23].
These dMNPs present a dose-sparing advantage by efficiently delivering vaccines intradermally, making them particularly useful in low-resource settings where cost, cold-chain logistics, and availability of trained staff are major constraints. Beyond achieving immune responses comparable to conventional IM injection, these patches provide practical benefits—including self-administration, elimination of sharps waste, reduced pain, and improved thermostability—which enhance feasibility, acceptance, and scalability, aligning with the WHO’s “Zero by 30” target of eliminating human deaths from dog-mediated rabies.
Given these findings, rabies vaccine–loaded dMNPs have emerged as a promising next-generation immunization strategy with strong translational potential. Further work should focus on large-scale manufacturing, long-term stability studies under field-relevant conditions, and clinical trials to confirm safety, immunogenicity, and usability in human populations. Importantly, while the current study primarily assessed humoral protective responses (RVNA), future investigations should include cellular immune profiling, such as cytokine analysis (e.g. IFN-γ, IL-4), to elucidate the underlying mechanisms of immune induction. Adoption of such a platform could meaningfully expand rabies vaccination coverage and contribute to closing the global equity gap in life-saving prophylaxis.

5. Conclusion 
This study shows that rabies vaccine–loaded dissolving MN patches (dMNPs) elicit protective humoral immune responses comparable to conventional IM vaccination, while providing reliable mechanical strength, effective dermal delivery, and preservation of vaccine integrity. The dMNP platform offers important advantages, including dose sparing, improved thermostability, ease of administration, and reduced reliance on cold-chain logistics and trained personnel—features particularly valuable for low-resource and endemic settings. Overall, these findings highlight dMNPs as a promising, scalable vaccination strategy that could support global rabies elimination efforts. Further studies addressing large-scale manufacturing, long-term stability, and clinical evaluation are warranted to advance translation to human use.

Acknowledgements
The authors would like to thank Mohammad Mahdi Ghasemi for his valuable assistance in designing the schematic illustrations in this manuscript.

Compliance with ethical guidelines
This study was approved by the research Ethics Committee of Iran University of Medical Sciences, Tehran, Iran (Code: IR.IUMS.AEC.1402.039).

Data availability
All data supporting the findings of this study are presented within the article.

Funding
The present study was funded and supported by Iran University of Medical Sciences, Tehran, Iran (Grant No.: 25591). 

Authors' contributions
Conceptualization, supervision, and funding acquisition: Hossein Keyvani and Angila Ataei-Pirkooh; Methodology: Mehrnaz Hosseini Tehrani, Reza Aramideh Khouy, and Atefeh Malek-Khatabi; Investigation: Mehrnaz Hosseini Tehrani, Reza Aramideh Khouy, Atefeh Kachooei, Babak Peirovi, and Leila Mousavizadeh; Formal analysis: Mehrnaz Hosseini Tehrani and Reza Aramideh Khouy; Visualization: Mehrnaz Hosseini Tehrani, Reza Aramideh Khouy, and Maryam Shahali; Writing the original draft: Mehrnaz Hosseini Tehrani, Reza Aramideh Khouy, Atefeh Malek-Khatabi, and Angila Ataei-Pirkooh; Review and editing: Mehrnaz Hosseini Tehrani, Reza Aramideh Khouy, Atefeh Malek-Khatabim Mazda Rad-Malekshahim Hossein Keyvani, and Angila Ataei-Pirkooh; Validation: Mehrnaz Hosseini Tehrani, Reza Aramideh Khouy, Atefeh Malek-Khatabi, and Mazda Rad-Malekshahi; Resources: Atefeh Malek-Khatabi, Mazda Rad-Malekshahi, and Hossein Keyvani; Project administration: Angila Ataei-Pirkooh; Final approval: All authors.
Mehrnaz Hosseini Tehrani and Reza Aramideh Khouy equally contributed.

Conflict of interest
The authors declared no conflict of interest.

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