Proniosomes: A provesicular system in ocular drug delivery

The eye is the most beautiful and sensitive organ of our body that helps us to perceive and understand our surroundings. It is situated in the orbital cavity of the skull. The eye is spherical in shape, with 24 mm anterior diameter. In terms of drug delivery eye can be divided into four target sites: (a) cornea, (b) anterior and posterior chamber and associated tissue, (c) posterior eye segment (including the retina and posterior cavity), and (d) preocular structure (conjunctiva and eyelids). The cornea, crystalline lens, iris, and pupil make up the anterior part of the eye. Aqueous fluid fills both the anterior and posterior chambers  [1].
A growing proportion of people worldwide suffer from ocular illness. Possible significant vision problems caused by some pathological conditions of the eyes, such as diabetic retinopathy, age-related macular degeneration (AMD), HIV infections, and glaucoma may cause complete loss of vision [2]. Eyes disorders are cured or managed using either a topical or systemic delivery of drugs. Although the administration of drugs through the systemic route offers the advantage of delivering the medicine to the eye more conveniently, it has the disadvantage of unexpected side effects and insufficient therapeutic efficacy. The various delivery approaches, as given in Table 1, have been used for ocular administration. The diagrammatic representation is given in Figure 1 depicting the comparison between conventional and novel drug delivery systems in ocular administration. Some conventional forms of drug delivery, like ocular gels, solutions, suspensions, etc., have significant drawbacks in the form of tears turnover, poor corneal permeability, drug loss on eyelids and eyelashes, nasolacrimal drainage, inaccurate dosing associated with eye drops, blinking, and blurred vision. The above-mentioned challenges have raised the demand for innovative approaches to delivering drugs to the eyes [3]. Eye drops are the most common and widely used ocular dosage forms. However, the major drawback of conventional dosage forms in ocular delivery is that only 5% of the drug reaches the target site because of the various ocular barriers present in the eye [4]. Furthermore, the use of nanotechnology and other emerging drug delivery systems is widely regarded as a means of overcoming the drawbacks of conventional dosage forms and avoiding the various obstacles present in the eye [5–7]. The vesicular system encloses the drug within surfactant vesicles to achieve targeted drug delivery at the corneal surface resulting in enhanced bioavailability [8].  Ideal characteristics for vesicular ocular drug delivery include easy penetration of the drug through the corneal membrane, longer residence time in the eye to achieve a desired therapeutic effect, reduced dosing frequency, minimum drug loss, avoidance of blurred vision, and minimal adverse reaction/side effects [9]. Proniosomes are a novel class of drug delivery system that has gained considerable attention in various applications in recent years [10–13]. Proniosomes gained popularity over liposomes and niosomes because of their excellent stability and better bioavailability [14]. This current review discussed various aspects of proniosomes in ocular drug delivery.

Proniosomes are a form of lipid-based drug delivery system consisting of a dry mixture of surfactants and cholesterol that form vesicles in an aqueous phase. Proniosomes are solid colloidal particles that can be quickly hydrated before use to make aqueous niosome dispersions that are comparable to those made using more laborious traditional procedures [15]. The proniosomes reduce the issues with niosomes' physical stability, including aggregation, fusion, and leakage. Additionally, they make transportation, distribution, storage, and dosage more convenient. The shape, particle size, particle size distribution, and drug release of the proniosome-derived niosomes are superior to those of traditional niosomes [16]. Based on their processing, proniosomes can be classified as either dry granular or liquid crystalline. Dry granular proniosomes are prepared by coating a water-soluble carrier (maltodextrin or sorbitol) with a surfactant. Based on the type of carrier used, dry granular proniosomes are further classified into two types of maltodextrins and sorbitol dry granular proniosomes [17]. Dry granular proniosomes provide advantages such as improved stability and convenient storage. On the other hand, liquid crystalline proniosomes possess a highly organized liquid crystalline structure, which enhances drug encapsulation, sustained release, and permeation through the biological membranes [18].
Proniosomes are designed to improve the solubility and bioavailability of drugs and were investigated for a range of applications, including ocular delivery [19]. Proniosomes exhibit favorable characteristics for gene delivery as they are easy to formulate, economical, stable, and nontoxic because of non-ionic surfactant inclusion [20]. Drug targeting is one of the most advantageous properties of proniosomes. Proniosomes are employed in transdermal drug delivery systems to deliver hypertension drug captopril [21]. Frusemide is also delivered non-invasively by proniosomes [22]. In hormonal therapy, levonorgestrel, an emergency contraceptive, has been tested for transdermal distribution using proniosomes [23]. Peptide delivery is also possible with proniosomes. Oral peptide breakdown by gastrointestinal enzymes is problematic [24]. Nevertheless, peptides were shielded successfully using niosomes against peptide degradation in the digestive tract [25]. An entrapment inside the vesicles considerably boosted the stability of the peptide when it was given orally as a vasopressin derivative in niosomes [26]. Proniosomes have been widely used to deliver antibiotics and anti-inflammatory agents [27]. There are different methods available for the preparation of proniosomes, including the slurry method, coacervation phase separation method and slow spray coating method. In the slurry method, a non-ionic surfactant is dissolved in a volatile organic solvent to form a slurry, to which the drug is added followed by carrier material like lactose or mannitol. The organic solvent is evaporated under reduced pressure resulting in dry proniosomal powder [28]. In the coacervation method proniosomes are prepared by mixing non-ionic surfactant and carrier in a solvent and then adding a coacervation-inducing agent such as calcium or magnesium ions which leads to the formation of coacervate [29]. The slow spray coating method involves the use of a spray dryer to coat the proniosomes with a thin layer of a polymer or other coating material. Proniosomes produced by the spray coating method are stable. However, it is a tedious and time-consuming process. Each method offers its advantages and can be chosen based on specific requirements of the formulation [30].

The eye's unique physiological and anatomical features make the ocular drug delivery system a challenging field. The eye has a limited surface area, and the tear film and the blood-retinal barrier limit the penetration of drugs into the eye [31]. Thus, developing a drug delivery system that can overcome these barriers and deliver drugs effectively to the eye is critical [32]. Proniosomes offer a promising solution for ocular drug delivery due to their unique properties [33]. Proniosomes are formulated using a range of surfactants, including non-ionic, cationic, and anionic surfactants and other excipients, as given in Table 2. The physiochemical parameters of drugs and the intended proniosomes features determine the surfactant to be used. Surfactant and cholesterol mixture is typically dried and reconstituted with an aqueous phase to form Niosomes. Niosomes can then be further processed to form proniosomes by lyophilization or spray drying [34]. Proniosomes can protect drugs from degradation by enzymes and pH changes in the body, improving their stability and shelf-life. The surfactant in proniosomes also helps to increase the solubility of the drug, which can improve its bioavailability  [35].

Factor affecting the formulation of proniosomes                                                                                                             

The formulation of proniosomes depends on several processing factors such as surfactant chain length, cholesterol content, pH of the hydration medium, total lipid concentration and charge of the lipids as shown in Figure 2 and discussed below-
a) Surfactant chain length: A longer alkyl chain length results in greater drug entrapping effectiveness. For example, Spans are widely used in the production of proniosomes. The order of entrapment efficiency for surfactant is Span 60(C18)>Span 40(C16) >Span 20(C12) [36].
b) Cholesterol content: Depending on the type of surfactant or its concentration in the formula, cholesterol either improves or decreases the percentage of encapsulation efficiency. A higher amount of cholesterol in the formulation increases the rigidity of the bilayer leading to a decrease in the drug release from the encapsulated formulation.  
c) pH of the hydration medium: A decrease in the pH of the hydration medium leads to an increase in the percentage encapsulation efficiency. 
d) Total lipid concentration: As the concentration of lipids rises, the fraction of lipids participating in encapsulation decreases.
e) Charge of the lipids: The incorporation of a positive charge (Stearyl amine and stearyl pyridinium chloride) or a negative charge (diacetyl phosphate and phosphatidic acid) decreases the percentage encapsulation efficiency of proniosomes [37].

The evaluation of proniosomes is essential to ensure their quality, safety, and effectiveness in delivering drugs to the eyes. Several key parameters are mentioned in Table 3 and below, which are required in evaluating the performance of proniosomes in the ocular route.

Surface morphology
It can be determined using various microscopy methods like scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Such techniques allow visualization of the size, shape, and surface characteristics by providing high-resolution images of proniosomes [38]. The surface morphology of proniosomes can also be determined using fluorescence microscopy by incorporating fluorescent dyes in proniosomes to provide results of the size, shape, and distribution of proniosomes [39]. The vesicle size of proniosomes intended for ocular delivery is an important parameter that can influence their stability, drug release kinetics and ocular tissue penetration. Vesicle size range for proniosomes used in ocular delivery should be less than 10 μm. For example, Fouda et al. 2018 determined the surface morphology of dorzolamide proniosomes using TEM after negative staining with potassium phophotungustate [40]. TEM image showed that the proniosomes formed were spherical in shape [40].
Abaoli et al. (2020) investigated the surface morphology of curcumin proniosomes by SEM and TEM [41]. TEM image confirmed the smooth and spherical shape of proniosomes without aggregation. SEM results showed that the surface was smoother and more compact with no apparent pores suggesting good entrapment efficiency and a vesicle size of 212 nm [41].

Encapsulation efficiency
To determine encapsulated drugs in the formulation, it is necessary to isolate free drugs using methods like column chromatography, dialysis, ultra-centrifugation, freeze-thawing, gel filtration, etc. The effectiveness of trapped drugs can be assessed in two ways: proniosomal vesicle destruction with a triton (0.1%) or propane (50%) and identification of the drug trapped inside [42]. Alternatively, untapped drugs can be measured after the vesicle has been destroyed. The amount of drug entrapped can calculate by using the formula:
                                         EE (%) = [(Ct - Cf)/ Ct] × 100
Where, Ct = the concentration of total drug 
Cf= the concentration of the unentrapped drug    

    
The ideal entrapment efficiency can vary depending on the specific drug, therapeutic goal, and formulation requirements. Generally, a higher entrapment efficiency (>50% or above) is desirable for effective ocular delivery. High entrapment efficiency ensures that a significant amount of drug is entrapped inside the vesicle leading to efficient drug delivery to the target ocular tissues and reducing potential drug loss or wastage.

Rheological Measurement
Rheological measurements of proniosomes for ocular delivery are valuable for understanding their physical behaviour, stability, and suitability for application to the ocular surface. Rheological measurements such as viscosity can provide information about the formulation’s consistency and its ability to spread and adhere to the ocular surface. For ocular delivery, the viscosity should be low enough to facilitate easy spreading and administration on the ocular surface but high enough to ensure sufficient retention and contact time. Viscosity for ocular preparation should range between 2 to 3 mPa.s. [43,44].

Isotonicity and osmolarity
Isotonicity of the ocular formulations is important to minimize potential discomfort or irritation to the ocular surface. For ocular application, the ideal isotonicity range for proniosomes should be between 250-350 mOsm/kg as this range is close to the osmolarity of tears which is 308 mOsm/kg. Formulating proniosomes within this isotonic range help to minimize the risk of irritation or adverse effect on the ocular tissue. An osmolarity of less than 100 mOsm/kg or greater than 640 mOsm/kg is considered an eye irritant [44,45]. 

In vitro studies
The drug release from proniosomes can be determined using a dialysis membrane, Franz diffusion cell, reverse dialysis, and USP dissolution apparatus type 1.  It is possible to analyze the drug release kinetics from the in-vitro drug release data. The release date is fitted into various kinetic models like- zero order, first order, Higuchi, Korsmeyer-Peppas, and Hixson Crowell models to find the order and mechanisms of drug release [46]. The “best-fit model” for drug release/dissolution can be selected based on a variety of factors like the coefficient of determination (R2), R2Adjusted, Sum of squares of residue (SSR), Akaike information criteria (AIC), Mean square error (MSE), and Correlation coefficient (R) [47].

Ocular irritation 
The Draize test is an effective and accurate method for accessing the irritation potential of proniosomes. It involves applying a test substance to the eyes of an animal, usually a rabbit (because of their wide eyes, ease of handling, and well-described anatomy). The responses are observed to determine any irritation caused due to proniosomes. The Draize test evaluates ocular irritation based on the study score. The score ranges from 0 to 3 for no irritation to redness and highest irritation. Nowadays, non-animal approaches such as computer modelling, and in-vitro experiments employing human cells and tissues are being used [48]. Eldeeb et al., 2019 performed the Draize test on male albino rabbits which were subjected to Brimonidine Tartrate proniosomal formulation and marketed formulation (Alphagan) to evaluate irritancy [49]. The marketed formulation showed irritancy for the first 1 h whereas the test formulation showed no sign of irritation indicating the safety of the formulation for ocular delivery [49].

Zeta potential
The zeta potential determines both the stability of a colloidal system and a vesicle's surface charge. Zeta potential measures the electrical potential at the shear plane around a particle or vesicle [50]. The Zeta potential of proniosomes can affect drug loading, stability, and drug release characteristics. The ideal zeta potential for proniosomes used in ocular delivery should be around +30 mv. Higher zeta potential prevents particle aggregation and coalescence, ensuring dispersion stability. A low zeta potential indicates a risk of aggregation. Zeta potential can be measured using a Malvern zeta sizer [51].

Stability
According to ICH recommendations, Stability studies on proniosomes for dry powder meant for reconstitution should be conducted as per climatic conditions and climatic zones (WHO, 1996). For accelerated stability, a temperature and relative humidity (RH) of 40 0C/75% RH is required, and for long-term studies, 25 0C/ 60% RH is required for Zone 1 and Zone 2, respectively, whereas Zone 3 and Zone 4 must maintain a condition of 30 0C /65% RH and measured over time for in vitro drug release, poly dispersibility index (PDI), zeta potential, pH, sterility, pyrogenicity, etc [52]. Whereas stability studies for proniosomal gel should be conducted at a refrigeration temperature of 2-80C, room temperature of 25±0.5° and an elevated temperature of 45±0.50° for 90 days. Conduction stability studies are vital to ensure the safety and efficacy of the product during its shelf life [53]. According to a stability study performed by Li et al. (2014) [54] on Tacrolimus-derived proniosomes for topical ocular delivery, studies were performed at 40 ± 2 0C/75 ± 5%RH; 4±2 0C/75 ± 5%RH; 25 ± 2 0C/60 ± 5%RH by storing proniosomes in a glass container. Physical stability studies were carried out to investigate the leakage of drug from the proniosomes during storage after 1,2 and 3 months of storage. Results indicated that proniosomes remain visually unchanged and entrapment efficiencies slightly reduce from 95% to 93% after 3 months of storing at 4 0C [54]. 

Toxicity of proniosomes 
The eye is a very sensitive and very useful organ of humans. Therefore, attention should be given to the safety of any dosage form administered to the eye. There are limited research reports on the toxicity of proniosomes. Researchers need to assess the possible toxicity of ocular formulations and the materials utilized to prepare these formulations during repeated and prolonged application in the eye before their clinical use and commercial production. Most of the biocompatibility and safety studies of proniosomes are performed on animal eyes. However, the structure of animal eyes is quite different from human eyes. Unfortunately, the results of these preclinical studies are sometimes not validated in humans. Most preclinical findings concluded no signs of toxicity and altered pharmacological effects of proniosomes in the eyes.  Proniosomes are composed of surfactants and cholesterol. Surfactants were used in proniosomal formulation to increase drug permeability through the cell membrane [55]. However, the chemical nature of surfactants imparts toxicity. Various studies have shown that the toxicity of proniosomes in ocular delivery depends on several factors, including the type of surfactant used, the concentration of surfactant, and the duration of exposure. A study by Govindarajan et al., 2022 [56] has shown that the ester derivative surfactant is harmful compared to the ether derivative. Surfactants like tween 80, can cause ocular irritation and damage to the cornea and conjunctiva when used at high concentrations [56]. CTAB (cetyltrimethylammonium bromide), a commonly used cationic surfactant, has been associated with cytotoxicity and disrupts cell membranes [57,58]. The most preferred constituents of proniosomes Span 60 and cholesterol are safe and do not produce any ocular toxic effect [59]. Overall potential toxicity of surfactants should be carefully considered, and at the same time, it is essential to conduct thorough safety evaluations of proniosomes before using them for ocular delivery in humans [60].

Proniosomes have gained significant attention in the pharmaceutical field due to their potential application in various medical fields. One of those areas is the treatment of various ocular diseases as shown in Table 4.

Glaucoma
Glaucoma is an eye disorder marked by elevated intraocular pressure that eventually results in optic nerve damage, leading to vision loss or blindness. Glaucoma is the primary cause of blindness worldwide [61]. Dorzolamide (Carbonic anhydrase inhibitor) is used in the treatment of glaucoma. The proniosomes of dorzolamide showed decreased intraocular pressure with enhanced therapeutic efficacy in the in-vivo studies in male albino rats [62,63]. Brimonidine tartrate (α-2 adrenergic agonist) proniosomes decrease the production of aqueous humour and reduce the ischemia-induced optic nerve damage by enhancing the ocular bioavailability of the drug [49]

Conjunctivitis 
Infection of the conjunctiva is known as conjunctivitis, commonly referred to as pink eyes. Conjunctiva is a thin transparent membrane covering the white part of the eyes. Proniosomes may use to encapsulate and deliver anti-inflammatory and antibiotic drugs directly to the eye to treat conjunctivitis [64,65]. Various studies have demonstrated the benefits of proniosomes conjunctivitis treatment. Lomefloxacin-proniosomal formulation was found effective in reducing the severity of bacterial conjunctivitis in rabbits [66]. In another study, Levofloxacin-loaded proniosomal formulation showed increased ocular residence time by providing sustained drug release [67].

Fungal infection 
Fungal keratitis/mycotic/keratomycosis is a severe eye infection that affects the cornea of the eyes leading to corneal damage and vision loss. Candida parapsilosis, candida albicans, candida tropicalis, and candida glabrata mainly causes ocular fungal infections [68]. Proniosomes may be used to encapsulate and deliver antifungal medication for fungal keratitis. One of the studies indicated that proniosomes loaded with Amphotericin B were able to effectively treat fungal keratitis in rabbits [69].

Uveitis 
Uveitis, or chorioretinitis, is an inflammation of the middle layer of the eyes causing inflammation of eyes. Proniosomes can be used to encapsulate and deliver anti-inflammatory medication, such as corticosteroids or NSAIDS, directly to the eye to treat uveitis [70]. Tacrolimus-loaded proniosomes gave higher precorneal permeation and retention in an in vitro study in rabbit cornea for 21 days [71].

Despite the development of proniosomes and nanoparticles in general being successful, there are still many obstacles to overcome, such as complicated regulatory issues, scale-up practicability, and reproducibility. As a result, producing nanomaterials on a large scale might be difficult [59]. To produce proniosomes on an industrial scale for commercial use, it is necessary to integrate new methods and technology transfer. However, any preparation procedure may fail to translate from a laboratory scale to an industrial scale due to process restrictions in small-scale preparation. Proniosomes’ characteristics, such as particle size, drug encapsulation, process leftover materials, stability, and surface properties, are most affected by scaling up. Moreover, the scale-up process may decrease the drug loading in the proniosomes. Therefore, a well-designed scale-up procedure is required which can guarantee the effectiveness, affordability, and fast production of these nanomedicines [59]. The stability of the materials used for the production and the use of toxic solvents (such as the use of chloroform or dichloromethane as an organic phase) are some of the process limitations. Therefore, new techniques utilizing aqueous solvents or solvents with low toxicity must be developed for the pharmaceutical industry to produce nanomedicines.
It is critical to demonstrate the ability to transfer the technology to a development facility or contract manufacturing business where a practical, scalable, and cost-effective process can be established to produce large batch sizes under good laboratory practices (GLP) and ultimately good manufacturing practices (GMP) conditions [72]. 
Due to their simple fabrication method and flexibility in drug delivery, proniosomes have the potential to be manufactured on a large scale. Proniosomes were investigated as potential replacements for liposomes and other carrier systems for entrapping both hydrophobic and hydrophilic or polar and nonpolar pharmaceuticals. Proniosomes also have the advantage of minimal toxicity due to their non-ionic nature and the lack of any specific production or processing requirements. Additionally, it is an easy approach for producing proniosomes regularly and in big quantities without the use of undesirable solvents [73]. 

Proniosomes have been proven as an efficient delivery method that offers several benefits over conventional delivery methods in the form of better stability, bioavailability, and targeted delivery of therapeutic molecules. Further potential future applications of proniosomes include their use in gene therapy, vaccine delivery, and cancer treatment. Moreover, using proniosomes in combination with other drug delivery technologies, such as nanotechnology could lead to the formation of even more effective drug delivery systems. However, the formulation of proniosomes may present several challenges due to their complex nature and the need for precise control over their properties to ensure efficient drug delivery. Scaling up proniosomes from laboratory to large-scale production can be difficult, and it is important to ensure consistent product quality for regulatory approval. Tackling these challenges will be essential to developing and adopting proniosomes as a drug delivery system. Some patented formulations of proniosomes in various delivery systems are given in Table 5.

In conclusion, proniosomes represent a promising drug delivery platform for ocular drug delivery (Figure 3). These are essentially dry formulations that consist of a blend of surfactant and carrier material. The ability of proniosomes to improve drug absorption and retention in the eyes, along with their ease of handling and stability, makes them an attractive option for developing novel ocular therapies. These are widely applicable for the delivery of various drugs, including antibiotics, anti-cancer agents, and anti-inflammatory drugs. Furthermore, research is needed to optimize the formulation and delivery of proniosomes for clinical use.  

None

This manuscript was drafted by MNK and NN. MYA provided supervision throughout the manuscript preparation. Other authors contributed directly or indirectly to the revision and publication of this manuscript.

There is no conflict of interest among the authors.