Polysorbate-Based Drug Formulations for Brain-Targeted Drug Delivery and Anticancer Therapy

: Polysorbates (PSs) are synthetic nonionic surfactants consisting of polyethoxy sorbitan fatty acid esters. PSs have been widely employed as emulsiﬁers and stabilizers in various drug formulations and food additives. Recently, various PS-based formulations have been developed for safe and efﬁcient drug delivery. This review introduces the general features of PSs and PS-based drug carriers, summarizes recent progress in the development of PS-based drug formulations, and discusses the physicochemical properties, biological safety, P-glycoprotein inhibitory properties, and therapeutic applications of PS-based drug formulations. Additionally, recent advances in brain-targeted drug delivery using PS-based drug formulations have been highlighted. This review will help researchers understand the potential of PSs as effective drug formulation agents.


Introduction
Drug delivery systems are one of the mainstream strategies for achieving targeted delivery and controlled release of therapeutic drugs in the body. Various drug carriers composed of polymers, dendrimers, metal oxides, hydrogels, cell-derived membranes, exosomes, and surfactants have been developed to improve the therapeutic efficacy of drugs and alter their pharmacokinetics [1]. Despite the several advantages of drug carriers, some drug carriers have limitations, including toxicity, low physicochemical stability, immune responses, poor cell specificity, unwanted drug leakage, and high manufacturing cost, which make them unsuitable for practical applications [2]. Extensive research efforts have been made to develop drug carriers for safe, efficient, and targeted drug delivery by addressing the aforementioned issues [2]. Recently, drug carriers composed of surfactants have garnered great attention owing to their enhanced bioavailability and high biocompatibility [3]. Surfactants have been widely used in pharmaceutical formulations because they can enhance drug solubility and prevent protein aggregation [4]. Nonionic surfactants with charge-free head groups are an important subclass of surfactants. They have been intensively utilized as emulsifiers and stabilizers in the cosmetic industry [5]. Nonionic surfactants are commercially available in various chemical structures. Various types of nonionic surfactants have been used to form emulsions, micelles, and niosomes, all of which can efficiently encapsulate hydrophilic and hydrophobic molecules [6,7]. The features of nonionic surfactant-based formulations are similar to those of liposomes, and thus, they are suitable alternatives to phospholipids. Low cost, bulk abundance, high physical stability, and ease of storage are advantages of using nonionic surfactants as pharmaceutical formulations. Moreover, the rich phase separation behavior and low critical micellization temperature values are important features of nonionic surfactants that make them attractive for pharmaceutical applications. Polysorbates (PSs), which are commercially known as Tween, are synthetic nonionic surfactants that consist of polyethoxy sorbitan fatty acid esters. PSs have numerous advantages, such as commercial abundance and ease of chemical modification, which render them suitable for various biomedical applications. Therefore, PSs have been widely employed as emulsifiers and stabilizers in various drug formulations and food additives [6,7]. PSs have also served as wetting agents, foaming agents, and dispersants [8][9][10][11]. Recently, various PS-drug conjugates and PS-conjugated drug carriers have been developed for safe and efficient drug delivery [12][13][14]. PSs have also attracted great attention as brain-targeting coating materials that can facilitate the transport of drug carriers across the blood-brain barrier (BBB) [15]. This review introduces the general features of PSs and PS-based drug carriers and summarizes recent progress in the development of PS-based drug carriers for the treatment of various diseases. Special emphasis is placed on PS-based drug carriers for brain-targeted drug delivery. In addition, the mechanism of action, current limitations, and perspectives of PS-based drug carriers are presented.

Chemical Structures and Physicochemical Properties of PSs
The common backbone structure of PSs is a sorbitan ring (sugar alcohol) with poly (ethylene oxide) (PEO) conjugated to hydroxyl groups. The different numbers of ethylene oxide subunits are conjugated to the four hydroxyl groups of the sorbitan ring, but the overall number of repeating units of ethylene oxide is approximately 20. The chemical preparation of PSs involves a two-step process: sorbitan is ethoxylated, followed by esterification with fatty acids (e.g., lauric acid, palmitic acid, stearic acid, and oleic acid). The synthesis scheme of PSs is shown in Figure 1. features of nonionic surfactants that make them attractive for pharmaceutical applications. Polysorbates (PSs), which are commercially known as Tween, are synthetic nonionic surfactants that consist of polyethoxy sorbitan fatty acid esters. PSs have numerous advantages, such as commercial abundance and ease of chemical modification, which render them suitable for various biomedical applications. Therefore, PSs have been widely employed as emulsifiers and stabilizers in various drug formulations and food additives [6,7]. PSs have also served as wetting agents, foaming agents, and dispersants [8][9][10][11]. Recently, various PS-drug conjugates and PS-conjugated drug carriers have been developed for safe and efficient drug delivery [12][13][14]. PSs have also attracted great attention as brain-targeting coating materials that can facilitate the transport of drug carriers across the blood-brain barrier (BBB) [15]. This review introduces the general features of PSs and PS-based drug carriers and summarizes recent progress in the development of PS-based drug carriers for the treatment of various diseases. Special emphasis is placed on PS-based drug carriers for brain-targeted drug delivery. In addition, the mechanism of action, current limitations, and perspectives of PS-based drug carriers are presented.

Chemical Structures and Physicochemical Properties of PSs
The common backbone structure of PSs is a sorbitan ring (sugar alcohol) with poly (ethylene oxide) (PEO) conjugated to hydroxyl groups. The different numbers of ethylene oxide subunits are conjugated to the four hydroxyl groups of the sorbitan ring, but the overall number of repeating units of ethylene oxide is approximately 20. The chemical preparation of PSs involves a two-step process: sorbitan is ethoxylated, followed by esterification with fatty acids (e.g., lauric acid, palmitic acid, stearic acid, and oleic acid). The synthesis scheme of PSs is shown in Figure 1. The number following the "polysorbate" refers to the type of fatty acid ester associated with the polyoxyethylene sorbitan portion of the molecule. Fatty acid ester moieties of PS 20, PS 40, PS 60, and PS 80 are laurate, palmitate, stearate, and oleate, respectively. Figure 2 illustrates the chemical structures of various PSs (i.e., PS 20,PS 40,PS 60,and PS 80). The PEO segments in PS are hydrophilic, and the fatty acid segments are hydrophobic. Therefore, amphiphilic PSs can serve as effective surfactants. The amphiphilic nature of PSs allows them to self-assemble into micelles in aqueous solutions after reaching critical micellar concentrations. PS-based micelles have been widely employed for drug delivery because of their nanoscale size and high drug-loading capacity [16][17][18]. PSs have been used as emulsifiers and solubilizers for hydrophobic drugs and as protein-stabilizing agents [19]. For example, PS 80 was used as a stabilizer in the Janssen COVID-19 vaccine formulation [20]. Emulsion-based formulations containing PSs as emulsifiers have been intensively developed and have shown potential as effective drug carriers for both hydrophobic and hydrophilic drugs [21][22][23]. The behavior of protein aggregation in the absence or presence of PS 80 as an excipient was evaluated [24]. The addition of PS 80 substantially inhibited protein aggregation during agitation [24]. PS 80 and PS 20 have been used in the formulation of therapeutic monoclonal antibodies owing to their biocompatibility, low toxicity, and good protein-stabilizing properties [25]. PSs The number following the "polysorbate" refers to the type of fatty acid ester associated with the polyoxyethylene sorbitan portion of the molecule. Fatty acid ester moieties of PS 20, PS 40, PS 60, and PS 80 are laurate, palmitate, stearate, and oleate, respectively. Figure  2 illustrates the chemical structures of various PSs (i.e., PS 20,PS 40,PS 60,and PS 80). The PEO segments in PS are hydrophilic, and the fatty acid segments are hydrophobic. Therefore, amphiphilic PSs can serve as effective surfactants. The amphiphilic nature of PSs allows them to self-assemble into micelles in aqueous solutions after reaching critical micellar concentrations. PS-based micelles have been widely employed for drug delivery because of their nanoscale size and high drug-loading capacity [16][17][18]. PSs have been used as emulsifiers and solubilizers for hydrophobic drugs and as protein-stabilizing agents [19]. For example, PS 80 was used as a stabilizer in the Janssen COVID-19 vaccine formulation [20]. Emulsion-based formulations containing PSs as emulsifiers have been intensively developed and have shown potential as effective drug carriers for both hydrophobic and hydrophilic drugs [21][22][23]. The behavior of protein aggregation in the absence or presence of PS 80 as an excipient was evaluated [24]. The addition of PS 80 substantially inhibited protein aggregation during agitation [24]. PS 80 and PS 20 have been used in the formulation of therapeutic monoclonal antibodies owing to their biocompatibility, low toxicity, and good protein-stabilizing properties [25]. PSs have been utilized to increase the permeability of various drugs in vivo and in vitro [26][27][28][29]. Additionally, PSs exhibit high biocompatibility and low toxicity and are thus advantageous for use as drug carriers.
In particular, nonionic surfactants are less toxic and more biocompatible and have lower hemolytic potential than charged surfactants (cationic, anionic, or amphoteric) [30]. have been utilized to increase the permeability of various drugs in vivo and in vitro [26][27][28][29]. Additionally, PSs exhibit high biocompatibility and low toxicity and are thus advantageous for use as drug carriers. In particular, nonionic surfactants are less toxic and more biocompatible and have lower hemolytic potential than charged surfactants (cationic, anionic, or amphoteric) [30].

Biosafety Studies of PSs
Biosafety is an umbrella term that reflects the damage of the material to biological integrity [31]. Any material used for biomedical applications should possess desirable biosafety. To achieve desirable biosafety, materials should be degraded in biological environments without causing any adverse effects after their therapeutic use. Enzymatic degradation is a crucial function of the human body that involves the breakdown or elimination of unnecessary materials. Food additives are generally excreted from the body without causing adverse effects. PSs have been utilized as emulsifiers in a variety of food and cosmetic products [32]. PSs were approved as a food additive in the USA in 1960 and the EU in 1995 [33]. The Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Committee on Food Additives established an acceptable oral daily intake of PSs (e.g., PS 20, PS 60, PS 65, and PS 80) of 25 mg/kg by adults [34,35]. Recently, the pediatric safety of PSs in drug formulations has also been investigated [36]. The amount of parenterally administered PSs in formulations was calculated using a progressive pediatric safety factor through an age-and weight-based estimation. For example, the maximum total daily doses of parenterally administered PS 20 and PS 80 for 2-year-old children are 3.37 and 50.6 mg, respectively [36].
Over the past few decades, the cytotoxicity and metabolism of various PSs have been investigated to demonstrate their potential for biomedical applications. Upon parenteral administration, PSs are degraded readily via hydrolysis by esterases in the plasma [37]. The fatty acids generated by the hydrolysis of PSs are expected to be further metabolized via beta-oxidation, leading to the formation of carbon dioxide, which can be exhaled. Polyoxyethylene sorbitan, which is generated by the hydrolysis of PSs, is excreted via urine and feces [38]. An in vivo study showed that PSs were efficiently hydrolyzed by esterase in the digestive tract after oral administration in a rat model [39,40]. Free fatty acids are then readily absorbed from the gastrointestinal tract and further metabolized to carbon dioxide in exhaled breath, similar to the phenomena observed in normal fatty acid metabolism [39]. The absorption of polyoxyethylene sorbitan into the digestive tract was found to be very low. Approximately 87% of PS 20 and 91% of PS 80 were excreted in the feces. [40]. Negligible systemic toxicity was observed when PS 80 (22 g/kg body weight) was orally administered to rats. In addition, liver or kidney dysfunction was not observed [34]. An in vivo rabbit eye model was used to evaluate the irritation effect of aqueous and oily solutions (Miglyol 812) of PS 20 and PS 80. In vitro tests using fibroblast cells demonstrated that cytotoxicity of PSs depended on surfactant concentrations (0.1, 1, or 5%) and types of solvents (water or oil). Treatment with 1% aque-

Biosafety Studies of PSs
Biosafety is an umbrella term that reflects the damage of the material to biological integrity [31]. Any material used for biomedical applications should possess desirable biosafety. To achieve desirable biosafety, materials should be degraded in biological environments without causing any adverse effects after their therapeutic use. Enzymatic degradation is a crucial function of the human body that involves the breakdown or elimination of unnecessary materials. Food additives are generally excreted from the body without causing adverse effects. PSs have been utilized as emulsifiers in a variety of food and cosmetic products [32]. PSs were approved as a food additive in the USA in 1960 and the EU in 1995 [33]. The Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Committee on Food Additives established an acceptable oral daily intake of PSs (e.g., PS 20, PS 60, PS 65, and PS 80) of 25 mg/kg by adults [34,35]. Recently, the pediatric safety of PSs in drug formulations has also been investigated [36]. The amount of parenterally administered PSs in formulations was calculated using a progressive pediatric safety factor through an age-and weight-based estimation. For example, the maximum total daily doses of parenterally administered PS 20 and PS 80 for 2-year-old children are 3.37 and 50.6 mg, respectively [36].
Over the past few decades, the cytotoxicity and metabolism of various PSs have been investigated to demonstrate their potential for biomedical applications. Upon parenteral administration, PSs are degraded readily via hydrolysis by esterases in the plasma [37]. The fatty acids generated by the hydrolysis of PSs are expected to be further metabolized via beta-oxidation, leading to the formation of carbon dioxide, which can be exhaled. Polyoxyethylene sorbitan, which is generated by the hydrolysis of PSs, is excreted via urine and feces [38]. An in vivo study showed that PSs were efficiently hydrolyzed by esterase in the digestive tract after oral administration in a rat model [39,40]. Free fatty acids are then readily absorbed from the gastrointestinal tract and further metabolized to carbon dioxide in exhaled breath, similar to the phenomena observed in normal fatty acid metabolism [39]. The absorption of polyoxyethylene sorbitan into the digestive tract was found to be very low. Approximately 87% of PS 20 and 91% of PS 80 were excreted in the feces [40]. Negligible systemic toxicity was observed when PS 80 (22 g/kg body weight) was orally administered to rats. In addition, liver or kidney dysfunction was not observed [34]. An in vivo rabbit eye model was used to evaluate the irritation effect of aqueous and oily solutions (Miglyol 812) of PS 20 and PS 80. In vitro tests using fibroblast cells demonstrated that cytotoxicity of PSs depended on surfactant concentrations (0.1, 1, or 5%) and types of solvents (water or oil). Treatment with 1% aqueous solutions of PS 20 and PS 80 induced the cell death of approximately 20%. Only 10% of cells were dead when the oily solution of the surfactant was added. The difference in cell viability between aqueous and oily solutions was larger when the concentration of PSs increased from 1 to 5%. In vivo tests showed that aqueous (0.9% NaCl) solutions of PSs were nonirritant, whereas small changes in conjunctiva were observed after administration of oily PSs [41]. The in vitro cytotoxicity of PSs against human fibroblast cells has also been studied. The half-maximal inhibitory concentration (IC 50 ) values of aqueous solutions of PS 60 and PS 80 were found to be 70.8 and 65.5 mg/mL, respectively [42].
Although PS 20 and PS 80 have been recognized as safe and nonirritant food additives and drug-formulating agents [36], some rare cases of hypersensitivity [43] and hepatotoxicity [44] have been reported, possibly owing to highly dosed PSs as excipients. These reports suggest that safe and acceptable doses of PSs in food additives and drug formulations should be regulated or defined. Various toxicological studies of PSs are highly required to develop safe PS-based drug formulations.

P-glycoprotein (P-gp) Inhibitory Property of PS
P-gp is a membrane transport protein present in normal tissues, such as the intestine, brain, liver, placenta, kidney, and cancer cells [45]. The crucial role of P-gp is to maintain intracellular drug concentrations via influx and efflux pumps. Therefore, P-gp plays a key role in the absorption, localization, and elimination of various drugs. Drug-resistant tumors overexpress P-gp, which can eliminate cancer chemotherapeutic agents. Multidrug resistance (MDR) is a major issue in cancer treatment [46]. The inhibition of P-gp is one of the ideal steps to reverse MDR.
Recently, nonionic surfactants were found to be effective P-gp inhibitors to overcome P-gp-mediated MDR in various types of cancer cells [47]. Working mechanisms of P-gp inhibition by PSs have been investigated using fluorescence polarization techniques [47]. The results indicated that PSs can alter the membrane fluidity of cells, thereby changing the conformation of transporters in the membranes [47]. Effects of nonionic surfactants such as PS 20 and Cremophor EL on breast cancer resistance protein (BCRP) transporters in Caco-2 cell monolayers were analyzed [48]. PS 20 increased the membrane fluidity of the inner lipid bilayers of the intestine. Therefore, PS 20 effectively improved the intestinal absorption of drugs topotecan in rats via the transcellular pathway by inhibiting the function of BCRP.
The effects of PS as a pharmaceutical excipient on the intracellular localization, transport kinetics, and intestinal absorption of epirubicin were evaluated in both human colon adenocarcinoma (Caco-2) cells and rat jejunum and ileum [49]. PS 20 and PS 80 substantially increased apical to basolateral absorption and reduced the basolateral to apical efflux of epirubicin across Caco-2 cell monolayers [49]. In addition, PS drastically enhanced the mucosal to serosal absorption of epirubicin in the rat jejunum and ileum. These results suggest that the inhibition of intestinal P-gp by PSs significantly improves the oral absorption of drugs in multidrug-resistant cancer cells.
The effect of PS 80 on the absorption of lipophilic digoxin was also investigated in vitro and in vivo. PS 80 enhanced the bioavailability of digoxin by modulating P-gp systems in cancer cells [29]. When rats were orally administered digoxin (0.2 mg/kg) containing 1% (v/v) and 10% (v/v) PS 80, the in vivo oral absorption of digoxin increased by 30% and 61%, respectively, compared to the control groups [26]. The significantly increased absorption of digoxin after oral administration was attributed to the inhibition of P-gp in the gut.
To inhibit the effect of P-gp, a clay-based nanocomposite using montmorillonite and PS 20 was developed [50]. The particles consisted of montmorillonite, the nonionic surfactant PS 20, and the P-gp substrate digoxin. In vivo results showed that compared to administration of free digoxin, administration of montmorillonite-surfactant hybrid particles resulted in an increased digoxin concentration in the plasma. This increase in digoxin concentration might be attributed to the increase in mucosa-localized concentrations of both digoxin and PS 20.

PS-Based Formulations for Various Routes of Drug Administration
Drugs are administered to human bodies through various routes, depending on their formulations and disease sites [51]. For instance, ointment, emulsion, and solutions are suitable for ocular and intranasal delivery; pills and capsules are suitable for oral delivery; and patches, gels, and ointments are effective for transdermal delivery. For all formulations, surfactants play a key role in enhancing the therapeutic index of drugs. In particular, various PS-based formulations, including emulsions [52], micro/nanoemulsions [53], suspensions [54], and niosomes [55], have been utilized as drug carriers and delivered into the body via various routes of drug administration.

PS-Based Formulations for Ocular Delivery
Most eye-related problems, including dry eye, fungal infection, inflammation, ocular hypertension, and glaucoma, are generally treated with topical ophthalmic drugs [56]. Ophthalmic drug formulations include nanosomes, micelles, and emulsions; these formulations show high drug-loading efficiency, ocular bioavailability, and prolonged drug effects in the eyes [57]. Compared to other charged surfactants, PSs offer more efficient ocular drug delivery owing to their properties such as smooth penetration, drug-solubilizing capability, and effective drug protection. Moreover, nonionic surfactants are known to be less harmful and irritating than charged surfactants [58]. PSs increase ocular bioavailability because they can act as penetration enhancers that can remove mucus layers and destroy junctional complexes [59].
DUREZOL ® is a commercially available corticosteroid formulation used for the treatment of inflammation and pain associated with ocular surgery. This Food and Drug Administration (FDA)-approved formulation contains PS 80 as a nonionic emulsifier (4% w/v) [60]. Similarly, cyclosporine emulsions containing 1% PS 80 were developed and sold under the brand name Restasis ® [61]. Many PS-based formulations with various ocular drugs, including flurbiprofen [62], ibuprofen [63], indomethacin [64], dexamethasone [65], chloramphenicol [66], and hydrocortisone [67], have been developed and evaluated in preclinical studies. These PS-based formulations exhibit low irritation, high drug solubility, efficient penetration across cell membranes, and high bioavailability.
Recently, niosomes composed of cationic lipids and PSs have been developed for nonviral gene delivery. Niosomes are nonionic surfactant-based vesicles formed via selfassembly in an aqueous solution [68,69]. Niosomes have bilayer structures that can encapsulate both hydrophilic and hydrophobic drugs, such as liposomes. Because niosomes are biodegradable, stable, and inexpensive, they have great potential as alternatives to liposomes [70,71]. Niosome formulations comprising the cationic lipid N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA) and nonionic PS 60 were developed for retinal gene delivery [72]. The incorporation of natural lipids (i.e., lycopene) resulted in high transfection efficiency in the human retinal pigment epithelial cells (ARPE-19) through the caveolae-mediated endocytic pathway. The DOTMA/PS 60/lipid niosomes administered into the rat retina via subretinal and intravitreal routes efficiently transfected the outer segments of the retina. The same group studied the effects of PS (PS 20, PS 80, and PS 85) on the transfection efficiency of niosomes in the rat retina [73]. The graphical representation of niosomes is shown in Figure 3. Niosomes formulated with PS 20 showed more efficient transfection in retinal cells than the niosomes containing PS 85. The greater hydrophilicity of PS 20-containing niosomes than the PS 85-containing niosomes facilitated caveolae-mediated endocytosis in retinal cells, thereby preventing lysosomal degradation [73]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 24

PS-Based Formulations for Transdermal Delivery
Transdermal drug delivery involves the topical administration of drugs across the skin barrier. The topical delivery of active compounds is hampered by the limited skin permeability [74]. Surfactants, such as PSs, enhance the permeation of drugs through the skin [75]. Microemulsions composed of eucalyptol and PS 80 were fabricated as transdermal delivery vehicles for curcumin [76]. The curcumin permeation rate of the microemulsion was 15.7-fold higher than that of the control (eucalyptol only). The microemulsions containing 25% water, 25% ethanol, 37.5% ethanol, and 12.5% PS 80 exhibited the highest curcumin permeation rate [77]. Microemulsions composed of PS 80, medium-chain glycerides, and propylene glycol were developed for the efficient transdermal delivery of hydrophilic (i.e., adenosine) and hydrophobic (i.e., progesterone) drugs [78]. Microemulsions with 47% (w/v) PS and 63% (w/v) PS were prepared. Microemulsions with 63% (w/v) PS exhibited higher efficacy in transdermal delivery of hydrophilic agents than those with 47% (w/v) PS. In addition, various PS-based microemulsions have been developed for the transdermal delivery of testosterone [79], meloxicam [80], capsaicin [81], fluorouracil [82], and ketoprofen [83].

PS-Based Formulations for Oral Delivery
Intestine, brain, liver, placenta, kidney, and cancer tissues express ATP-binding cassettes and solute carrier transporters (e.g., P-gp, MDR1, and ABCB1) [84,85]. These membrane transport proteins avoid the uptake of toxins and xenobiotics from the cells and maintain the bioavailability of active drug ingredients [86]. Previous studies have demonstrated that PS-based formulations enhance the intestinal absorption and bioavailability of drugs by modulating membrane transport proteins [87,88] and etoposide [89,90].
To improve the bioavailability of erythromycin for an in vivo fish model, three types of microemulsified erythromycin formulations were prepared and administered to fish over seven consecutive days. The results showed that emulsified erythromycin improved oral bioavailability and achieved a higher therapeutic plasma concentration than erythromycin powder [91]. Salmon calcitonin has low bioavailability and stability in the gastrointestinal tract [92]. PS-based microemulsions containing salmon calcitonin improved its oral bioavailability as well as pharmacological activity compared to those of free drugs [92].
The oral bioavailability of curcumin is poor owing to its low aqueous solubility, alkaline instability, and rapid elimination. Curcumin was encapsulated into ionic-gelated

PS-Based Formulations for Transdermal Delivery
Transdermal drug delivery involves the topical administration of drugs across the skin barrier. The topical delivery of active compounds is hampered by the limited skin permeability [74]. Surfactants, such as PSs, enhance the permeation of drugs through the skin [75]. Microemulsions composed of eucalyptol and PS 80 were fabricated as transdermal delivery vehicles for curcumin [76]. The curcumin permeation rate of the microemulsion was 15.7-fold higher than that of the control (eucalyptol only). The microemulsions containing 25% water, 25% ethanol, 37.5% ethanol, and 12.5% PS 80 exhibited the highest curcumin permeation rate [77]. Microemulsions composed of PS 80, medium-chain glycerides, and propylene glycol were developed for the efficient transdermal delivery of hydrophilic (i.e., adenosine) and hydrophobic (i.e., progesterone) drugs [78]. Microemulsions with 47% (w/v) PS and 63% (w/v) PS were prepared. Microemulsions with 63% (w/v) PS exhibited higher efficacy in transdermal delivery of hydrophilic agents than those with 47% (w/v) PS. In addition, various PS-based microemulsions have been developed for the transdermal delivery of testosterone [79], meloxicam [80], capsaicin [81], fluorouracil [82], and ketoprofen [83].

PS-Based Formulations for Oral Delivery
Intestine, brain, liver, placenta, kidney, and cancer tissues express ATP-binding cassettes and solute carrier transporters (e.g., P-gp, MDR1, and ABCB1) [84,85]. These membrane transport proteins avoid the uptake of toxins and xenobiotics from the cells and maintain the bioavailability of active drug ingredients [86]. Previous studies have demonstrated that PS-based formulations enhance the intestinal absorption and bioavailability of drugs by modulating membrane transport proteins [87,88] and etoposide [89,90].
To improve the bioavailability of erythromycin for an in vivo fish model, three types of microemulsified erythromycin formulations were prepared and administered to fish over seven consecutive days. The results showed that emulsified erythromycin improved oral bioavailability and achieved a higher therapeutic plasma concentration than erythromycin powder [91]. Salmon calcitonin has low bioavailability and stability in the gastrointestinal tract [92]. PS-based microemulsions containing salmon calcitonin improved its oral bioavailability as well as pharmacological activity compared to those of free drugs [92].
The oral bioavailability of curcumin is poor owing to its low aqueous solubility, alkaline instability, and rapid elimination. Curcumin was encapsulated into ionic-gelated alginate-PS 80 nanoparticles (NPs) to increase its oral bioavailability. In healthy human volunteers, administration of the NPs showed five-fold higher bioavailability and higher plasma concentrations of curcumin than administration of curcumin suspensions [93]. 9-Nitrocamptothecin (9-NC) is a potent topoisomerase I inhibitor for the treatment of pancreatic cancer. However, its bioavailability after oral administration is poor. To address the low bioavailability of 9-NC, 9-NC microemulsions were prepared using a selfmicroemulsifying drug delivery system (SMEDDS). Different types of SMEDDS formulations of 9-NC were prepared using ethyl oleate as an oil phase, cremophor EL or PS 80 as a surfactant, and PEG-400/ethanol as cosurfactants. The antitumor effects of the resulting microemulsions were evaluated against human ovarian cancer cells in vitro and in vivo. In SKOV-3 cells, PS 80-based nanoemulsions showed the most efficient growth inhibition. Orally administered PS 80-based nanoemulsions also showed efficient tumor growth suppression in mice bearing human ovarian cancer xenografts [94].
Rosuvastatin is a prescription medicine used to prevent cardiovascular diseases. Microemulsion formulations were used to improve the oral bioavailability of rosuvastatin calcium. The microemulsion of rosuvastatin calcium was prepared using a mixture of peceol oil, PS 20 as a surfactant, and transcutol as a cosurfactant. Stability studies showed that the emulsion was stable for at least 3 months, with a high drug encapsulation efficiency of 91.6%. The microemulsion increased the oral bioavailability of rosuvastatin calcium by increasing the effective surface area of drug exposure in the physiological medium [95].
Fenofibrate is a lipid-regulating agent that controls the levels of cholesterol and triglycerides (fatty acids) in the blood. A fenofibrate-loaded emulsion was formulated by mixing olive oil, surfactants (a mixture of PS 20 and PS 80), and distilled water; a smallsized emulsion was prepared with a 5% olive oil concentration, and the mixture of PS 20 and PS 80 at 1:2 ratios showed high fenofibrate solubility [96]. The formulations were physically stable for up to 4 weeks, with no significant change in particle size. In contrast to the limited dissolution rate of the drug, the SMEDDS formulation showed effective and rapid drug release. The comparative pharmacodynamic evaluation was investigated in terms of lipid-lowering efficacy using a Triton-induced hypercholesterolemia model in rats [97]. The SMEDDS formulation substantially lowered serum lipid levels in phases I and II of the Triton test compared with plain fenofibrate. This result demonstrates that the SMEDDS formulation is an effective alternative to traditional oral formulations of fenofibrate to improve its bioavailability [97].

PS-Based Formulations for Intranasal Delivery
Intranasal delivery is a noninvasive administration that can bypass the BBB and thus facilitate the transport of drugs to the brain. However, intrinsic drug accumulation in the brain after intranasal delivery may not be sufficient to achieve clinical efficacy. PS 80 was used to increase the drug accumulation in the brain. In vivo rat model studies showed that the intranasal delivery of 2,3,5,6-tetramethylpyrazine (TMPP) along with PS 80 increased the concentration of TMPP in the brain [98]. The nasal mucosa is known to be the entry site of respiratory syncytial virus (RSV) into the body. The results showed that intranasal administration of a formulation containing virucidal lipids along with PS 20 significantly lowered the RSV load in the nasal mucosa of infected rat models compared to saline-treated groups [99].
Neurotoxin-II (NT-II) is an analgesic peptide, which has limited permeability across the BBB via intravenous injection. Intranasal delivery of NT-II into brain using PS 80-coated poly(lactic acid) (PLA) NPs was demonstrated. In vivo mouse model studies revealed PS 80-coated PLA NPs promoted the biodistribution of NT-II into brain via intranasal delivery [100].

PS-Conjugated Drugs or Drug Carriers
Substituents conjugated to functional groups of polymeric drug carriers can alter their physicochemical properties and drug loading capacity, consequently affecting their drug delivery efficiency [101][102][103][104][105]. For example, polyglycerols (PGs) possessing multiple hydroxyl groups, which can be functionalized with other polymers and drugs, have been utilized as versatile drug carriers [103]. Drug-conjugated polymers have been developed to enhance drug stability in systemic circulation and increase drug encapsulation [103]. Conjugation of drugs to polymers via stimuli-responsive linkers has also achieved controlled drug release [104,105]. For example, hyperbranched PG-polycaprolactone block copolymer (PG-co-PCL)-based NPs were developed for controlled delivery of gemcitabine (GEM) [104]. GEM was conjugated to the PG-co-PCL through polycarbonate blocks modified with pH-sensitive tertiary amine. The GEM-conjugated NPs were stable at pH 7.4 and released the GEM in pH-dependent manner. Amine-functionalized dendritic PG was also prepared and conjugated to paclitaxel (PTX) via bioresponsive ester linkages [105]. PTX was rapidly released under hydrolytic or esterase-rich conditions. The examples mentioned above regarding the functionalization of polymeric drug carriers suggest the strategies for chemical modifications of PSs [103][104][105]. PSs bear three hydroxyl groups at the end of the ethylene oxide chains. These hydroxyl groups can be easily functionalized or transformed into various functional groups, including esters, ethers, carbonates, and carbamates. The possible chemical modifications of the PSs are summarized in Figure 4. These functional groups can be used to incorporate chemical drugs and proteins to increase the therapeutic effects and pharmacokinetics of PS-based formulations. Some bioresponsible linkers have also been utilized to achieve controlled drug release. Herein, we summarize recent progress regarding PS-conjugated drugs or drug carriers for biomedical applications.

PS-Conjugated Drugs or Drug Carriers
Substituents conjugated to functional groups of polymeric drug carriers can alter their physicochemical properties and drug loading capacity, consequently affecting their drug delivery efficiency [101][102][103][104][105]. For example, polyglycerols (PGs) possessing multiple hydroxyl groups, which can be functionalized with other polymers and drugs, have been utilized as versatile drug carriers [103]. Drug-conjugated polymers have been developed to enhance drug stability in systemic circulation and increase drug encapsulation [103]. Conjugation of drugs to polymers via stimuli-responsive linkers has also achieved controlled drug release [104,105]. For example, hyperbranched PG-polycaprolactone block copolymer (PG-co-PCL)-based NPs were developed for controlled delivery of gemcitabine (GEM) [104]. GEM was conjugated to the PG-co-PCL through polycarbonate blocks modified with pH-sensitive tertiary amine. The GEM-conjugated NPs were stable at pH 7.4 and released the GEM in pH-dependent manner. Amine-functionalized dendritic PG was also prepared and conjugated to paclitaxel (PTX) via bioresponsive ester linkages [105]. PTX was rapidly released under hydrolytic or esterase-rich conditions. The examples mentioned above regarding the functionalization of polymeric drug carriers suggest the strategies for chemical modifications of PSs [103][104][105]. PSs bear three hydroxyl groups at the end of the ethylene oxide chains. These hydroxyl groups can be easily functionalized or transformed into various functional groups, including esters, ethers, carbonates, and carbamates. The possible chemical modifications of the PSs are summarized in Figure 4. These functional groups can be used to incorporate chemical drugs and proteins to increase the therapeutic effects and pharmacokinetics of PS-based formulations. Some bioresponsible linkers have also been utilized to achieve controlled drug release. Herein, we summarize recent progress regarding PS-conjugated drugs or drug carriers for biomedical applications. Photo-responsive microgels composed of polymeric β-cyclodextrin (Pβ-CD) and PS 20-coumarin conjugates (TCCs) have been developed [106]. TCC was prepared via covalent conjugation of coumarin to the hydroxyl groups of PS 20. In contrast, Pβ-CD was prepared by reacting epichlorohydrins with the hydroxyl groups of β-CDs. The addition of TCC to aqueous solutions of Pβ-CD led to the formation of microgels. The coumarin residue of TCC underwent dimerization and de-dimerization processes upon irradiation, which caused size variation in the particles. The size of the microgels was controlled by UV irradiation at different wavelengths. Irradiation with λ = 365 nm decreased the mi- Photo-responsive microgels composed of polymeric β-cyclodextrin (Pβ-CD) and PS 20-coumarin conjugates (TCCs) have been developed [106]. TCC was prepared via covalent conjugation of coumarin to the hydroxyl groups of PS 20. In contrast, Pβ-CD was prepared by reacting epichlorohydrins with the hydroxyl groups of β-CDs. The addition of TCC to aqueous solutions of Pβ-CD led to the formation of microgels. The coumarin residue of TCC underwent dimerization and de-dimerization processes upon irradiation, which caused size variation in the particles. The size of the microgels was controlled by UV irradiation at different wavelengths. Irradiation with λ = 365 nm decreased the microgel size, whereas the size of the microgels was increased in response to UV irradiation at 254 nm. This photo-responsive microgel can be used as a drug carrier, and the drug release could be controlled using UV irradiation [107].
Cinnamic acid (CA) was identified as a UV protecting molecule because it absorbs a broad range of UV light. CA was covalently attached to a nonionic PS to obtain a UV absorbing emulsifier [108]. After covalent conjugation with a PS, the nature of molar extinction coefficient of CA residue did not change. This result indicates that CA could absorb the UV light after conjugation to the PS. In vitro cytotoxicity of various surfactant-CA conjugates were evaluated, and the cell viability was higher than 80% at the concentration of 0.2% for all the conjugates tested [108,109].
Amphotericin B (AmB), a polyene antibiotic for the treatment of fungal and leishmanial infections, is insoluble in water and exhibits severe toxicity. PS 20 was activated by p-nitrophenyl chloroformate to form a stable carbonate intermediate, followed by the direct nucleophile-catalyzed reaction with mycosamine of AmB to form PS 20-AmB conjugates [110]. Chemical synthesis of PS 20-AmB conjugates is illustrated in Figure 5. PS 20-AmB conjugates demonstrated good water-solubility and negligible hemolytic potentials. The conjugates showed potent antifungal activity against C. albicans, C. parapsilosis, and C. neoformans. They also showed anti-leishmanial activity against promastigotes of L. donovani in vitro [110].
crogel size, whereas the size of the microgels was increased in response to UV irradiation at 254 nm. This photo-responsive microgel can be used as a drug carrier, and the drug release could be controlled using UV irradiation [107].
Cinnamic acid (CA) was identified as a UV protecting molecule because it absorbs a broad range of UV light. CA was covalently attached to a nonionic PS to obtain a UV absorbing emulsifier [108]. After covalent conjugation with a PS, the nature of molar extinction coefficient of CA residue did not change. This result indicates that CA could absorb the UV light after conjugation to the PS. In vitro cytotoxicity of various surfactant-CA conjugates were evaluated, and the cell viability was higher than 80% at the concentration of 0.2% for all the conjugates tested [108,109].
Amphotericin B (AmB), a polyene antibiotic for the treatment of fungal and leishmanial infections, is insoluble in water and exhibits severe toxicity. PS 20 was activated by p-nitrophenyl chloroformate to form a stable carbonate intermediate, followed by the direct nucleophile-catalyzed reaction with mycosamine of AmB to form PS 20-AmB conjugates [110]. Chemical synthesis of PS 20-AmB conjugates is illustrated in Figure 5. PS 20-AmB conjugates demonstrated good water-solubility and negligible hemolytic potentials. The conjugates showed potent antifungal activity against C. albicans, C. parapsilosis, and C. neoformans. They also showed anti-leishmanial activity against promastigotes of L. donovani in vitro [110]. Hydrogels can be used as drug delivery carriers owing to their high drug-loading capacity. PS 20-trimethacrylate was synthesized and used as a flexible crosslinker for photopolymerizable hydrogels. The aqueous solutions of PS 20-trimethacrylate were photo-polymerized within 30 min of exposure to UV light. Aqueous PS 20trimethacrylate was then coupled with N-vinyl-2-pyrrolidone to form hydrogels whose swelling ratios and swelling rates were tuned by varying the amount of PS 20trimethacrylate. These hydrogels showed potential for controlled drug release [111].
Hydrogel NPs containing poly(ethylenimine) (PEI)-graft-PS (PEIP) copolymers have been developed as drug carriers. A schematic of the preparation of PEIP and hydrogel formation is shown in Figure 6. The PEIP-based hydrogel NPs showed good stability and efficient cellular internalization owing to the stabilizing ability and drug-absorption-enhancing activity of PS. The cytotoxicity of PEIP was considerably lower than that of unmodified PEI in retinal ganglion cell 5 and human embryonic kid- Hydrogels can be used as drug delivery carriers owing to their high drug-loading capacity. PS 20-trimethacrylate was synthesized and used as a flexible crosslinker for photopolymerizable hydrogels. The aqueous solutions of PS 20-trimethacrylate were photo-polymerized within 30 min of exposure to UV light. Aqueous PS 20-trimethacrylate was then coupled with N-vinyl-2-pyrrolidone to form hydrogels whose swelling ratios and swelling rates were tuned by varying the amount of PS 20-trimethacrylate. These hydrogels showed potential for controlled drug release [111].
Hydrogel NPs containing poly(ethylenimine) (PEI)-graft-PS (PEIP) copolymers have been developed as drug carriers. A schematic of the preparation of PEIP and hydrogel formation is shown in Figure 6. The PEIP-based hydrogel NPs showed good stability and efficient cellular internalization owing to the stabilizing ability and drug-absorptionenhancing activity of PS. The cytotoxicity of PEIP was considerably lower than that of unmodified PEI in retinal ganglion cell 5 and human embryonic kidney HEK293 cells. The highly branched structure of PEIP enhances the sustainability of protein release by increasing the degree of chain entanglement. These results demonstrate that PEIP-based hydrogel NPs are promising drug carriers for protein delivery [112]. ney HEK293 cells. The highly branched structure of PEIP enhances the sustainability of protein release by increasing the degree of chain entanglement. These results demonstrate that PEIP-based hydrogel NPs are promising drug carriers for protein delivery [112].

PS-Coated NPs for Efficient BBB Transport
PS-coated NPs are known to efficiently deliver therapeutic molecules to the brain by crossing the BBB [113][114][115]. PS-coated NPs can interact strongly with the brain blood vessel endothelial cells. PS 80 enables the adsorption of apolipoproteins (e.g., apolipoprotein E (ApoE)) onto NPs [116], and the NPs interact with lipoprotein receptors on the luminal surface of the BBB, triggering the receptor-mediated transcytosis of the PS-coated NPs (Figure 7). Moreover, PS-coated particles reduce nonspecific uptake by the reticuloendothelial system [117].
In recent years, various PS-coated NPs have been developed to improve brain-specific delivery of drugs, such as tacrine [118], doxorubicin [119], hexapeptide dalargin [120], loperamide [121], and tubocurarine [122], by increasing LDL-mediated endocytosis [123]. Kreuter et al. extensively investigated PS-coated nanoparticle systems for the brain-targeted delivery of drugs [113][114][115]. Transport of dalargin across the BBB was facilitated using PS 80-coated poly(butyl cyanoacrylate) (PBCA) NPs. Intravenous injection of PS 80-coated PBCA NPs encapsulating dalargin in mice showed an analgesic effect [113]. PBCA NPs encapsulating the anti-nociceptive peptide dalargin were coated with apolipoproteins for efficient brain targeting [115]; additionally, the NPs were coated with PS 80. The formulations were administered intravenously into mice, and the mean percentage of the maximum possible effect (MPE) was calculated. It was shown that PS

PS-Coated NPs for Efficient BBB Transport
PS-coated NPs are known to efficiently deliver therapeutic molecules to the brain by crossing the BBB [113][114][115]. PS-coated NPs can interact strongly with the brain blood vessel endothelial cells. PS 80 enables the adsorption of apolipoproteins (e.g., apolipoprotein E (ApoE)) onto NPs [116], and the NPs interact with lipoprotein receptors on the luminal surface of the BBB, triggering the receptor-mediated transcytosis of the PS-coated NPs ( Figure 7). Moreover, PS-coated particles reduce nonspecific uptake by the reticuloendothelial system [117].
In recent years, various PS-coated NPs have been developed to improve brain-specific delivery of drugs, such as tacrine [118], doxorubicin [119], hexapeptide dalargin [120], loperamide [121], and tubocurarine [122], by increasing LDL-mediated endocytosis [123]. Kreuter et al. extensively investigated PS-coated nanoparticle systems for the brain-targeted delivery of drugs [113][114][115]. Transport of dalargin across the BBB was facilitated using PS 80-coated poly(butyl cyanoacrylate) (PBCA) NPs. Intravenous injection of PS 80-coated PBCA NPs encapsulating dalargin in mice showed an analgesic effect [113]. PBCA NPs encapsulating the anti-nociceptive peptide dalargin were coated with apolipoproteins for efficient brain targeting [115]; additionally, the NPs were coated with PS 80. The formulations were administered intravenously into mice, and the mean percentage of the maximum possible effect (MPE) was calculated. It was shown that PS 80 precoating achieved a significantly higher MPE than that in the control groups. PS 80-coated NPs adsorb apolipoproteins from the blood after injection, thus mimicking lipoprotein particles that can be efficiently internalized by the brain capillary endothelial cells via receptor-mediated endocytosis. Additionally, tail-flick test results using ApoE-deficient mice demonstrated that ApoE plays a critical role in the transport of NPs across the BBB.
These findings explain the mechanism of PS 80-coated drug carriers for brain-targeted delivery [115]. Recent progress in the use of PS-coated NPs for brain-targeted delivery is summarized in Table 1. 80 precoating achieved a significantly higher MPE than that in the control groups. PS 80-coated NPs adsorb apolipoproteins from the blood after injection, thus mimicking lipoprotein particles that can be efficiently internalized by the brain capillary endothelial cells via receptor-mediated endocytosis. Additionally, tail-flick test results using Ap-oE-deficient mice demonstrated that ApoE plays a critical role in the transport of NPs across the BBB. These findings explain the mechanism of PS 80-coated drug carriers for brain-targeted delivery [115]. Recent progress in the use of PS-coated NPs for brain-targeted delivery is summarized in Table 1. Several studies have demonstrated that PS 80-coated NPs can improve drug accumulation in the brain [124][125][126][127][128][129][130][131]. It has been reported that brain targeting of PS 80-coated NPs is closely associated with the interaction between PS 80 coating and brain microvessel endothelial cells [132]. Efficient penetration of the hexapeptide dalargin across the BBB was achieved using PS 80-coated PBCA NPs [124]. Intravenous administration of this formulation to mice exerted analgesic effects due to efficient phagocytic uptake by the brain blood vessel endothelial cells. This study demonstrated that PS-coated NPs facilitate the transport of large peptides that cannot penetrate the BBB in their native form. When dalargin bound to PS 80-coated NPs was intravenously administered to mice, a strong analgesic effect was observed; however, its administration with other surfactants, such as poloxamers 184, 188, 338, 407, poloxamine 908, cremophor EZ, cremophor RH 40, and Brij 35, showed negligible effects [125]. Rivastigmine-loaded PBCA NPs have been developed for the treatment of Alzheimer's disease [126]. Animal model results showed that drug uptake in the brain increased four-fold for PBCA NPs coated with 1% PS 80 compared to free drugs.  Several studies have demonstrated that PS 80-coated NPs can improve drug accumulation in the brain [124][125][126][127][128][129][130][131]. It has been reported that brain targeting of PS 80-coated NPs is closely associated with the interaction between PS 80 coating and brain microvessel endothelial cells [132]. Efficient penetration of the hexapeptide dalargin across the BBB was achieved using PS 80-coated PBCA NPs [124]. Intravenous administration of this formulation to mice exerted analgesic effects due to efficient phagocytic uptake by the brain blood vessel endothelial cells. This study demonstrated that PS-coated NPs facilitate the transport of large peptides that cannot penetrate the BBB in their native form. When dalargin bound to PS 80-coated NPs was intravenously administered to mice, a strong analgesic effect was observed; however, its administration with other surfactants, such as poloxamers 184, 188, 338, 407, poloxamine 908, cremophor EZ, cremophor RH 40, and Brij 35, showed negligible effects [125]. Rivastigmine-loaded PBCA NPs have been developed for the treatment of Alzheimer's disease [126]. Animal model results showed that drug uptake in the brain increased four-fold for PBCA NPs coated with 1% PS 80 compared to free drugs. PS 80-bound poly(lactic-co-glycolic acid) (PLGA) NPs were prepared to increase the localization of acetylpuerarin (AP) in the brain of mice [133]. AP-loaded PLGA NPs showed more efficient protective effects against cerebral ischemia-reperfusion injury in rats compared to the free drug. The antitumor effects of PS 80-coated PBCA NPs encapsulating GEM were demonstrated using a rat brain tumor model. The 1% PS 80-coated GEM-loaded PBCA NPs were able to cross the BBB, leading to necrosis. As a result, the NPs significantly inhibited the growth of C6 glioma cells in the rat brain tumor model. Furthermore, they significantly extended the survival time compared to the saline-treated control groups [134].
Gelperina et al. evaluated the toxicity of PS 80-coated NPs encapsulating doxorubicin (DOX) in glioblastoma-bearing rat models. PS 80-coated NPs did not show any changes in toxicity compared to free DOX or DOX-encapsulated NPs. The in vivo toxicity results showed similar effects in both healthy and tumor-bearing rats [135].
Ren et al. synthesized PS 80-coated AmB/PLA-b-PEG NPs for transport across the BBB. The BBB efficiency was significantly enhanced when NPs were coated with PS 80.
The drug concentration in mouse brain was greatly enhanced compared to non-coated NPs [136]. In vivo albino mice [141] PLGA siRNA Traumatic brain injury PS 80 In vivo C57BL/6J mice [142] Super paramagnetic iron oxide NPs (SPIONs) coated with polyethylene glycol (PEG), PEI, and PS 80 were synthesized for effective drug delivery to the brain [137]. A detailed illustration of the BBB transport of PS 80-coated SPIONs is presented in Figure 8. An in vivo study using rat models demonstrated that PS 80-coated SPIONs efficiently crossed normal BBB under an external magnetic field (EMF). The energy dispersive spectroscopy (EDS) results showed that SPIONs accumulated in the cortex near the magnet. Transmission electron microscope (TEM) images and elemental identification through EDS showed efficient accumulation of PS 80-coated SPIONs in the brain. This study demonstrates that both PS conjugation and EMF play crucial roles in the effective passage of SPIONs across the intact BBB [137].
Appl. Sci. 2021, 11, x FOR PEER REVIEW 13 of 24 magnet. Transmission electron microscope (TEM) images and elemental identification through EDS showed efficient accumulation of PS 80-coated SPIONs in the brain. This study demonstrates that both PS conjugation and EMF play crucial roles in the effective passage of SPIONs across the intact BBB [137]. PS 80-coated redox-sensitive micelles were fabricated using hydrophilic hyaluronic acid-conjugated curcumin through disulfide linkers [138]. The micelles were coated with PS 80 to achieve effective brain targeting. The PS 80-coated redox-sensitive micelles exhibited redox sensitivity in the presence of glutathione. CD44 is a cell surface hyaluronic acid-binding glycoprotein that is overexpressed in cancer cells. Therefore, cellular uptake of hyaluronic acid-curcumin conjugates encapsulated into PS 80-coated redox-sensitive micelles was higher than that of free curcumin in CD44 receptor-expressing G422 glioblastoma cells. The increased cellular internalization of PS 80-coated redox-sensitive micelles incorporating curcumin induced higher cytotoxicity against G422 cells than free curcumin [138]. The redox-sensitive micelles also exhibited high plasma stability and did not induce hemolysis in erythrocytes.
PS 80-coated PBCA NPs were developed for the delivery of brain-impermeable imaging agents into the brain. The results showed that PS 80-coated PBCA NPs delivered imaging agents into the brain of mice for in vivo imaging of neuronal and glial nuclei. Neuropathological changes in neurodegenerative diseases can be visualized using NPs. Notably, BBB-permeable NPs did not induce nonspecific disruption of the BBB [139].
PS 80-coated PLGA NPs loaded with methotrexate-transferrin conjugates were developed for efficient BBB migration [140]. Figure 9 shows the BBB crossing details in the pictorial view. The nanoformulation coated with PS-80 crossed the BBB through the endocytosis pathway and inhibited the P-gp efflux pump to avoid drug elimination [140]. PS 80-coated particles showed higher anticancer efficacy than free drugs or PS 80-free formulations. Alzheimer's disease-induced albino mice were treated with PS 80-coated PLGA NPs encapsulating thymoquinone [141]. These NPs enhanced the bioavailability of thymoquinone, reduced the immobility time (39.45 ± 3.32 s), and increased superoxide dismutase functioning (8.33 ± 2.61 units/mg) upon injection into mice (5 mg/kg concentrations). Thymoquinone reduced oxidative stress and thus restricted the accumulation of Aβ and hyperphosphorylated τ-protein tangles via the antioxidant cascade [141]. PS 80-coated redox-sensitive micelles were fabricated using hydrophilic hyaluronic acid-conjugated curcumin through disulfide linkers [138]. The micelles were coated with PS 80 to achieve effective brain targeting. The PS 80-coated redox-sensitive micelles exhibited redox sensitivity in the presence of glutathione. CD44 is a cell surface hyaluronic acid-binding glycoprotein that is overexpressed in cancer cells. Therefore, cellular uptake of hyaluronic acid-curcumin conjugates encapsulated into PS 80-coated redox-sensitive micelles was higher than that of free curcumin in CD44 receptor-expressing G422 glioblastoma cells. The increased cellular internalization of PS 80-coated redox-sensitive micelles incorporating curcumin induced higher cytotoxicity against G422 cells than free curcumin [138]. The redox-sensitive micelles also exhibited high plasma stability and did not induce hemolysis in erythrocytes.
PS 80-coated PBCA NPs were developed for the delivery of brain-impermeable imaging agents into the brain. The results showed that PS 80-coated PBCA NPs delivered imaging agents into the brain of mice for in vivo imaging of neuronal and glial nuclei. Neuropathological changes in neurodegenerative diseases can be visualized using NPs. Notably, BBB-permeable NPs did not induce nonspecific disruption of the BBB [139].
PS 80-coated PLGA NPs loaded with methotrexate-transferrin conjugates were developed for efficient BBB migration [140]. Figure 9 shows the BBB crossing details in the pictorial view. The nanoformulation coated with PS-80 crossed the BBB through the endocytosis pathway and inhibited the P-gp efflux pump to avoid drug elimination [140]. PS 80-coated particles showed higher anticancer efficacy than free drugs or PS 80-free formulations. Alzheimer's disease-induced albino mice were treated with PS 80-coated PLGA NPs encapsulating thymoquinone [141]. These NPs enhanced the bioavailability of thymoquinone, reduced the immobility time (39.45 ± 3.32 s), and increased superoxide dismutase functioning (8.33 ± 2.61 units/mg) upon injection into mice (5 mg/kg concentrations). Thymoquinone reduced oxidative stress and thus restricted the accumulation of Aβ and hyperphosphorylated τ-protein tangles via the antioxidant cascade [141].
PS 80-coated PLGA NPs were developed for the efficient transport of small interfering RNA (siRNA) across the BBB in traumatic brain injury (TBI) [142] (Figure 10). siRNA-based therapeutics can mitigate disease progression in TBI treatment, but siRNA suffers from poor BBB transport. The surface of siRNA-loaded PLGA NPs was coated with five different coating materials (e.g., PS 80, Pluronic F-68, DSPE-PEG, DSPE-PEG-glutathione (GSH), DSPE-PEG-transferrin (Tf)) to identify a surface coating material and coating density that can maximize the penetration of siRNA-loaded NPs across intact BBB. Among the NPs with different coatings materials, PS-coated NPs showed the most efficient in vitro gene silencing efficiency and BBB penetration. As a result, tau siRNA-loaded PS 80-coated PLGA NPs significantly suppressed tau expression in cultured primary neural cells. Additionally, they achieved efficient tau silencing in TBI mice with no systemic toxicity when administered during the early or late injury period [142].
PS 80-coated PLGA NPs were developed for the efficient transport of small interfering RNA (siRNA) across the BBB in traumatic brain injury (TBI) [142] (Figure 10). siRNA-based therapeutics can mitigate disease progression in TBI treatment, but siRNA suffers from poor BBB transport. The surface of siRNA-loaded PLGA NPs was coated with five different coating materials (e.g., PS 80, Pluronic F-68, DSPE-PEG, DSPE-PEG-glutathione (GSH), DSPE-PEG-transferrin (Tf)) to identify a surface coating material and coating density that can maximize the penetration of siRNA-loaded NPs across intact BBB. Among the NPs with different coatings materials, PS-coated NPs showed the most efficient in vitro gene silencing efficiency and BBB penetration. As a result, tau siRNA-loaded PS 80-coated PLGA NPs significantly suppressed tau expression in cultured primary neural cells. Additionally, they achieved efficient tau silencing in TBI mice with no systemic toxicity when administered during the early or late injury period [142]. ) and different coating densities were prepared. The gene silencing efficiency and BBB permeability of siRNA-loaded PLGA NPs with various surface coatings were assessed in TBI mice when administered during early injury or late injury periods, corresponding to physically breached BBB and intact BBB, respectively. Upon neuronal uptake of NPs, siRNA is released and silences the harmful proteins involved in TBI pathophysiology. Reproduced with permission from Reference [142].

Anticancer Therapy Using PS-Based Formulations
Cisplatin is widely used for treating bladder cancer [143,144]. Because of its high toxicity, direct therapeutic usage is avoided. Cisplatin formulation developed with nonionic surfactants for i.v. administration. The nanoemulsions showed sustained release of the drugs and better activity against bladder cancer cells.
GEM, a chemotherapeutic agent, has limitations to in vivo cancer therapy owing to Figure 10. Schematic illustrating the design and mechanism of BBB pathophysiology-independent delivery of siRNA in TBI using siRNA-loaded PLGA NPs. Various siRNA-loaded PLGA NPs with five different surface coating materials (e.g., PS 80, Pluronic F-68, DSPE-PEG, DSPE-PE G-GSH, and DSPE-PEG-transferrin (DSPE-PEG-Tf)) and different coating densities were prepared. The gene silencing efficiency and BBB permeability of siRNA-loaded PLGA NPs with various surface coatings were assessed in TBI mice when administered during early injury or late injury periods, corresponding to physically breached BBB and intact BBB, respectively. Upon neuronal uptake of NPs, siRNA is released and silences the harmful proteins involved in TBI pathophysiology. Reproduced with permission from Reference [142].

Anticancer Therapy Using PS-Based Formulations
Cisplatin is widely used for treating bladder cancer [143,144]. Because of its high toxicity, direct therapeutic usage is avoided. Cisplatin formulation developed with nonionic surfactants for i.v. administration. The nanoemulsions showed sustained release of the drugs and better activity against bladder cancer cells.
GEM, a chemotherapeutic agent, has limitations to in vivo cancer therapy owing to its hydrophilic nature. The mixture of Tween 80, Span 80, 0.9 % sodium chloride solution was used to prepare GEM-loaded nanoemulsions [145]. The results showed that the release of GEM at pH 6.5 (45.19%) was higher than that at pH 7.4 (13.62%). The cytotoxicity study showed that the optimized nanoemulsion containing GEM induced cytotoxicity towards A549 cells compared to a control group (i.e., GEM solution). Docetaxel (DTX) is a widely used anticancer drug for the treatment of several types of cancers, including metastatic breast cancer, metastatic non-small cell lung cancer, hormonerefractory prostate cancer, gastric adenocarcinoma, and squamous cell carcinoma of the head and neck [146]. DTX-encapsulated, rod-like micelles were fabricated using PS 80 and phospholipids. The size of micelles was 13 nm with narrow size distribution. Pharmacokinetics results suggested that the micelles increased the residence amount of DTX in kidney, spleen, ovary, uterus, heart, and liver. The hemolytic potentials study revealed that the mixed micelles were safe for intravenous injection [147].
PS-based nanoemulsions were developed for efficient intracellular delivery of photosensitizers [148]. Indocyanine green (ICG) is a near-infrared light-absorbing dye approved by the FDA [149]. ICG has been employed as an NIR fluorescence-imaging tracer and photosensitizer. ICG was loaded into nanoemulsions composed of cationic lipid stearylamine (SA) and surfactant mixtures of PS 80 and Span 85. Loading ICG into SA-incorporated nanoemulsions more effectively reduced the aggregation and degradation of ICG compared to loading in SA-free nanoemulsions. SA incorporation also enhanced tumor cell uptake of ICG-loaded nanoemulsions due to positive charges of the SA-incorporated nanoemulsions. As a result, compared to SA-free nanoemulsions encapsulating ICG, SA-incorporated nanoemulsions encapsulating ICG exhibited higher phototoxicity upon NIR light irradiation. After subcutaneous injection into the footpad of mice, SA-incorporated nanoemulsions exhibited higher concentrations of ICG in popliteal lymph nodes compared to SA-free nanoemulsions [148].
PS 80-based micelles were prepared for combination chemo/photothermal/photodynamic cancer therapy via effective intracellular delivery of piperlongumine (PL) and ICG [150] ( Figure 11). PL was used as a cancer-specific anticancer agent by inducing selective oxidative stress in cancer cells. ICG was utilized as an NIR light-absorbing photothermal and photodynamic agent. PS 80-based micelles demonstrated successful dual drug encapsulation. The micelle formulations enhanced the cellular uptake, photothermal effects, and photodynamic effects of ICG. ICG-loaded PS 80-based micelles demonstrated NIR light-triggered photothermal and photodynamic effects. ICG-and PL-loaded PS 80-based micelles exhibited cancer-specific cytotoxicity because PL selectively induces oxidative stress in cancer cells. This study confirmed that PL-ICG-T80 micelles are an effective platform to achieve efficient, cancer-targeted chemo-photodynamic combination therapy [150]. demonstrated successful dual drug encapsulation. The micelle formulations enhanced the cellular uptake, photothermal effects, and photodynamic effects of ICG. ICG-loaded PS 80-based micelles demonstrated NIR light-triggered photothermal and photodynamic effects. ICG-and PL-loaded PS 80-based micelles exhibited cancer-specific cytotoxicity because PL selectively induces oxidative stress in cancer cells. This study confirmed that PL-ICG-T80 micelles are an effective platform to achieve efficient, cancer-targeted chemo-photodynamic combination therapy [150].

Conclusions and Perspectives
PSs are considered important excipients in pharmaceutical formulations. Several studies and reviews have reported the multimodal use of PSs as solubilizers, emulsifiers, stabilizers, microemulsions, nanosomes, self-assembled micelles, and drug carriers for pharmaceutical applications. In this review, we summarized recent progress in PS-based drug formulations and drug delivery systems. We also summarized various PS-based drug formulations in terms of different routes of administration, such as ocular, transdermal, oral, and nasal delivery. PS was found to be safe and effective for all the administration routes. PSs are used as emulsifiers and solubilizers for hydrophobic drugs and proteins. PS-based drug formulations improve the solubility, stability, and pharmacokinetic properties of drugs, as well as control drug release and drug dissolution rates. In addition, PS can inhibit the P-gp efflux transporter, resulting in increased drug accumulation. Notably, PS-coated drug carriers can cross the BBB via receptor-mediated Figure 11. PL-and ICG-loaded PS 80-based NPs for combination chemo/photothermal/photodynamic therapy. Reproduced with permission from Reference [150]. Copyright 2020, Elsevier B.V.

Conclusions and Perspectives
PSs are considered important excipients in pharmaceutical formulations. Several studies and reviews have reported the multimodal use of PSs as solubilizers, emulsifiers, stabilizers, microemulsions, nanosomes, self-assembled micelles, and drug carriers for pharmaceutical applications. In this review, we summarized recent progress in PS-based drug formulations and drug delivery systems. We also summarized various PS-based drug formulations in terms of different routes of administration, such as ocular, transdermal, oral, and nasal delivery. PS was found to be safe and effective for all the administration routes. PSs are used as emulsifiers and solubilizers for hydrophobic drugs and proteins. PSbased drug formulations improve the solubility, stability, and pharmacokinetic properties of drugs, as well as control drug release and drug dissolution rates. In addition, PS can inhibit the P-gp efflux transporter, resulting in increased drug accumulation. Notably, PS-coated drug carriers can cross the BBB via receptor-mediated transcytosis, resulting in brain-targeted drug delivery. The biocompatible nature of PSs is an additional advantage for drug delivery systems.
PSs have been widely used in formulations and have demonstrated high therapeutic performance in preclinical studies [3]. In addition to the emulsifying and stabilizing properties, the advantages of using PSs as drug formulation agents are low cost, commercial availability, and ease of handling. Additionally, PSs are relatively low in toxicity, and their characteristic features can be achieved at low concentrations. However, complex mixtures of PSs, lack of spectral/quantitative characterization, and chemical/enzymatic degradation have hampered the use of PSs as active drug formulations. Commercial PSs are heterogeneous mixtures of structurally related compounds. PSs are available in three different grades according to their purities, including multi-compendial, super-refined, and ultrapure grades. The multi-compendial-grade PS 80 is a mixture of various fatty acid esters, with ≥58% oleic acid content. In contrast, super-refined and ultrapure PS grades are esterified with at least 98% of their corresponding fatty acids. Ultrapure PS 80 is a highly purified PS that was prepared to meet the standards of Chinese pharmacopeia for human use [151,152]. Commercial PS 80 contains impurities such as 12-tricosanone, which leads to the occurrence of visible 12-tricosanone particles in drug formulations and decreases the activity of drugs [153]. The impurities of PSs can affect their performance in drug formulations. However, the challenges in characterization of PS heterogeneity have aroused significant interest in the development of new analytical methods to characterize PS heterogeneity. A recent study addressed the limitations in the characterization of PS heterogeneity using high-resolution mass spectrometry (HRMS) [154]. The molecular heterogeneity of PS 80 was analyzed using HRMS coupled with hydrogen/deuterium (H/D) exchange in deuterated methanol. The terminal hydroxyl groups of PSs have labile protons that can undergo H/D exchange in deuterated solvents. The resultant mass shifts indicate structural differences of isomers. Mass variation was identified to predict the structure and heterogeneity of PSs. This method was successfully applied to profile PS 80 samples from different suppliers or of different purity grades [154].
One of the major concerns involved in PS use is the degradation of PSs. The two main routes of PS degradation are oxidation and hydrolysis [155]. Degradation of PSs leads to protein aggregation, particle formation, lowered efficacy of drugs, and immune responses. Therefore, formulators need to characterize and understand PS degradation because impurities and degradation products strongly influence the physicochemical properties and therapeutic efficacies of PS-based drug formulations. Reversed-phase ultrahigh-performance liquid chromatography was also used to collect and characterize the ester fractions of PSs such as sorbitan-polyoxyethylene (POE)-monoester, isosorbide-POEmonoester, and sorbitan-POE-diester [155]. Various physicochemical properties of the ester fractions of PSs, including surface tension, micelle size, critical micelle concentration, and agitation protection for a monoclonal antibody, have been evaluated [155]. This method can provide information regarding the pattern of PS degradation, thus enabling formulators to better understand the risk of particle formation caused by degradation.
The purity of PS plays a crucial role in the physicochemical properties of PS-based drug formulations. Therefore, new synthesis strategies to minimize impurities and impurityinduced PS degradation should be exploited. The bulk synthesis of PSs should be monitored carefully using recently developed characterization techniques such as HRMS. The generation of byproducts from PS synthesis should be minimized by using suitable purification methods. Precise and less time/cost-consuming characterization techniques should be investigated to easily identify impurities and degradation products of PSs. To avoid PS degradation, the temperature, types of buffers, and types of coexcipients should be carefully considered.
Owing to the amphiphilic nature, PSs have been used as solubilizers of hydrophobic drugs, proteins, and inorganic nanoparticles. Therefore, PS-based drug carriers are able to carry multiple drugs for combination therapy. By incorporating inorganic nanoparticles as molecular imaging probes, PS-based drug carriers can be used as theranostic nanoplatforms for combined imaging and therapy. Particularly, PS-drug conjugates and PS-coated drug carriers have great potential to treat central nervous system-related diseases because PS coatings can improve the crossing of the BBB. In addition to the development of PS-based drug formulations, biosafety evaluations, such as systemic clearance, must be performed prior to their clinical use. These efforts will facilitate the successful translation of PS-based drug formulations into clinical settings.