Optimized spray-drying of zinc sulfate-loaded liposomes: physicochemical characterization and in vitro release assessment

Abstract

Zinc, a vital trace element plays a crucial role in various physiological functions, faces challenges in bioavailability due to dietary and pharmaceutical interactions. To enhance its delivery and stability, this study formulated spray-dried zinc sulfate-loaded liposomes using Maltodextrin as a stabilizing agent. Zinc-loaded liposomes were prepared via the thin-film hydration method and spray-dried under optimized conditions. The spray-dried liposomes exhibited a mean particle size of 18.35 ± 7.42 μm and a polydispersity index (PDI) of 0.32 ± 0.18, indicating size uniformity. A notably high encapsulation efficiency (EE%) of 88.24 ± 0.98% was achieved, demonstrating effective zinc entrapment. Thermal stability analysis revealed significant improvement, with TGA confirming enhanced structural integrity. XRD and FTIR analyses indicated successful encapsulation and interaction between zinc sulfate and phospholipids, while in vitro release studies showed sustained zinc release, with 94.98% cumulative release over 12 h compared with the rapid dissolution of free zinc sulfate. Stability tests over 90 days highlighted minimal changes in particle size (Δ = 0.22 μm) and EE% (Δ = 0.13%) under ambient storage conditions. These findings demonstrated the potential of spray-dried liposomes as an effective delivery system for zinc sulfate, offering enhanced stability and controlled release for pharmaceutical and nutraceutical applications.

Introduction

Zinc (Zn) is a necessary trace mineral that plays a pivotal role in the human body¹. It is essential for a wide range of physiological functions, participating in several aspects of cellular metabolism such as immune function, protein synthesis, wound healing, DNA and RNA synthesis, signal transduction, cell division, hair growth, and skin health^2,3. Zinc is also prescribed for the long-term management of Wilson’s disease, a genetic disorder characterized by the accumulation of copper in different organs^4,5. Its versatile functions highlight the importance of maintaining adequate zinc levels for overall health and well-being¹. Despite the valuable role of zinc, its bioavailability was affected by different factors. Specific food components, such as phytates, calcium, phosphate, and some medications, including antibiotics and diuretics can reduce zinc bioavailability and inhibit its absorption⁶. Additionally, the consumption of zinc supplements has been observed to potentially elicit gastrointestinal distress in a subset of individuals^7,8. To diminish the problem of minerals bioavailability and their unpleasant side effects, different delivery systems have been developed^9,10.

The coacervation technique and nanoencapsulation were previously employed to prepare zinc sulfate and other mineral particles to improve bioavailability^11,12. Additionally, liposome can also encapsulate zinc and protect it from interactions with food compounds that negatively affect its absorption¹³. Liposomes have been shown to effectively encapsulate therapeutic agents, including zinc complexes, enhancing their stability and bioavailability¹⁴.

Despite the advantageous characteristics of liposomes, including biocompatibility, a lipid bilayer structure, appropriate particle sizes, and significant applications such as the encapsulation of various pharmaceutical agents, the protection of drugs from degradation, and the enhancement of unfavorable pharmacokinetics, the stability and storage of liposomes encounter numerous challenges¹⁵. These issues can hinder their efficacy as drug carriers. Physical or chemical reactions and environmental factors such as temperature and pH could affect the liposome’s storage condition^16,17. Indeed, loosening of the bilayer structure can lead to increased size, decreased stability, and the loss of encapsulated substances, ultimately reducing the efficacy of liposomes at the target point. The destabilization can even result in the liposome’s structural breakdown and leakage, degradation, or decomposition of the encapsulated core material¹⁸. Modifying liposomes through biopolymers or implementing drying techniques has become a powerful approach to enhance liposomes’ storage stability and physicochemical specifications^19,20.

Common drying methods, such as freeze drying, spray drying, and spray-freeze drying, have successfully improved the stability and bioavailability of liposomes^21–23. The most often used, and highly efficient method in drug delivery systems for converting aqueous materials into a dried and powdery form is the spray drying process²⁴. A bioavailability study of spray-dried Lopinavir and Simvastatin loaded in liposomes showed that they can be more effective than free drugs^25,26. The effect of carrier type and lipid-to-carrier ratio on the properties of Salbutamol loaded up in liposome in the spray drying process produced stable inhalable dry liposomes powder²⁷. Dried nanoliposomes loaded with Dapsone using a spray drying method presented site-specific delivery to the lung for prolonging drug retention²⁸. The physicochemical properties of spray- dried Silymarin and Letrozole loaded in liposomes were evaluated^29,30. Moreover, different natural compounds such as, clotrimazole, tacrolimus, docetaxel and ciprofloxacin loaded up liposome were dried and studied by this technique^31–33.

As the most related literature review in the case of zinc sulfate, Polekkad et al.‍³⁴ demonstrated optimization of spray drying conditions for microencapsulation of zinc sulfate by several wall materials. Nagy et al.⁴ explored zinc sulfate loaded in liquid liposomal formulation for treating Wilson’s diseases. Sulwesky et al.³⁵ have investigated the interaction between different Zn^+ 2 concentrations with multilayers of phospholipids. Recent work by Zhang et al.³⁶ have reported the preparation of zinc sulfate gel to promote wound healing. The present research work bridges crucial gaps in the existing literature by developing a first-of-its-kind formulation specifically for zinc sulfate loaded in solid liposomal formulation using optimized spray-drying technique, providing comprehensive physicochemical characterization including SEM, EDX, XRD, TGA, and FTIR analyses; and demonstrating the superior bioavailability through in vitro release and stability studies that have not been previously conducted for spray-dried zinc-loaded liposomes.

So, the present study investigates the application of a liposomal drug delivery system to enhance the bioavailability of zinc sulfate. By combining liposomal encapsulation with spray-drying, we achieved an optimal balance of stability, controlled release, and uniformity, creating a promising platform for enhancing zinc supplementation in pharmaceutical applications.

Materials and methods

Materials

Zinc sulfate heptahydrate (CAS No. 7446-20-0) was supplied by Sigma Aldrich. The soy lecithin (purity 85%), cholesterol and maltodextrin (CAS 32671-12) were purchased from Kimia Exir Jam (Tehran, Iran). Absolute ethanol (CAS No. 64-17-5) was obtained from Merck (Tehran, Iran). Ultra-pure water was obtained from KFLOW (KFRO-150GPD) water purification systems. 2.2. Method.

Preparation of liposomes

Liposomes were prepared using the thin film hydration method as described by ref³⁷. Briefly, a lipid composition consisting of 90 mg soybean-derived L-α-phosphatidylcholine (PC) (Avanti Polar Lipids, Inc.) and 10 mg cholesterol (molar ratio 9:1) was dissolved in 20 mL of absolute ethanol. The resulting solution was transferred to a 50 mL round-bottom flask, and the organic solvent was completely evaporated using a rotary evaporator at 45 °C under controlled vacuum conditions (160 mbar) for 15 min to form a uniform thin lipid film on the flask’s inner surface. The lipid film was further dried under nitrogen gas flow overnight in a desiccator to ensure complete removal of residual organic solvent. For preparation of empty liposomes, the dried lipid film was hydrated by spraying 30 mL of 10 mM NaCl solution (pH 5.5) onto the film for 15 min at 40 °C, a temperature above the phase transition temperature of the phospholipids, to facilitate lipid hydration and vesicle formation. For zinc-loaded liposomes, the hydration medium consisted of 30 mL of 10 mM NaCl solution (pH 5.5) containing zinc sulfate at a concentration of 10 mg·mL^− 1 (expressed as zinc ion concentration). Following hydration, the multilamellar vesicle suspension was transferred to an ice-water bath (4 ± 1 °C) to stabilize the formed liposomes. Size reduction was subsequently achieved by sonication in an ultrasonic bath at room temperature for 5 min to produce smaller, more homogeneous liposomal vesicles suitable for further characterization and application.

Characterization of liposome

Particle size distribution and Poly dispersity index (PDI) value of liposomes

Measurement of mean diameter and size distribution of particles was carried out by Mastersizer 2000 (Malvern Instruments, Malvern, United Kingdom) at 25° C. The Scirocco dry dispersion unit was used to assess the particle size distribution of powdered coated liposomes. The dried coated liposomes were interspersed in absolute ethanol to keep away from possible accumulation. Then, the vessel containing liposome suspension was placed into an ice- water bath (4 ± 1 °C), and sonicated by an ultrasonic bath (Model 031 S, Farasot Company, Tehran, Iran) for 10 min to remove the accumulation of particles. Evaluation of average particle size and PDI value was performed from 16 runs for each record.

Zinc ion assay by flame atomic absorption spectroscopy (FAAS)

Liposome suspension and other samples were tested according to the described method in USP 42 to assess zinc ion concentration. Briefly, Zinc ion content was analyzed using a Flame Atomic Absorption Spectrophotometry (FAAS) system (AAnalyst 300, Perkin Elmer AAnalyst, United States). This instrument consisted of an automatic sampler (AS90, Perkin Elmer, United States) and a wide-range photomultiplier detector. The spectroscopic condition was optimized by recording a steady base line using 0.125 N hydrochloric acid as blank. The flame was provided by acetylene-air as fuel gas with a flow rate of 1600 mL. min^− 1. The result of absorbance of blank, standards and samples were collected at 213.8 nm wavelength by applying zinc hallow-cathode lamp. Standard stock solution (50 µg. mL^− 1) was prepared by diluting zinc standard stock solution (1000 µg. mL^− 1) and applied to prepare zinc standard solution. 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the standard stock solution were transferred to the separate 100-mL volumetric flasks and diluted the contents of each flask with 0.125 N hydrochloric acid to volume to get concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 µg. mL^− 1 of zinc. The standard calibration curve (y = 0.2576x + 0.043; R² = 0.9996) was made by plotting absorbance quantity versus zinc concentration in a graph. The amount of zinc was calculated using standard a calibration curve.

Assessment of encapsulation efficiency (%) and loading capacity (%)

Encapsulation efficiency (EE%) and loading capacity (LC%) are calculated by determining free zinc ion concentration in aqueous liposome suspension that is centrifuged using cooling microcentrifuge instrument at speed 12,000 rpm for 20 min at 4 °C. The supernatant fluid was diluted 100 times by 0.125 N hydrochloric acid and the amount of free zinc ion was evaluated according to Sect. 2.5³⁹.

Energy dispersive X-ray analysis (EDX)

An energy-dispersive X-ray instrument (EDX) (SAMx EDS, SAMx, France) was applied to identify and determine the presence of zinc ion in zinc sulphate-loaded liposomes. The images were recorded and evaluated through the instrument software.

Fourier transform infrared spectroscopy analysis (FTIR)

To verify the interaction between zinc ions and phospholipids in the liposomal structure, FTIR spectra were recorded using a Fourier transform infrared (FTIR) instrument (Nicolet Avatar 360, Thermo Nicolet Corp., United States) equipped with an attenuated total reflectance (ATR) accessory. The FTIR scans were performed over a wavelength range of 400 cm⁻¹ to 4000 cm⁻¹ at a resolution of 1 cm^− 1.

Thermal gravimetric analysis (TGA)

The thermogravimetric behaviors of specimens were analyzed using TGA tool (STA 6000, Perkin Elmer, USA). The thermal gravimetric tests were conducted out at a heating rate of 10 °C min^− 1 under a nitrogen flow of 60 ml. min^− 1. 6.0 mg of Zinc sulfate heptahydrate salt and zinc sulfate loaded liposomes were weighed precisely in a pan, and the device temperature was increased from 50 to 400 °C.

X-ray diffraction analysis (XRD)

The geometry and crystal structure of the coated liposomes were studied using an XRD device (XRD XPert Pro, Malvern PANalytical, United Kingdom) with Cu Kα radiation at 15 mA and 40 kV. X-ray diffraction spectra were recorded at ambient temperature, ranging from 0° to 80° (2θ) at a scan speed of 1° per minute.

Preparation of spray-dried liposomes

A maltodextrin solution (10% w/v) was prepared in purified water and homogenized using a temperature-controlled magnetic stirrer at 50 °C for 2 h. Zinc sulfate-loaded liposomes were incorporated into the maltodextrin solution at a 1:1 (v/v) ratio by dropwise addition at a flow rate of 1.0 mL. min^− 1 under continuous stirring at ambient temperature. The resulting dispersion underwent spray drying using a Büchi Mini Spray Dryer B-191 (Büchi Labortechnik AG, Flawil, Switzerland). The spray drying parameters were selected based on a combination of preliminary experiments and existing literature on liposomal formulations containing thermolabile components. The rationale for our specific parameter selection is as follows: Inlet temperature (90 °C) was deliberately chosen as the optimal balance between efficient drying and preservation of liposomal integrity. Higher temperatures (> 100 °C) led to excessive thermal stress on the phospholipid bilayers, resulting in increased leakage of zinc sulfate. In comparison, lower temperatures (< 80 °C) resulted in insufficient moisture removal and poor powder flow properties. This is consistent with the data reported in the literature^34,39which demonstrated that liposomal stability is compromised at inlet temperatures exceeding 100 °C. The outlet temperature range of 57–62 °C was maintained to ensure the product temperature remained below the phase transition temperature of the phospholipids used in our formulation. Our preliminary studies showed that outlet temperatures above 65 °C resulted significantly increased particle size distribution and polydispersity index. The pump flow rate of 3%, a relatively low feed rate, was selected to ensure adequate residence time in the drying chamber, allowing for effective encapsulation of the liposomes within the maltodextrin matrix. It should be noted that the higher flow rates resulted in incomplete drying and agglomeration of particles. Atomizing gas flow rate (550 L.h^− 1) and aspirator rate (80%): These parameters were optimized to achieve the desired particle size distribution (primarily in the 2–5 μm range) while minimizing particle agglomeration. The lower gas flow rates resulted in larger, less uniform particles, while higher rates increased the risk of premature liposome rupture due to shear forces. The selected parameters represent the optimal processing conditions for maintaining zinc stability within the liposomal structure while achieving suitable powder characteristics. It should be noted that the liposomal dispersion was maintained under continuous agitation throughout the spray drying process to ensure a homogeneous distribution of the active components.

Characterization of spray-dried liposome

Assay of zinc in spray-dried liposome

The zinc content in the spray-dried liposomes was quantified using the flame atomic absorption spectrophotometric method. Briefly, the formulation was dissolved in methanol at a concentration of 1 mg. mL^− 1, and zinc concentration was determined by measuring the absorbance according to the previously described method by ref⁴⁰. The drug loading was expressed as mg of zinc per mg of spray-dried liposomes. All measurements were performed in triplicate, and results were reported as mean ± relative standard deviation.

Morphology of spray-dried liposome

The morphological characteristics and dimensions of the spray-dried liposomes were analyzed using a scanning electron microscopy instrument (SEM; SU 3500, Hitachi, Japan). Samples were set on aluminium stubs and sputter-coated with a 60-nm gold layer under vacuum conditions. The SEM analysis was performed in secondary electron mode at an accelerating voltage of 15 kV.

In vitro release of liposome formulations

An in vitro release study evaluated and compared the zinc sulfate release profiles, conventional liposomal and spray-dried liposomal formulations. The dissolution test was performed under controlled conditions using phosphate buffer (pH = 6.8) as the dissolution medium, maintained at 37 ± 0.5 °C with continuous stirring at 50 rpm. To prevent evaporation of the medium, the dissolution vessel was sealed throughout the experiment. Liposomal formulations containing 23.5 mg of zinc were introduced into 5 mL of phosphate buffer, dispersed, and then added to a post-treated dialysis bag with a molecular weight cut-off (MWCO) of 12,000–14,000 Da (Spectrum Laboratories, Inc., USA). The dialysis bag was transferred to 100 mL of dissolution medium. Aliquots (1 mL) were collected at predetermined time intervals (0, 0.25, 0.5, 1, 2, 3, 6, 9, 12, 24, and 48 h) and analyzed using flame atomic absorption spectrophotometry. Following each sampling, the withdrawn volume was replaced with a fresh dissolution medium at 37 °C to maintain constant volume conditions. Zinc concentrations were quantified using a linear calibration curve (y = 36.096x + 227.800; R² = 0.9996) within the range of 0.5–2.5 µg. mL^− 1, with measurements performed in triplicate. The percentage of zinc released was calculated using the following equation:

1

Where Z_t, Z_r, and Z₀ represent the dissolved zinc amount (mg), the removed zinc amount (mg), and the total initial zinc amount (mg), respectively.

Storage stability test

The stability profile of the formulations was systematically investigated using accelerated stability protocols in compliance with the ICH Q1A (R2) 2003 guidelines for the stability test of new drug substances and products⁴¹. Samples were stored in borosilicate glass containers under two distinct temperature conditions: room temperature (25 ± 2 °C) and refrigerated storage (4 ± 0.5 °C) for 90 days. Periodic stability assessments were conducted at predetermined time points (days 7, 15, 30, 60, and 90) to monitor the critical quality parameters, specifically drug encapsulation efficiency and particle size distribution. This longitudinal analysis facilitated the quantitative evaluation of formulation stability and provided insights into temporal variations in physicochemical characteristics.

Statistical analysis

An analysis of variance (ANOVA) was employed to assess how the variables interact to influence the results. One-way ANOVA analysis followed by Tukey’s post-hoc test was used to determine statistically significant differences between the means of two or more independent groups. The probability value (P-value) was calculated to determine the significance of the interaction of the process variables. A P-value < 0.05, means that the interactions between the variables are significant. In repeated measurements such as our release profiles and stability data, ANOVA analysis was performed.

Results and discussion

Particle size and PDI analysis

The preparation of homogeneous pharmaceutical formulations is essential for effective drug delivery. Particle size uniformity directly influences the biodistribution and bioavailability of liposomal systems. The polydispersity index (PDI), ranging from 0 to 1, quantifies particle size heterogeneity, with lower values indicating more uniform populations. In pharmaceutical science, liposome preparations with a PDI < 0.3 are considered sufficiently homogeneous, representing a critical quality parameter for advanced drug delivery systems. The particle size and PDI values are presented in Table 1. As can be observed, the integration of zinc sulfate into the liposomal formulations significantly increased the mean particle diameter. This observation suggests the efficient incorporation of hydrophilic zinc sulfate within the phospholipid bilayers, leading to expanded vesicular dimensions compared to blank liposomes. Furthermore, the subsequent surface modification with maltodextrin caused an additional increase in particle diameter to 18.35 ± 3.52% µm, attributed to the formation of a supplementary coating layer on the liposomal surface, thereby enhancing the overall vesicular thickness. The PDI results provide a quantitative assessment of particle size distribution uniformity, as the higher the PDI values, the broader the size distribution profiles within the microparticulate system. After spray drying, the formulations exhibited a moderate increase in PDI to 0.32. However, they remained within the acceptable threshold of 0.5, indicating that the size homogeneity was reasonably preserved in the resultant powder formulation.

Table 1.

Particle size and PDI for prepared liposomes.

Formulation		Particle size (µm) (n = 3)	PDI (n = 3)
Zinc loaded liposomes		9.42 ± 3.52	0.15 ± 0.12
Spray-dried zinc-loaded liposomes		18.35 ± 7.42	0.32 ± 0.18
Blank liposomes		4.27 ± 2.11	0.12 ± 0.08

Zinc ion assay by FAAS, EE and LC

The zinc calibration curve demonstrated excellent linearity across the tested concentration range, yielding a regression equation of y = 0.2576x + 0.043 with a coefficient of determination (R²) of 0.9961. The EE represents a crucial quality attribute in liposomal formulations, defined as the percentage ratio of entrapped drug to the total drug content in the formulation, and also, The LC, which represents the amount of drug loaded per unit weight of the lipid and indicates the percentage of the mass of lipids that contains the encapsulated drug⁴². The determined EE values for zinc-loaded liposomes and spray-dried liposomes were 86.4 ± 1.05% and 88.24 ± 0.98%, respectively. The observed enhancement in EE following polymer coating can be attributed to the additional encapsulation of free zinc ions initially present in the liposomal suspension within the interfacial space between the liposomal bilayer and the polymeric coating matrix. The LC of zinc loaded in liposome before and after the spray drying process were calculated as 7.72 ± 0.91% and 6.38 ± 0.83%.

TGA analysis and XRD

Structural analysis of drug/lipid membranes utilizing X-ray scattering techniques provides valuable insights into the organization of the drug within the membrane. This information can aid in optimizing the lipid matrix to enhance its solubilizing capacity for a specific drug⁴³. Figure 1b shows the X-ray diffraction (XRD) spectrum of crystalline ZnSO₄_·7 H₂O. The sharp peaks (between 20–30° 2θ and 30–50° 2θ) indicated a highly ordered crystalline structure and pattern matched typical zinc sulfate heptahydrate crystal structure. The X-ray Diffraction (XRD) Spectrum of zinc Sulfate-loaded liposomes in Fig. 1a represented broader, less defined peaks (around 15° 2θ and 25° 2θ). The broadening of peaks indicated reduced crystallinity, a more amorphous nature and incorporation into liposomal structure. Generally, XRD pattern changes showed the loss of many sharp characteristic peaks of zinc sulfate and broad peaks characteristic of lipid bilayer vesicle. It suggested interaction between zinc sulfate and phospholipids, successful incorporation of zinc sulfate into liposomes and possible molecular-level interactions between zinc sulfate and phospholipid. These changes confirmed that zinc sulfate had been effectively incorporated into the liposomal structure, resulting in a more amorphous arrangement compared to its pure crystalline form and is corroborated by TGA data in Fig. 1a, which indicates a reduction in crystallinity that occurs upon encapsulation within liposomes. This decrease in crystallinity, attributed to liposomal encapsulation, has been documented as indicative of molecular interactions occurring within the lipid bilayers that enclose the cargo molecule⁴⁴.

Fig. 1.

XRD patterns of (a) zinc-loaded liposome and, (b) pure zinc sulfate heptahydrate.

Figure 2 presents the Thermogravimetric Analysis (TGA) of the thermal decomposition patterns of zinc sulfate, lecithin, and zinc sulfate-loaded liposomes. The TGA curve of pure zinc sulfate heptahydrate showed an initial rapid weight loss (~ 30%) up to 100 °C, which corresponds to the loss of water molecules. This was followed by a smaller weight loss of around 300 °C, and, a total weight loss of approximately 60%, indicating the stable conversion to anhydrous zinc sulfate. For lecithin, the TGA curve showed a gradual initial weight loss up to 200 °C due to the loss of bound water and volatile components. A steep decline was observed between 300 and 400 °C, corresponding to the primary decomposition of the phospholipid structure, breakdown of fatty acid chains, decomposition of phosphatidylcholine head groups, and complete degradation of organic components. The TGA curve of zinc sulfate-loaded liposomes exhibited a more gradual initial weight loss than pure zinc sulfate, with better stability observed between 300 and 500 °C than pure lecithin. The final weight was also higher than lecithin but lower than zinc sulphate. These results indicated that the zinc sulfate-loaded liposome exhibited unique thermal behavior, distinct from both components. The liposomes showed improved thermal stability compared to lecithin and more controlled water loss than zinc sulfate. The TGA analysis confirmed the successful encapsulation of zinc sulfate into liposomes, with modified thermal properties, the enhanced thermal stability of the lipid component, and distinct water-binding characteristics compared with the pure components.

Fig. 2.

TGA thermogram of zinc loaded in liposome, lecithin, and pure zinc sulfate.

FTIR analysis

FTIR spectroscopic analysis investigated the interactions between zinc sulfate and phospholipids in liposomal formulations. Figure 3 shows the spectra of blank liposomes, zinc sulfate, and liposomes containing zinc sulfate recorded in the range of 400 to 4000 cm^− 1. It must be noted that to eliminate the effect of free zinc sulfate ions on the spectrum, the centrifugation method was applied. Zinc ions do not exhibit absorption in the mid-IR region (4000 –500 cm^− 1), as metals have rigid crystal structures, and their bonds do not absorb infrared light³⁴. However, for zinc sulfate heptahydrate, absorption was observed due to functional groups such as the sulfate and hydrate groups. Broad peaks around 3525 –3229 cm^− 1 corresponded to O-H stretching vibrations from crystallization of water molecules, with the broadness suggesting hydrogen bonding. Multiple peaks in this region (3525.94 and 3229.33 cm^− 1) indicated different types of water coordination to the zinc ion. The peak at 1614 cm^− 1 (1614.55 cm^− 1) was attributed to the H-O-H bending vibration of water molecules, with sharpness typical for coordinated water. Peaks in the 1200 –900 cm^− 1 range corresponded to the S-O stretching vibrations of the sulfate group (SO₄²⁻), with multiple peaks (around 1102, 1144, 878, 631 cm^− 1) suggesting that sulfate ions were involved in coordination, acting as ligands. The peak around 460 cm^− 1 can be attributed to Zn-O vibrations from the coordination of water molecules to the zinc ion. The spectrum confirmed the presence of both hydration water and sulfate groups, consistent with the structure of zinc sulfate heptahydrate. The FTIR spectrum of lecithin (phosphatidylcholine) displayed broad peaks around 3300 –3077 cm⁻¹ due to O-H stretching vibrations, with a peak at 3077.55 cm^− 1 attributed to = C-H stretching from unsaturated fatty acid chains. The peak at 2956.63 cm^− 1 represented asymmetric CH₃ stretching, while the 1750 –1500 cm^− 1 region showed peaks around 1652 –1546 cm^− 1 for C = O stretching (carbonyl group) of ester linkages, as well as C = C bonds from unsaturated fatty acids. Peaks at 1448.16 and 1403.72 cm^− 1 corresponded to CH₂ bending vibrations, and the peak at 1247.15 cm^− 1 was characteristic of P = O stretching from the phosphate group. The peak at 923.06 cm^− 1 was attributed to N⁺(CH₃)₃ stretching from the choline group. For studying the formation of liposome, focused on the O-H stretching (3500 –3000 cm^− 1), carbonyl/phosphate (1800 –1500 cm^− 1), phosphate/sulfate (1500 –1000 cm^− 1), and lower frequency (1000 –400 cm^− 1) regions. A comparison of zinc sulfate, lecithin, and zinc-loaded liposomes revealed significant spectral modifications, indicating successful zinc incorporation and complex formation. Key changes included shifts in water binding patterns, carbonyl peak positions, and phosphate vibrations, suggesting specific Zn²⁺-phospholipid coordination. These results demonstrated modified lipid packing and bilayer structure, confirming the formation of zinc-phospholipid complexes within the liposomal system.

Fig. 3.

Fourier Transform Infrared Spectra of zinc sulfate heptahydrate (a), blank liposome (b) and zinc loaded liposome (c).

SEM and EDX of the spray dried zinc loaded liposome

Liposomes primarily comprise amphiphilic lipid or phospholipid molecules that form a bilayer, with hydrophobic tails inward and hydrophilic heads outward⁴⁵. They can be classified based on size and bilayer number⁴⁶. While liposomes are typically spherical, some may be oval or tubular. These morphological variations affect bioavailability and in-vivo behavior. Figures 4 and 5 present the morphological characterization and dimensional analysis of zinc-loaded liposomal particles using SEM and EDX results of spray-dried zinc-sulfate loaded liposomes, respectively. Figure 4a, b and c revealed predominantly spherical particles with amorphous characteristics, exhibiting particle dimensions ranging from 5 to 15 μm. Figure 4b also demonstrated occasional larger particulates, attributable to the encapsulation of multiple liposomal vesicles within a single polymeric matrix during the coating process. This observation consequently manifested as heterogeneity in particle size distribution following the dehydration process. The quantitative and qualitative analysis of zinc ion incorporation within spray-dried liposomal formulations was performed using EDX. The EDX spectral profile (Fig. 5) confirmed the successful encapsulation of zinc ions within the liposomal vesicles. Quantitative analysis (Table 2) revealed a zinc content of 4.45% (w/w) in the analyzed specimens. The characteristic phosphorus signal observed in the spectrum corresponds to the phosphate moieties of the constituent phospholipids. Additionally, the presence of sulfur and oxygen peaks in the spectrum can be attributed to the sulfate counter-ions associated with the incorporated zinc sulfate⁴⁷.

Fig. 4.

Scanning electron microscopy (SEM) photographs of spray dried zinc loaded liposome under different magnification: (a) spray dried liposome, magnification 500×, (b) spray dried liposome, magnification 1000×, (c) spray dried liposome, magnification 2000×.

Fig. 5.

Energy dispersive X-ray (EDX) spectrum of spray-dried zinc sulfate-loaded liposomes. The EDX spectrum from a selected area of the liposome highlights the elemental composition, confirming the presence of zinc sulfate within the liposomal formulation.

Table 2.

Weight% of elements obtained from energy dispersive X-ray (EDX) analysis of the spray-dried zinc sulfate-loaded liposomes.

Map Sum Spectrum
Element	Line Type	Weight%	Weight% Sigma	Atomic%
O	K series	90.36	0.28	96.08
P	K series	0.17	0.03	0.10
S	K series	5.02	0.17	2.66
Zn	K series	4.45	0.18	1.16
Total		100.00		100.00

The table shows the elemental composition, including the weight% of oxygen (O), phosphorus (P), sulfur (S), and zinc (Zn) present in the liposomal formulation.

In vitro release study

An in vitro release study evaluated and compared the zinc sulfate release profiles from conventional liposomal formulations, spray-dried liposomal formulations, and free zinc sulfate solution. As can be found from Fig. 6 there are distinct patterns among the formulations. Free zinc sulfate exhibited rapid dissolution, with complete (100%) release occurring within 20 min. In contrast, both liposomal formulations demonstrated sustained-release characteristics, with cumulative release reaching 95.32% and 94.98% for uncoated and spray-dried formulations over 12 h. The marked difference in release profiles between free zinc sulfate and the liposomal formulations suggests successful encapsulation of zinc sulfate within the phospholipid bilayer, effectively modulating the release profile. The absence of burst release in the liposomal formulations further supports the stable incorporation of zinc sulfate within the lipid matrix⁴.

Fig. 6.

In vitro drug release profiles of zinc sulfate salt, zinc sulfate-loaded liposomes, and spray-dried zinc sulfate-loaded liposomes in PBS (pH 6.8) over 48 h (n = 3). According to the one-way ANOVA analysis a significant change was considered at P-value < 0.0046, which means that the interactions between the groups are significant.

Stability study

The efficiency of spray-dried liposomes in stabilizing zinc sulfate was assessed by storing the samples at two different temperatures. Stability at room temperature is a key challenge in liposome formulations due to vesicle aggregation and potential drug leakage from the phospholipid bilayer, especially in the liposome with micron sized particles. The stability of the spray-dried liposomes was evaluated by monitoring the critical quality attributes, specifically particle size distribution and EE%, over 90 days. The results of the physical stability of the samples at various time points are shown in Table 3. Samples stored at 4 °C exhibited minimal variation in particle size, changing from 18.35 μm at day 0 to 18.39 μm at day 90 (Δ = 0.04 μm), while those stored at 25 °C showed a slightly larger increase from 18.35 μm at day 0 to 18.57 μm at day 90 (Δ = 0.22 μm). This size increment might be attributed to partial destabilization of the liposomal structure, potentially resulting from membrane swelling or phase transitions during rehydration. The encapsulation efficiency remained stable, with marginal reductions of 0.09% and 0.13% for samples stored at 4 °C and 25 °C, respectively, under the controlled relative humidity conditions. The slight reduction in EE% could be due to moisture absorption during storage. Statistical analysis revealed no significant changes (p-value > 0.05) in either particle size or encapsulation efficiency. The minimal variation in these critical parameters and the absence of considerable degradation or aggregation suggest excellent physicochemical stability of the formulation. It is also worth noting that any significant oxidative degradation or leakage would necessarily manifest as a reduction in EE, which remained remarkably consistent throughout our 90-day study. Also, maintaining particle size distribution provides compelling evidence of structural stability. It was reported that significant lipid oxidation would manifest as changes in particle size distribution owing to the membrane disruption and vesicle aggregation⁴⁸ The spray-drying process itself offers inherent protection against oxidative degradation by significantly reducing water content. Chen et al.⁴⁹ demonstrated that for spray-dried liposomal formulations, the EE% and particle size distribution serve as reliable surrogate markers for lipid oxidation and stability. Goldbach et al.²¹ established that in solid-state liposomal formulations, constant EE% over extended periods strongly correlated with minimal lipid oxidation and negligible drug leakage. These findings indicated that the spray-dried liposomal system maintained both its physical integrity and functional characteristics during long-term storage.

Table 3.

The stability results containing particle size and EE% of spray-dried zinc sulfate-loaded liposome over 90 days (a) at 4° C, (b) at 25° C.

Time (day)	Stability condition	Particle size (µm) (n = 3)	Encapsulation Efficacy (%) (n = 3)
0	At 4 °C/ 60 ± 5% RH (n = 3)	18.35 ± 7.42%	88.24% ± 0.98%
7	At 4 °C/ 60 ± 5% RH (n = 3)	18. 38 ± 7.42%	88.21% ± 0.85%
15	At 4 °C/ 60 ± 5% RH (n = 3)	18.37 ± 7.42%	88.23% ± 1.02%
30	At 4 °C/ 60 ± 5% RH (n = 3)	18.41 ± 7.42%	88.17% ± 0.92%
60	At 4 °C/ 60 ± 5% RH (n = 3)	18.39 ± 7.42%	88.14% ± 1.21%
90	At 4 °C/ 60 ± 5% RH (n = 3)	18.39 ± 7.42%	88.15% ± 1.07%
0	At 25 °C/ 60 ± 5% RH (n = 3)	18.35 ± 7.42%	88.24% ± 0.98%
7	At 25 °C/ 60 ± 5% RH (n = 3)	18.39 ± 7.42%	88.19% ± 1.04%
15	At 25 °C/ 60 ± 5% RH (n = 3)	18.42 ± 7.42%	88.16% ± 1.16%
30	At 25 °C/ 60 ± 5% RH (n = 3)	18.48 ± 7.42%	88.17% ± 0.93%
60	At 25 °C/ 60 ± 5% RH (n = 3)	18.51 ± 7.42%	88.14% ± 1.12%
90	At 25 °C/ 60 ± 5% RH (n = 3)	18.57 ± 7.42%	88.11% ± 0.67%

Note: According to the ANOVA analysis the stability condition between two groups including “at 4°C/ 60 ± 5% RH (n = 3)” and “at 25°C/ 60 ± 5% RH (n = 3)” over time [0–90 day] for particle size and EE% were not significant based on the fact that p-value > 0.05, (p-value = 0.063, p-value = 0.408).

Conclusion

In this study, we successfully formulated spray-dried zinc sulfate-loaded liposomes, demonstrating significant improvements in encapsulation efficiency, stability, and sustained release profiles compared to free zinc sulfate. The spray-drying process resulted in liposomes with a mean particle size of 18.35 ± 7.42 μm and a high encapsulation efficiency of 88.24% ± 0.98%, highlighting the effectiveness of this delivery system in protecting zinc and enhancing its bioavailability. The physicochemical characterization, including XRD, TGA, FTIR, and SEM, confirmed the successful incorporation of zinc sulfate into the liposomal structure, with modifications to the lipid bilayer that improved thermal stability and controlled drug release. The in vitro release studies demonstrated sustained release over 12 h, compared to the rapid dissolution of free zinc sulfate. Furthermore, the spray-dried liposomes exhibited excellent stability under room temperature and refrigerated conditions over 90 days, with minimal variations in particle size and encapsulation efficiency. These findings suggest that spray-dried liposomal systems are a promising approach to improving the bioavailability of zinc sulfate, offering a potential solution for overcoming the limitations of traditional zinc supplementation. Future studies should focus on evaluating the in vivo performance of this formulation to assess further its therapeutic potential and practical applications in pharmaceutical and nutraceutical formulations.

Author contributions

Monireh Abbassi: Conceptualization, Methodology, Investigation, Data analysis, Writing – original draft. Masoud Rahimi: Supervision, Conceptualization, Writing – review & editing. Samad N. Ebrahimi: Validation, Writing – review & editing, Funding acquisition, Project administration.

Data availability

All data generated or analyzed during this study are included in this article (and related files).Please address all correspondence concerning this manuscript to me at: ma_rahimi@sbu.ac.ir.

Declarations

Competing interests

The authors declare no competing interests.