Erythromycin (ERY) is a therapeutical compound belonging to the class of macrolide antibiotics, originally discovered by McGuire et al. in 1952^[1] and later produced through biosynthesis during fermentation from species of the Gram-positive Saccharopolyspora erythraea, formerly classified as Streptomyces erythraeus^[2][3]. The erythromycin group generated by bacterial strains encompasses numerous structural variations, that is, ERY A, B, C, D, E, F and G (Figure 1).

Out of the variety of ERY compounds, ERY A (ilotycin) is the primary constituent while ERY E and F are its metabolites^[4]. The other forms are intermediates generated during the biosynthesis of ERY A^[5].

ERY is a wide spectrum antibiotic, possessing significant therapeutic value both internally and externally. In the first case, it is administered with regard to the treatment of infectious diseases caused by mostly Gram-positive, but also Gram-negative^[6] pathogens, such as Corynebacterium diphteriae, Staphylococcus aureus (S. aureus), Streptococcus spp., Bordetella pertussis, Chlamydia trachomatis, Mycoplasma pneumoniae, Legionella pneumophila, Neisseria, Campylobacter fetus jejuni, and Bartonella henselae ^{[7][8][9][10][11][12][13]}. When administrated externally, it is used for the treatment of acne vulgaris caused by Propionibacterium acnes (P. acnes), being the most widely used antibiotic for this skin disease, due to its ability to inhibit the expression of lipase in P. acnes at concentrations below minimum inhibitory concentration (MIC) values^[14].

Furthermore, ERY has recently proven itself useful for other types of treatment, due to its anti-inflammatory effects and ability to inhibit the formation of osteoclasts^[15], being commonly used in bone cement for preventing infections^[16]. Considering its role as a motilin receptor agonist, it is also used to treat patients necessitating the acceleration of gastric emptying^[17][18]. It has a wide body distribution, with preponderant accumulation in the lungs; therefore, many lung-targeted formulations are being studied, especially since ERY has proven to be effective for the treatment of chronic obstructive pulmonary disease^[19].

However, ERY therapy has a series of shortcomings, such as poor aqueous solubility due to its hydrophobic nature^[20], instability in acidic medium (the majority of the dose is degraded at the gastric level), its relatively short half-life of about 1–1.5 h, and low bioavailability. It has been documented that ERY produces substantial gastrointestinal side effects, as well as liver toxicity, due to its instability and chemical conversion under acidic conditions^[21]. Elderly patients are often unable to tolerate the gut-motility activity of the drug, whereas children find the taste unacceptable^[22]. Another crucial drawback is related to its widespread distribution, which causes drug accumulation at other sites than target ones, provoking a series of undesirable adverse effects^[23]. The latter-mentioned limitation was one of the driving forces in recent development of novel targeted ERY formulations.

Considering that the development of new antibiotics has reached a plateau, another issue which makes the design of improved ERY formulations a current priority is the rapidly increase antibiotic resistance. Therefore, lowering the effective therapeutic dosages by amplifying the antibiotic’s potency has become a necessity.

2. Vesicles

The literature reveals a large variety of vesicular systems used to deliver drugs, such as lipid particles, liposomes, niosomes, sphingosomes, pharmacosomes, transferosomes, polymeric micelles, etc.^[24]. Generally speaking, vesicular carriers are formulated with lipid materials for the purpose of overcoming challenges of drug delivery and improving therapeutic outcomes. Encapsulation of both hydrophilic and lipophilic drugs, enhancement of bioavailability, increasing the drug’s circulation time, resolving toxicity issues and lowering therapeutic dosages are some of the major benefits of lipid-based drug delivery. In the following, ERY vesicle formulations developed over a long time; their preparation and performances will be presented.

2.1. Liposomes

Liposomes are sphere-shaped vesicles of small diameter, ranging from a few nanometres to several micrometres, consisting of aqueous compartments surrounded by one or more lipid bilayers (Figure 2)^[25]. The lipid layer is usually formed by a natural or synthetic phospholipid, but other lipids, such as cholesterol, can be incorporated in order to improve a specific property (e.g., the stability in media of different pH values of biological fluids), but also for reducing the fluidity of the bilayer membrane^[26][27][28]. Reported first by the Bangham group in 1965^[29][30], the liposomes were developed as useful vehicles for protection, delivery and specific targeting of various drugs. They can encapsulate either hydrophilic medicinal substances in the aqueous compartments or hydrophobic ones in the lipid bilayers, increasing their bioavailability and circulation time in the bloodstream, therefore preventing their premature disruption by blood components. Furthermore, the lipid layer confers biocompatibility and biodegradability to the encapsulated compounds. Their potential as drug delivery systems has been demonstrated through their successful clinical applications and many preclinical trials^[31][32]. Nevertheless, the investigation of ERY liposomes appears to still be in its infancy. Liposomal ERY was first reported in 1991, within an exhaustive study which focused on the influence of the phospholipid type over the encapsulation efficiency (EE). Using the film dehydration/rehydration vesicle (DRV) method for preparation (consisting in the rehydration of the lipid/ERY film in phosphate-buffered saline (PBS) and sepharose CL-4B column chromatography for purification), the researcheuthors obtained liposomes with diameters between 250–350 nm^[33]. They established that phosphatidylglycerol (PG) has a superior ability to retain ERY compared to phosphatidylcholine (PC) or phosphatidylethanolamine (PE). This was attributed to the least bulky head group of PG, therefore creating smaller steric barriers for ERY diffusion into the hydrophobic layers. When the phospholipid was composed of mixed fatty acids (saturated and unsaturated ones), the ability to retain ERY significantly increased. This was justified by the capacity of unsaturated fatty acids to improve the fluidity of the hydrophobic part of the phospholipid, making it more favourable for encapsulation of hydrophobic ERY (Figure 3). It was also discovered that the number of hydroxyl groups in the polar head of the phospholipid did not play a critical role in ERY encapsulation. The Omri group proposed a modified protocol of the DRV method for ERY liposomal preparation, named “active loading technique”, consisting of loading the drug into blank vesicles, which were previously prepared via dehydration–rehydration (Scheme 1Scheme 2)^[34]. Using a fatty acid-based phospholipid (1,2-dipalmitoyl-sn-glycero-3phosphocholine) combined with cholesterol (2/1, w/w), they prepared blank liposomes by rehydration of the thin lipid film in water/sucrose (1/1, w/w, sucrose to lipid) followed by sonication, in order to obtain small unilamelar vesicles. Subsequently, liposomes with ERY were prepared by loading the blank liposomes with the antibiotic, washing with PBS and lyophilization. Selection of the phospholipid was justified not only by its higher ability to retain the antibiotic, but also by its ability to improve the liposome’s stability in PBS compared to other phospholipids, such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine or distearoylphosphatidylcholine^[35]. Sucrose was used in order to prevent liposomes deterioration during lyophilization, choosing an optimal concentration to avoid reduction of the encapsulation rate. It appears that this protocol significantly improved the encapsulation efficiency to 32%, for liposomes with a diameter of 194 nm and excellent stability. ERY remained entrapped 100% at 4 °C in PBS, 80% at 37 °C in PBS and 50% in plasma. The researcheuthors remarked that sonication of the blank liposomes favoured the yielding of smaller loaded liposomes. They attributed this achievement to the (i) use of sucrose which prevented vesicle collision and to the (ii) lyophilization process which apparently eliminates large vesicles. It was assumed that sucrose interacts with the polar heads of the phospholipids, being a better liposome stabilizer than monosaccharides and polysaccharides. Moreover, there is proof that sucrose favoured a higher entrapment efficiency, especially when it was added during both lipid film hydration and hydration of the lyophilized liposomes^[36]. Nevertheless, decreasing the amount of cholesterol (1,2-dipalmitoyl-sn-glycero-3phosphocholine/cholesterol, molar ratio 6/1) and using the DRV method, the same group reported the preparation of liposomal ERY with a significant improvement in EE (~51%), but with a dramatical increase in liposomes size (~3 μm)^[37]. The increase in EE values was attributed to the direct dissolution of the lipophilic ERY in the organic lipid solution combined with a prolonged sonication time. No explanation for attaining such a high size for liposomes was given. However, one reason for this could be pinned down to the interactions between cholesterol and the lipid bilayer, inducing condensation effects^[38]. Compared to free ERY, the liposomal formulation showed a significant enhancement in the inhibitory and bactericidal activities against two Pseudomonas aeruginosa (P. aeruginosa) strains, with a MIC of four and eight-fold higher, which further improved when a broad-spectrum efflux pump inhibitor (phenylalanine arginine beta-naphthylamide—PABN) was added. Remarkably, the liposomal formulations showed a notable increase in the eradication of biofilms as well, indicating potential for the management of P. aeruginosa infections in cystic fibrosis patients. Recent investigations revealed the multi-inlet vortex mixer technology as a suitable method to reach liposomal ERY with sizes lower than 200 nm and an encapsulation efficiency around 35%. It was reported that this method allowed for a good control on liposome size and EE by simply tuning the aqueous/organic flow rate ratio and flow parameters^[39]. Controlling the permeability of liposome membranes is an important aspect for modulating drug release in a precise manner. Starting from the premise that nonionic liposomes facilitate the penetration of drugs through the skin barrier more efficiently than phospholipidic liposomes, liposomal ERY was prepared using a mixture of non-ionic lipids: cholesterol (CH), glyceryl dilaurate (GDL), and polyoxyethylene-10-stearyl ether (POE)^[41]. The preparation method involved the dissolution of ERY in a mixture of melted lipids (GDL:CH:POE, 57:15:28. w/w/w) followed by rehydration in isotonic HEPES buffer. Unfortunately, no information about the size, stability and EE of the resulting liposomes was provided, and the effectiveness of this preparation method could not be evaluated. They were more effective for ERY uptake into living skin compared to those based on phospholipids, due to the ability of GDL and POE to function as permeation enhancers, suggesting the nonionic liposomes as more suitable for topical formulations. With the purpose of being utilized in the sphere of pisciculture, liposomal ERY was prepared via a slightly modified DRV method, using a combination of lipids (phosphatidyl choline/cholesterol/phosphatidyl glycerol, 10/4/1, w/w/w)^[42]. The liposomes had micrometric size and high polydispersity degree, their diameters covering values in the range of 1–40 μm, with a prevalence of 2–3 μm sized liposomes. They were later bioencapsulated into brine shrimp Artemia franciscana with the intent of targeting Pacific salmon kidney disease of bacterial origin, creating a more palatable live feed. Even though no direct information was provided regarding the EE, the low ERY concentration in artemia indicated a low drug encapsulation within the liposomes. A recent rpapesearchr reported the preparation of liposomal ERY by an active loading technique method, followed by coating with chitosan^[43]. The resulted liposomes showed a hydrodynamic diameter of 311 nm and an EE of 55%. It appeared that a dramatic increase in Zeta potential was induced by chitosan coating (37 vs. 0.16 mV), suggesting the stabilization of the liposomes. The investigation of their biocompatibility, performed on mice, showed no toxic effects subsequent to oral administration. The modern trend in drug delivery is directed towards the development of smart targeted drug release in response to various stimuli, such as pH, temperature, light, ultrasound, magnetic field and specific receptors from disease sites^[44]. This can ensure the liposomes delivery to the targeted areas of disease, avoiding large drug dosages^[45]. Being intended for the treatment of acne, ERY-loaded liposomes decorated with a photosensitizer were designed in order to ensure a chemically controlled release under the influence of P. acnes lipases, as an environmental stimulus. Therefore, a pheophorbide photosensitizer was linked with pullulan via an ester linkage (Pu-Pheo) while 1,2 dipalmitoyl-sn-phosphoatidylcholine was chosen as a constituting lipid, knowing that lipase specifically breaks their ester bonds^[46]. The liposomes were prepared by DRV method, using erythromycin/1,2 dipalmitoyl-sn-phosphoatidylcholine/cholesterol (1/10/1.5, w/w/w). Preparation steps included rehydration in ammonium sulfate followed by decoration with PU-Pheo, and finally by lyophilization using sucrose lipoprotectant. The PU-Pheo-decorated liposomes had a quite small average hydrodynamic diameter of 93.6±3.1 nm. The in vitro release tests, carried out by dialysis in PBST (0.05 Tween 20 in PBS), showed a sustained release, achieving a 90% ERY cumulative release in 48 h, compared to free ERY which was released entirely within 12 h. The transdermal release evaluated via a Franz cell system using mouse skin demonstrated that ERY was preferentially delivered into infected inflammatory skin dermis, confirming the selective disruption of the liposomes in P. acnes-rich areas. Even more, the PU-Pheo-decorated liposomes proved a higher antibacterial effect in vitro under laser irradiation, indicating an additional antibacterial effect induced by the photosensitizer. These results were confirmed by in vivo tests on mice, which revealed that the combined treatment with both liposomes and laser irradiation decreased the number of residual P. acnes bacteria to an undetectable level, whereas each individual therapy was less effective. Importantly, no toxic effects were detected. These findings suggested that the combined therapy is a valuable route to achieve a reduction of the minimum therapeutic dose, which is promising for preventing the phenomenon of antibiotic resistance. The same researcheuthors reported other similar studies focused on designing liposomes bearing a polycationic photosensitizer, polyethyleneimine-hematoporphyrin (PEI-HP), which was chosen due to its potential to interact with negatively charged sites at the bacterial surface^[47]. The liposomes were prepared by an analogous procedure, with slight modifications, aside from the fact that the photosensitizer was mixed with the lipids and ERY in order to form the lipidic film. It was noted that this procedure led to liposomes with a higher average diameter, ~200 nm, a loading efficiency of ~85% and a loading content of ~7%. It was assumed that the high dimension of PEI determined the increase in liposome size, while its polycationic character improved the stability (Zeta potential approximately +52 mV). Following similar in vitro and in vivo drug release investigations, the researcheuthors proved that the ERY liposomes containing PEI-HP were effective in suppressing P. acnes by a dual phototherapeutic mechanism. Another stimulus which was examined for its potential to achieve a targeted release of ERY from liposomes was the magnetic field, considering that the mechanism of magnetic triggering is one of the most potent strategies for designing targeted release formulations^[48]. Hence, magneto-liposomes containing ERY were proposed as a combined strategy for controlled drug release, based on the premise that the electromagnetic field improves the permeability of the phospholipid membrane^[49]. Magneto liposomes were prepared using iron oxide nanoparticles which are FDA approved for medical applications^[50][51]. As for their preparation, the selected method was that of solvent injection, which involved dissolving the soybean phosphatidyl choline, ceteareth-25 edge activator, and ERY in ethanol at 30 °C, followed by the slow addition of an aqueous suspension of magnetic nanoparticles (Fe₃O₄) under continuous magnetic stirring and sonication^[49]. The resulted magneto-liposomes had a mean hydrodynamic diameter of 473±10 nm, which was significantly higher compared to that of the corresponding unmodified liposomal ERY (254 ± 10 nm). However, no difference was noted for the Zeta potential values, which were ~47 mV in both cases, indicating no influence of the magnetic nanoparticles over the liposome’s stability. Moreover, the encapsulation efficiency appeared to be favoured by the magnetic nanoparticles, from 87.9 ± 2.2% for the corresponding unmodified liposomal ERY to 97.8 ± 1.5% for magneto-liposomal ERY. In vitro drug release experiments demonstrated that the ERY release was induced at a low-intensity magnetic field, and the release rate was controlled by varying its intensity. The researcheuthors hypothesized that the integration of iron-based nanoparticles into the lipid layer produced an overall stiffening and decrease in homogeneity. The application of a magnetic field caused rotations and vibrations of the iron nanoparticles, generating mechanical forces, which induced deformations of the phospholipid membrane, altering its integrity. Amongst the macrolide liposomes, ERY ones appear to have the best chances for application. This was indicated by some computational investigation of the impact of macrolide liposomes on living organisms, which suggested that phospholipids can interact with cellular components, inhibiting enzyme activity and consequently leading to the lipid accumulation in cells, raising potential concerns for toxicity. Among the studied macrolides, ERY showed the lowest negative impact^[52].

2.2. Ethosomes

To improve drug delivery performance of liposomes, soft vesicles, mainly containing molecules with the capacity to induce more fluidity into the phospholipid bilayers, were proposed. When ethanol (20–50%) is entrapped into liposomes, the soft vesicles are called ethosomes^[53]. Compared to liposomes, which remain confined to the upper stratum corneum of the skin, ethosomes exhibit a higher potential to transport active therapeutic agents into deeper skin layers, due to the permeation enhancing capacity of ethanol^[54][55]. It was stated that ethanol can fluidize both the vesicles and the stratum corneum lipids, making the skin more penetrable, while the vesicles become more flexible. ERY ethosomes were prepared in a well-sealed container by dissolving phospholipon 90 and ERY in ethanol, followed by the slow addition of distilled water under constant stirring at 30 °C^[56]. They displayed a spherical unilamelar configuration with low size (116 and 123 nm), EE of ~78%, improved bilayer fluidity and excellent shelf-life, their size and configuration being almost unaffected after one year of storage under ambiental conditions (Figure 4). The investigation of the antibacterial properties against S. aureus and B. subtills showed improved activity when compared to standard hydroethanolic solutions, with remarkable reduction of the MIC of 2.5 times, against a clinically isolated S. aureus strain with known resistance to ERY. These results indicated an improvement of ERY delivery from the ethosomes to the bacteria, which was attributed to an enhanced permeability of microorganisms to ethosomal ERY, suggesting the necessity of administrating lower doses of the drug. Moreover, this researchtudy suggested that ethosomes are a good approach to overcome bacterial resistance to antibiotics. The ethosomes were not toxic to dermal fibroblasts and did not show any skin irritation. In vivo experiments on mice intradermally inoculated with live S. aureus revealed that the topical administration of ethosomal formulation (ethosomal ERY dispersed into a Carbopol gel) was as effective as systemically administrated ERY, resulting in efficient infection inhibition and healing S. aureus-induced deep dermal^[57]. Moreover, the histological investigations revealed no bacterial growth and a normal skin structure, whilst topical application of a hydroethanolic ERY revealed no subdermal healing and necrosis destroying the skin structure. All data indicated that the ethosomes are efficient carriers for ERY delivery into the deep skin strata for the eradication of staphylococcal infections.

2.3. Niosomes

Niosomes are slightly modified version of liposomes, in which the lipid is replaced by non-ionic surfactants (Figure 5)^[58]. They are biodegradable, relatively nontoxic, and more stable, yet less expensive compared to liposomes. Drug-loaded niosomes show interactions with the epidermal tissue without presenting an immediate or strong systemic action, thus being preferred for dermal applications. An exhaustive study was presented on the optimization of niosomal ERY preparation by the thin film hydration technique, focusing on the surfactant type, cholesterol/surfactant ratio, volume and time of hydration, stirring speed, vacuum intensity, temperature, annealing time, solvent system and solvent volume^[59]. The optimized niosomes (ERY/Cholesterol/Span 80 molar ratio of 1/1/2) reached a drug entrapment of ~90% and the smallest size of 4.5 µm. Their suspension presented good stability at 4 °C, with a drug leakage of 35% after 12 weeks, but low stability at room temperature with a drug leakage of 83% over a similar period. This was justified by the higher fluidity of the lipid bilayer at higher temperatures. Varying the previously mentioned parameters in a broader range, the same researcheuthors obtained niosomes with similar drug entrapment (88%) for a different composition (ERY/Span80/cholesterol molar ratio of 0.625/1.5/1), highlighting the high impact of preparation conditions^[60]. Applying a slightly different procedure, another group prepared niosomal ERY (Span60/Cholesterol in a mass ratio 70/30) and used them in clinical trials for the treatment of mild to moderate acne vulgaris.^[61] The trial showed the superior performance of the niosomal ERY (4%) in comparison to a control (ERY (4%) and zinc acetate (1.2%)), with more than 75% reduction in the number of lesions observed for 26.6% patients (Figure 6) and much less severe side effects than in the control group.

2.4. Micelles

Micelles are obtained by similar preparation methods and composition as niosomes, with the difference that cholesterol is absent (Figure 7). Liu et al. developed a micelle formulation encapsulating ERY with the use of Pluronic F-127 nonionic surfactant^[62]. The obtained micelles had an EE of 28.3%, and they displayed a spherical morphology with an approximate diameter of 193 nm. In vitro release studies concluded that over 90% of the drug was released over 8 h, but the antibacterial activity tested on strains of S. aureus and Escherichia coli (E. coli) revealed MIC values five times higher than those of simple ERY. Another group aimed to prepare Pluronic micelles incorporating ERY, generally focusing on the size distribution and aggregation of the particles in dilute aqueous media. Compared to the previous study, the particle size was considerably smaller (70 nm). The study demonstrated a strong pH dependence of the micelle behaviour. A pH increase from 4.65 to 6.1 led to the ionization of ERY and consequently to the formation of larger sized micelles, while an increase in pH to 11.36 triggered the micelle rupture and consequently the drug release^[63]. Using a block copolymer with amphiphilic properties (poly(ethylene glycol)-block-poly[2-(2-methoxyethoxy) ethyl methacrylate)], polymeric micelles encapsulating ERY were prepared via dialysis method. They had a loading degree (LD) of 16.8%, size of 741.2 nm, negative Zeta potential, and were able to release the drug over the timespan of 180 h^[64]. Using other amphiphilic copolymer, PCL-PEG, ERY micelles with LD of 6.5%, EE of 97%, size of 220 nm, and positive Zeta potential (+19 mV) were prepared. The formulation was proven to lack cellular toxicity and to successfully inhibit the proliferation of S. aureus species through a sustained release^[65].

2.5. Cubosomes

Cubosomes are discrete, sub-micron nanostructured particles comprising a bicontinuous cubic liquid crystalline phase, possessing a good ability to penetrate the skin even through the pilosebaceous units^[66]. They are obtained by the self-assembly of certain surfactants and lipids in water, enabling the entrapment of hydrophobic and hydrophilic drugs due to their bicontinuous nature (Figure 8). Cubosomes form a viscous optically clear material, suitable for cosmetic applications, and generally for transdermal drug delivery. In this context, the entrapment of ERY into cubosomes was regarded as an innovative alternative to the other lipid-based formulations aiming to minimize side effects while preserving its efficacy in acne treatment. Cubosomal ERY was prepared employing the emulsification method, by adding hot water, under stirring, to a mixture of glyceryl monooleate, poloxamer surfactant and ERY^[67]. The optimization of glyceryl monooleate and poloxamer concentrations, stirring speed and time led to ERY-loaded cubosomes with EE values of 95% and an average particle size of 264 nm. The particles were observed to have a spherical shape along with a smooth surface and forming a gel with pseudoplastic behaviour. ERY cumulative release was 75% in the first 7 h and reached 88% after 24 h (pH = 6.4).

2.6. Solid Lipid Nano(Micro)Particles

Literature data mentioned the preparation of solid lipid nanoparticles (SLN) and solid lipid microparticles (SLM), the classification being created by their dimension. Nevertheless, the similarity of the composition of SLNs and SLMs induce similar physicochemical properties, the main distinction being their application fields and administration routes that are dictated by their different size^[68]. Furthermore, the use of the term “nano” has varied along the course of time^[69][70]. Therefore, in this section, solid lipid nano(micro)particles shall be presented as they were reported, rather than their differentiation based strictly on size criteria. The distinct characteristic of the solid lipid nano(micro)particles is the presence of biodegradable lipid-based components found in a solid state at physiological temperatures (Figure 9). Some examples of such components are hard fats such as glycerol (mono-, di-, and/or triglycerides), complex glyceride mixtures, fatty acids, steroids or even waxes^[71]. One of the obvious advantages of solid lipid particles is that the lipid matrix is composed of physiological lipids, thus significantly decreasing the risk of toxicity^[72]. Therefore, the mentioned drug delivery system was promoted as a safer option, in addition to the higher stability of the solid lipid matrix compared to other nano(micro)particles, and the cheaper production costs than those of phospholipid-based liposomes developed thus far^[73]. They offer great potential for the administration of active molecules by any route, including tablets and suppositories. SLNs and SLMs consist of three main components: solid lipids, surfactants and water. Due to the solid nature of the lipid, the drug usually requires to be incorporated within the lipid after it is brought to a melted state. In this light, an important aspect that should be considered in the lipid selection is ERYs solubility in the solid lipid. A study of the partitioning behaviour of ERY in different lipids showed that it presents the best miscibility when added along with glycerol monostearate, followed by stearic acid, acetyl alcohol, and cetostearyl alcohol^[74]. Nonetheless, other solid lipids and mixtures of solid lipids were used with good results.

2.6.1. Solid Lipid Nanoparticles

An exhaustive study regarding the preparation and composition optimization of ERY loaded solid lipid nanoparticles (ERY-SLN) was realized by using the hot homogenization method choosing glycerol monostearate to act as the lipid phase, Poloxamer 188 as surfactant and soya lecithin as a cosurfactant^[74]. Even though no other component was mentioned, the DSC study makes reference to the presence of mannitol as a cryoprotectant. Considering that the surfactant concentration can shape the properties of SLNs, different compositions were investigated, and it was found that 2% (w/v) Poloxamer 188 is optimal to reach the smallest particle size of 138 nm, with the highest EE and DL (84.5% and 28.1%, respectively). Studying the effect of different variables on SLN properties via a miscellaneous design–response surface methodology, the researcheuthors concluded that the increase in lipid concentration produced a decrease in the mean particle size but also in the entrapment efficiency. Therefore, a minimum particle size of 153 nm and maximum EE of 88.4% were reached when using a lipid concentration of 15 mg/mL and surfactant/cosurfactant ratio of 1.07/1. The ERY-SLN presented negative Zeta potential values up to −19 mV, reflecting an excellent stability. No significant alteration of the particle size and EE was observed for the SLN dispersions stored at 4 or 25 °C, over a three-month period. The release profile, which was investigated in phosphate buffer media, showed a prolonged release during 24 h, up to 66.26 ± 2.83%. Pursuing the preparation of a topical formulation aimed at the treatment of acne, Dhillon et al. investigated the development and optimization of ERY-loaded SLNs. The study focused on selecting the best preparation method, lipid type, drug/lipid ratio, stirring, and sonication time^[75]. The findings led to the conclusion that the microencapsulation technique was superior to the solvent evaporation and the solvent emulsification diffusion ones, because it showed better results in terms of particle size, particle shape and drug entrapment. The screening of three lipids, stearic acid, glycerol monostearate and Compatriol 888, revealed that the use of glycerol monostearate significantly decreased the particle size into the nanometric range and favoured the highest ERY entrapment. Furthermore, it was discovered that an ERY/glycerol monostearate ratio of ½ was optimal for a maximum encapsulation of 78% into nanoparticles of 176 nm. The researcheuthors also reported that a 0.5% concentration of surfactant (Poloxamer 188) was enough to preserve the size of the nanoparticles and to ensure a high Zeta potential, required for particle stability. An increase in stirring time (from 30 to 60 min) produced a dramatic decrease in particle size (from 528 to 176 nm) and higher entrapment degree values, which reached 78.59%. A sonication time of 20 min was optimal to reduce the particle size. The average particle size, PDI, Zeta-potential, EE and LD of the optimized SLNs were found to be ~176 nm, 0.275, −34 mV, 73.56% and 69.74% respectively. They showed a prolonged release, reaching around 85% cumulative ERY release after 12 h and 93% after 24 h. Targeting the improvement of ERY’s penetration into bacterial cells, cationic SLNs were designed using a cationic lipid, quaternary ammonium salt and didecyldimethylammonium bromide^[76]. SLNs with mean diameter from 250 to 400 nm were prepared using the solvent injection method. The cationic lipid induced positive Zeta potential and great stability over time, with no change in the morphologic parameters being recorded after one year of storage. As proof of concept, a higher antimicrobial activity of SLNs compared to free ERY was evidenced, and the MIC decreased along with the increase in cationic lipid content, proving its synergic antibacterial activity.

2.6.2. Solid Lipid Microparticles

ERY-loaded SLMs were prepared for oral delivery, using a binary mixture of ½ ratio solid lipids (Softisan154 and Phospholipon90H or Softisan154 and beeswax) forming a solidified reverse micellar solution (SRMS) matrix. PVA, sorbitol and sorbic acid were also used as surfactants^[77]. Priorly, the SRMS matrix was prepared by fusion, and the SLMs were prepared by hot homogenization method, varying the ERY concentration in order to obtain SLMs with different drug contents. The DSC investigation suggested the occurrence of interactions between the two lipids, leading to a less crystalline morphology, envisioned to have a higher ability to retain ERY over time compared to a more crystalline matrix, which is prone to a faster drug expulsion. The SLMs showed a spherical morphology, with a smooth and non-porous surface, while the diameters increased proportionally from 13 to 17 µm along with the entrapped ERY amount, with no significant distinctions between those containing different lipid mixtures. The nature of the lipids influenced the EE and LD, i.e., the SLMs composed of Phospholipon90H displayed higher EE values, ranging from 89 to 95%, while those containing beeswax showed variations of the EE from 78 to 89%. They were stable over the span of 4 weeks; however, an aggregation tendency towards the end was observed, while a slight pH shift towards acidity suggested the degradation of the lipid component to fatty acids. The in vitro release studies revealed that all ERY-loaded SLMs possessed higher prolonged release properties and no burst effect or dose dumping, compared to the reference ERY-tablets. This suggests a reduction in the daily number of administrations (from 3–4 to 1–2) needed to reach and maintain the optimal plasma concentration. The release profile was dose dependent and was influenced by the nature of the lipid; SLMs containing Phospholipon90H exhibited a maximum release ranging from 90 to 75%, while those containing bee waxes ranged from 79 to 61%. The antimicrobial activity tests against S. aureus, S. paratyphii, P. aeruginosa, and E. coli confirmed a time- and dose-dependent anti-bacterial activity, higher for those containing Phospholipon90H compared to bee waxes and simple ERY. The findings suggest that the formulation of ERY as SLMs potentiates its activity, possibly due to the fusion between the lipid component and the bacterial cell membranes, promoting an easier penetration of the antibiotic. Pharmacokinetical studies on rats showed a steady and slow decrease in the drug concentrations or a gradual clearance, reaching a T_1/2 value for the SLMs of 8 h, compared to a T_1/2 of 4.5 h in the case of the neat ERY. ERY-loaded SLMs were also prepared using stearic acid as the lipid component and Tween 80 as surfactant, via hot homogenization method^[78]. By varying the lipid/surfactant ratio, the average hydrodynamic diameters ranged from 2.28 to 8.15 μm, the lowest particle size being reached for a lipid/surfactant ratio of 1 g/1 mL and 1 g/2 mL. The melting temperature of ERY was clearly identified in the DSC curves, indicating that it was dispersed as large particles into the lipid, attributed to the ERY insolubility into the lipid matrix. The microparticles showed much higher in vitro antimicrobial activity compared to neat ERY; however, those with the lowest MIC values were large in size (6.5 μm), at risk of not being able to pass into the lymphatic circulation^[79]. Furthermore, the microparticles containing higher amounts of surfactant and thus more stable in acidic environment, were selected for in vivo tests on model infected animals. It was proven that the administration of equal amounts of SLM and neat ERY led to almost similar bacteremia reduction (75% vs. 72% during day 3, and 95% vs. 94% during day 7), despite the small percentage of ERY entrapped into SLMs (21%). This suggested a significant increase in the antimicrobial activity of ERY when encapsulated into SLMs. Further, only low fluctuations in plasma concentrations were recorded in the case of SLMs, indicating a potential to reduce major side effects.