Lipid-based nanocarriers are influenced by the physicochemical properties of the drug, as well as by the composition and preparation techniques used. These carriers have been successfully developed and patented for the treatment of various diseases. The market for nano-based drug delivery is projected to reach $142.8 billion by [34]. According to [35], this drug delivery system is on the cusp of clinical translation. Given the hydrophobic nature of E2, lipid nanosystems show promise for its safe and effective delivery.

While these nanosystems may be suitable for the encapsulation of both hydrophilic and lipophilic drug substances and can circulate through the bloodstream effectively, they may also pose a risk of cytotoxicity due to non-specific cellular uptake [36].

Tables 1, 2 collectively present 13 studies on nano-based E2 delivery systems. Lipid-based and polymeric are the most commonly used nanoparticles in the medical field for drug delivery systems. Table 1 details lipid-based nanostructures for E2 delivery, whereas Table 2 focuses on polymeric nanoparticles. Among these studies, the routes of administration most frequently examined include oral and subcutaneous, each covered in two studies, as well as transdermal administration, also reported in two studies. Nasal and intravenous (IV) routes were each explored in one study. The administration routes were not specified in the remaining five studies. Specifically, Table 1 highlights five studies on lipid nano-based E2 delivery systems: two studies investigated liposomes (LP), which are bilayer vesicles composed of lipids and phospholipids; one study examined a lipid-ethanol-drug nanoparticle (LED) system; and two studies explored nanoemulsions (NE).

Liposomes, which are phospholipid vesicles encapsulating a hydrophilic space with at least one lipid bilayer, boast the distinct advantage of being able to incorporate both lipophilic and hydrophilic compounds. This trait broadens the spectrum of deliverable drug substances. The flexibility and width of the liposome bilayer, which are crucial factors affecting E2 encapsulation, are influenced by bilayer composition, as elucidated by Gallez et al. (2020) [37]. Notably, the inclusion or exclusion of cholesterol impacts the physical state and structural dynamics of phospholipid chains; cholesterol serves to densify the lipid bilayer. Glycero-3-phosphocholine (POPC), containing an unsaturated cis-9 lipid chain (oleic acid), was selected for E2 encapsulation due to its enhanced flexibility. POPC also demonstrated superior loading capacities compared to 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Furthermore, the saturation and length of the fatty acid chains are determinants of the lipid bilayer’s thickness and membrane robustness.

Innovative strategies utilizing novel materials for E2 lipid nanostructures have been shown to enhance therapeutic efficacy. Notably, LEDs have been developed with a new lipid class that includes heterocyclic head groups and oleyl chains, which promote superior transdermal penetration. Within this context, 1-Di-(Z)-octadec-9-en-1-yl) pyrrolidin-1-ium iodide (Cy5) demonstrated the most effective transdermal enhancement. E2 interaction with the bilayer is not limited to the phospholipid head but extends to the lipophilic core, primarily associated with the acyl chains of the lipid membrane. This interaction was exploited by Heger et al. (2015) [38] who replaced cholesterol in the hydrophilic part of the lipid bilayer with E2, preserving the hydrophilic properties while improving targeting to estrogen receptors (ERα and ERβ). E2 enhanced transfection efficiency by modulating plasma membrane properties and increasing liposome uptake into cells. The cationic nature of the liposomes further strengthened interactions with negatively charged cell membranes, enhancing the intracellular delivery of antisense oligonucleotides (ASOs). Additionally, E2 promoted the proliferation of estrogen receptor-positive cells, such as MCF-7, thereby increasing the overall therapeutic efficacy. This dual functionality of E2 improved both cell targeting and treatment efficiency.

Nanoemulsions (NEs) are oil-in-water nanocarriers that are heterogeneous and thermodynamically stable. These structures carry lipids that may function as surfactants, forming barriers that enhance the absorption of drug substances. NEs have been utilized to create promising mucoadhesive buccal films for E2 delivery, employing non-toxic and biocompatible components like PEG 400, polysorbate 80, and transcutol®P, which are commonly found in topical formulations. Additionally, ω-3 PUFA alpha-linolenic acid, a cardiovascular risk-lowering agent from flaxseed oil, was combined with E2 to form an NE with bioactive properties aimed at treating restenosis. Even in the absence of a drug substance, NEs demonstrated inherent bioactive effects, such as inhibiting cell proliferation. Egg lecithin was incorporated as a stabilizer, and DOTAP was used to impart a positive charge to enhance membrane transport. DSPE-PEG2000 played a role in prolonging the exposure of E2 to the vasculature by decelerating the clearance of the formulation.

The encapsulation of E2 within liposome formulations is characterized by a particle size range of 138–217 nm, a polydispersity index (PDI) spanning from 0.06 to 0.136, and a zeta potential fluctuating between −28.0 and +27.0 mV. The LED exhibits a mean particle size of 167.4 nm, a PDI of 0.51, and a zeta potential of 22.7 ± 6 mV. Additionally, two E2 NE have been reported with particle sizes ranging from 14.9 to 138 nm, PDIs between 0.14 and 0.487, and zeta potentials of −33.80 ± 2.45 mV.

The influence of particle size on the therapeutic efficacy of lipid nanoparticles is critical. Within the target tissue, the retention of lipid nanocarriers can be attributed to the size constraints imposed by capillary pores and the interstitial spaces. Optimal particle size for sustained-release antitumor drugs is typically within the range of 50–200 nm. In contrast, sizes starting from approximately 200 nm are indicated for intravenous (IV) or intramuscular (IM) administration. Lipid nanocarriers, such as liposomes, may exhibit prolonged tissue retention or phagocyte-mediated clearance when their size exceeds 100–150 nm after extravasation from the vasculature.

The PDI serves as an indicator of colloidal dispersion uniformity, with values approaching 0.01 signifying a uniform particle size distribution, and values near 1.0 indicating significant polydispersity. For lipid-based carriers, a PDI below 0.3 is generally accepted as indicative of a homogeneous particle population. Empirical data reveal a range of PDI from 0.06 ± 0.02 to 0.35 ± 0.04, although Marepally et al., 2013, reported a notably higher PDI of 0.51 for an E2 liposome formulation [39].

The zeta potential, an electrokinetic parameter, is used to assess the physical stability of nanoparticles, which includes their propensity for aggregation or agglomeration as a result of Ostwald ripening. Additionally, this parameter is influential in enhancing cellular uptake and determining the effectiveness of nanoparticle coatings. It is observed that lipid nanocarriers with positive zeta potentials ranged from 22.7 ± 6 to 27 ± 3 mV. The inclusion of a cationic surfactant such as DDAB can positively charge the nanocarrier, potentially increasing the encapsulation efficiency (EE) of negatively charged compounds like E2. Despite this, the highest EE of 97.50% was achieved using transcutol P^®, a solubilizing agent commonly employed in drug formulations [39]. Its chemical structure, containing diethylene glycol monoethyl ether, enhances the solubility of poorly soluble drugs. The hydrophilic-lipophilic balance of transcutol P^® plays a pivotal role in drug solubilization and encapsulation. A favorable balance increases EE by maintaining the drug in solution during formulation, thereby improving its encapsulation within liposomes. In contrast, Gallez et al. (2020) demonstrated successful in vivo results with a low EE of 3.0%, highlighting that effective drug delivery to the target site is possible even with suboptimal encapsulation [37].

The preparation method significantly influences the properties of lipid nanoparticles. The thin-film hydration technique, employed in the preparation of E2 liposomes in two out of five studies, is a time-honored and straightforward approach for creating multilamellar vesicles. However, this method’s reliance on organic solvents presents a challenge for complete solvent removal, which can be problematic particularly for scalability and achieving high entrapment efficiency. Yaseen et al. (2021) [40] highlight the environmental concerns associated with non-green solvent use in pharmaceutical manufacturing, advocating for the adoption of sustainable solvents to mitigate pollution and reduce costs and environmental impact.

The modified ethanol injection method, as presented by Marepally et al. (2013) [39], involves the injection of an ethanol-phospholipid solution into an aqueous phase, with the ethanol volume being a critical factor for uniform vesicle formation. This technique is noted for its simplicity, reproducibility, and the preservation of lipid integrity.

Deshpande et al. (2013) [41] utilized the microfluidization technique, a high-pressure homogenization process that relies on particle interaction mechanisms such as collision, cavitation, shear, and turbulent forces to achieve particle size reduction. Its primary advantage lies in the method’s scalability, which is important for pharmaceutical industry applications.

Lastly, ultrasonication, employed by Abdella et al. (2022) [42], utilizes ultrasonic energy to disaggregate large particles into smaller or more uniform ones. While probe-type sonicators are more efficient than bath-type, the localized effect of emulsification near the waveguide radiator limits the method’s suitability for industrial-scale production due to non-uniform particle size distribution.

The composition of E2 lipid nanoparticles and the methodology for their preparation provide efficacious drug delivery systems. These systems elicited a therapeutic response in human vascular culture cells [41] and exhibited heightened permeation efficacy [39, 42].

LED-E2 demonstrated enhanced permeation, significantly improving the delivery of E2 to the dermal layer [39] (Table 1). The LED nanoparticles, particularly those using the cationic lipid 1,1-Di-[(Z)-octadec-9-en-1-yl] piperidin-1-ium iodide (Cy5), showed superior permeation due to their 5-membered amine heterocyclic ring, which is more effective than 6-membered rings. These Cy5 nanoparticles, sized between 150 and 200 nm, efficiently penetrate the skin barrier, leading to a 1.54 to 22-fold increase in drug retention in the dermis compared to conventional enhancers like oleic acid. The permeation mechanism involves ionic interactions, membrane destabilization, and increased skin hydration, promoting the penetration of encapsulated drugs.

The permeation rate of NE E2 incorporated within a mucoadhesive film reached 15% over 10 h, which is threefold greater than the permeation expected from oral tablets. However, these results, derived from an ex vivo model and a predictive model, still demand validation through in vivo studies to establish a proof of concept and to deepen our understanding [42].

In a related experiment, the impact of E2 liposomes was assessed using ovariectomized female rats as a model. The presence of E2 influenced several parameters in the uterus, which is known to respond to ERα-genomic/nuclear E2 dependent pathways. These parameters included the uterus’s wet weight, the proportion of Ki67-positive epithelial cells, and the height of the luminal epithelium, effects that were absent in groups not receiving the hormone. These findings suggest that E2 liposomes are capable of being internalized and releasing their contents, thereby inducing the activation of the ERα genomic/nuclear pathway in a manner comparable to free E2. To summarize, the liposomes were specifically effective in activating the genomic/nuclear pathway of ERα, indicating that LP-E2 could serve as a valuable tool in elucidating molecular mechanisms associated with ERα. Additionally, the liposomes appeared to modulate specific action mechanisms of pathways.

It is also imperative to ascertain the activation pathways of E2 receptors. Gallez et al. (2020) [37] highlighted that E2 encapsulated in liposomes failed to activate the α receptor found in the cell membrane. Consequently, the liposome-encapsulated E2 is internalized and activates the genomic pathway without initiating membrane-mediated steroid signaling. Overall, cationic LP showed the highest E2 loading capacity and better cellular uptake. LED formulation presents a higher permeation and delivers E2 to the dermal layer. Also, a buccal transmucosal formulation could be a promising administration route for E2 delivery. The LP-E2 is a promising tool to study the molecular mechanisms related to E2 receptors; NE – E2 inhibited VSMC and supported endothelial cells in connection with MAPK signaling.

Lipid-based nanocarriers, including liposomes, NE, and LED systems, present effective methods for delivering E2, each offering unique advantages based on their composition and manufacturing processes. Liposomes, with flexible bilayers like POPC, enhance drug loading and release by accommodating both hydrophilic and lipophilic substances. LED systems, especially those with cationic lipids like Cy5, provide improved transdermal delivery through better skin penetration and drug retention. Nanoemulsions used in buccal films achieve superior permeation compared to oral tablets, making them effective for mucosal delivery. The selection of lipids and manufacturing techniques, such as thin-film hydration or microfluidization, critically influences particle size, PDI, and zeta potential, which in turn affect the carriers’ performance. These lipid-based systems offer promising routes for patient-friendly E2 administration, including transdermal, buccal, and potentially intravenous, enhancing E2’s solubility, absorption, bioavailability, and controlled release for effective drug delivery.

The utilization of polymeric nano-based drug delivery systems emerges as a compelling methodology for the sustained and controlled release of therapeutic agents. Advantages inherent to polymeric nanoparticles include biodegradability, biocompatibility, non-toxicity, and the capacity for surface modification. These nanoparticles are versatile in encapsulating a wide range of pharmaceuticals, encompassing both hydrophobic and hydrophilic drugs. However, their interaction with the reticuloendothelial system can precipitate rapid clearance from the bloodstream, potentially necessitating increased or more frequent dosing. Additionally, they may accrue within hepatic tissues, diminishing their efficacy in other organ systems [36].

In the realm of polymeric nanostructures, a dichotomy exists between nanocapsules (NCs) — reservoir systems — and nanospheres (NSs) — matrix systems — based on their respective formulations. NSs are characterized by a polymeric matrix that either entraps the therapeutic agent or adsorbs it on the surface [43]. In contrast, NCs consist of a drug solution encased within an oily core, enveloped by a polymeric shell that modulates drug release kinetics. NCs are predominantly synthesized via nanoprecipitation, a process entailing interfacial polymer deposition subsequent to the displacement of a semi-polar hydrophilic solvent. NSs, on the other hand, are fabricated through a series of processes including solvent evaporation, emulsification, solvent diffusion, reverse salting-out, and also nanoprecipitation. Both NSs and NCs can be formulated either by the polymerization of monomers or by the dispersion of pre-existing polymers or biopolymers, with the latter including naturally occurring, noncytotoxic, biocompatible, and biodegradable polymers such as chitosan and gelatin. These biopolymers are intrinsic to living tissues and may significantly enhance the absorption of drugs compared to their free, unencapsulated counterparts [43].

Table 2 delineates a comparative analysis of eight studies focusing on polymeric nano-based E2 delivery mechanisms. Within these studies, six pertain to nanosphere-based systems (NS), one utilizes a nanocapsule (NC) with an olive oil core, and the remaining three employ nanoparticles derived from biopolymers such as gelatin.

The analysis of Table 2 reveals that the majority of the reviewed studies (five out of eight) predominantly utilized poly (lactide-co-glycolide) (PLGA) as their primary polymeric compound in formulating nanoparticles, with polyethylene glycol (PEG), and poly (lactic acid) (PLA) following suit. Notably, PLGA and PLA are frequently selected polymers for nanoparticle synthesis, as evidenced by the literature [36, 43].

In a novel approach, a composite of PLA and PEG (specifically PLA17000-PEG2000) was employed to fabricate a distinct type of polymeric nanoparticle characterized by a bilayer membrane enveloping an aqueous core. E2 was effectively conjugated to the surface of these polymersomes [44].

Alternative polymers, including those that are synthesized or lesser known, have also been explored for nanoparticle preparation. For instance, the polymer PEG-PBA-PEG, in varying concentrations, has been demonstrated to modulate the release rate of E2 effectively. Moreover, this nanoparticle maintained stability with a minimal application of surfactant [45].

A singular study highlighted the use of a biopolymer, specifically gelatin nanoparticles (GNP), which encapsulated E2 utilizing the complexing properties of cyclodextrins (CD). This combination resulted in a GNP-CD system that exhibited a prolonged and sustained E2 release profile [46].

The particle sizes of these polymeric nanostructures ranged significantly, from 49.4 ± 2.2 nm to 418 ± 11 nm, a variation attributable to the differing compositions and methods of preparation. Notably, a larger particle size was observed with a polymer conjugate under hypoxic conditions (2% oxygen availability), resulting in particles measuring 695 ± 32 nm, whereas, under normoxic conditions, the size was reduced to approximately 168 nm [44].

Diverse techniques have been applied to measure particle size, with Prakapenka et al. (2020) [47] utilizing both transmission electron microscopy (TEM) and dynamic light scattering (DLS) for assessing size and hydrodynamic diameter, respectively. Each method possesses inherent advantages and limitations, but when used in conjunction, they provide a comprehensive understanding of particle dimensions. The FDA endorses such combined measurements for critical material attributes and emphasizes the importance of detailing the specifics of the analysis, including the distinction between hydrodynamic and projected radii, as well as comparing ensemble and single particle data [48].

Regarding PDI, values at or below 0.2 are generally deemed acceptable for polymeric nanoparticles [49]. The studies summarized in Table 2 largely conformed to this standard, with exceptions noted in the work of Guo et al. (2021) [50], which reported a PDI of 0.236 ± 0.008, and Khoee and Hossainzadeh (2010) [45], with a PDI over 0.204.

The zeta potential of the nanoparticles varied widely, from −58.2 ± 3.0 mV to +92.4 ± 3.2 mV, reflecting the diverse compositions (Table 2). Positive zeta potentials were specifically recorded for nanoparticles in the studies by Mittal et al. (2009) [51] and Joachim et al. (2020) [46], which were attributed to the incorporation of a quaternary ammonium salt, didodecyl dimethylammonium bromide (DMAB), as a stabilizing agent, and the use of type A gelatin, which is positively charged at physiological pH, respectively [46].

EE of the polymeric nanostructures was reported between 20% and 94.5% (Table 2). The highest EE was associated with nanoparticles having an olive oil core. It was observed that the ratio of polymer to olive oil critically influenced the EE, with a notable decrease in loading efficiency at a 1:2 ratio (53.94%), suggesting an optimal threshold for this proportion [45].

Remarkably, the highest EE was achieved by nanoparticles formulated with gelatin [46]. Additionally, Gil et al. (2018) [52] explored a novel assay for assessing the sustained release of active pharmaceutical ingredients, focusing on drug-albumin interactions. Nevertheless, this method has yet to be validated through in vivo testing.

The pursuit of polymeric nanostructures led to the execution of five studies employing various emulsion-based methodologies: emulsion-diffusion-evaporation [50, 51], emulsification-solvent diffusion [45], water/oil emulsion [52], and single-emulsion [47], the latter two of which were augmented with ultrasonication. Additionally, researchers utilized the solvent exchange method [45] and a modified desolvation technique [53], as delineated in Table 2.

A specific antisolvent diffusion approach, characterized by preferential solvation, yielded PLGA nanoparticles with protracted release profiles and enhanced dermal penetration, culminating in improved bone mineral density. These outcomes were notably superior to those attained through the conventional emulsification-solvent evaporation technique [53].

In the context of E2 nanoparticles, five in vivo and two in vitro assessments were conducted (Table 2). The application of E2 nanoparticles in rodent models with hyperlipidemia induced by a high-fat diet underscored the potential of these polymeric nanoparticles for oral administration. A comprehensive analysis of pharmacokinetic and pharmacodynamic parameters—including maximum plasma/serum concentration (Cmax), time to reach Cmax (Tmax), area under the plasma/serum concentration-time curve (AUC), absolute bioavailability, absorption rate constant (Ka), elimination rate constant (Kel), elimination half-life (T1/2), mean residence time, mean absorption time—revealed the efficacy of the treatment [54]. For instance, a dose of 500 μg/kg of E2 encapsulated in PLGA nanoparticles exhibited a Cmax of 476.02 pg/mL, Tmax of 18 h, T1/2 of 11.84 h, and AUC of 34,212.08 pgh/mL. In contrast, a drug suspension (DS) at an equivalent dosage demonstrated a Cmax of 757.03 pg/mL, Tmax of 4 h, T1/2 of 5.46 h, and AUC of 11,339.20 pgh/mL, respectively [51]. Consequently, the E2-nanoparticle formulation displayed a lower Cmax and a delayed Tmax relative to the DS, while delivering an approximately threefold higher drug concentration over time, as evidenced by the AUC, indicating a sustained release profile. Furthermore, when the same dosage was administered intravenously as a DS, the resultant AUC was 55,725.67 pg*h/mL with a T1/2 of 4.60 h, suggesting a more rapid elimination and greater bioavailability compared to oral administration of the E2-PLGA nanoparticles, thereby extending the potential dosing interval.

Additionally, intravenous administration of E2-PLGA nanoparticles was found to enhance bone density. In this study, ovariectomized female rats were employed as an animal model and treated over a course of 9 weeks. These findings corroborate the efficacy of E2-PLGA nanoparticles in the therapeutic management of osteoporosis in postmenopausal women, with specificity and targeting to bone tissues being potentiated by the co-administration of bisphosphonates or iron oxide [50].

Beyond oral and intravenous routes, the delivery of E2 polymeric nanoparticles was explored via intranasal, subcutaneous (SC), and transdermal pathways. Transdermal administration was specifically chosen to circumvent the first-pass metabolism associated with oral administration of E2 as an intervention for osteoporosis. Experimental models included excised rat abdominal skin for ex vivo analysis and female Sprague-Dawley rats for in vivo studies. The findings demonstrated that E2-PLGA nanoparticles, with a polyvinyl alcohol (PVA) coating, exhibited higher skin permeability compared to traditional nanoparticle formulations and enhanced bone mineral density [53].

For SC administration, a study on middle-aged ovariectomized female rats, which received weekly doses of E2-loaded PLGA nanoparticles, reported improved cognitive functions in learning and memory relative to the control group. Nevertheless, the therapy was also associated with notable adverse effects, such as increased uterine stimulation, when juxtaposed with the administration of free drugs [47].

The intranasal delivery of GNP conjugated with CD utilized a male C57BL/6 J mouse model. The outcomes indicated a reduction in the area of cerebral infarction, surpassing even that achieved with water-soluble E2 encapsulation, hence demonstrating promise for the treatment of ischemic stroke [46].

Polymeric nanoparticles are designed to provide controlled, sustained release, maintaining therapeutic levels over time and offering stable delivery across various routes. Their preparation involves well-established methods that may not require specialized equipment, though precise control of manufacturing processes is needed to achieve the desired performance. Despite the promising results from various studies, the progression toward a definitive proof of concept remains limited, highlighting the need for further in vivo research to validate and enhance their therapeutic applications.