Preparation and investigation of solid lipid nanoparticles for drug delivery

Accepted Manuscript Title: PREPARATION AND INVESTIGATION OF SOLID LIPID NANOPARTICLES FOR DRUG DELIVERY Author: Gokce Dicle Kalaycioglu Nihal Aydogan PII: DOI: Reference:

S0927-7757(16)30476-9 http://dx.doi.org/doi:10.1016/j.colsurfa.2016.06.034 COLSUA 20761

To appear in:

Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: Revised date: Accepted date:

4-2-2016 13-6-2016 14-6-2016

Please cite this article as: Gokce Dicle Kalaycioglu, Nihal Aydogan, PREPARATION AND INVESTIGATION OF SOLID LIPID NANOPARTICLES FOR DRUG DELIVERY, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2016.06.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

PREPARATION AND INVESTIGATION OF SOLID LIPID NANOPARTICLES FOR DRUG DELIVERY

Gokce Dicle Kalaycioglu, Nihal Aydogan*

Hacettepe University, Faculty of Engineering, Department of Chemical Engineering, 06800 Beytepe, Ankara, TURKEY.

Corresponding Author: Nihal Aydogan Fax: +90 312 2992124, Phone: +90 312 2976781 E-mail:[email protected]

Graphical abstract

Highlights  12 newly formulated SLNs were synthesized via microemulsion method.  Size and surface properties of SLNs were controlled by changing the composition.  Drug entrapment and release capacities of SLNs have been investigated.  Methylene blue have been used as model molecule in the drug incorporation studies.  Surface charge densities were correlated with entrapment/release capacities of SLNs.

Abstract Solid lipid nanoparticles (SLN), a promising drug delivery vehicle, offer an alternative system to traditional colloidal carriers. In our study 12 new SLN formulations were fabricated via the “microemulsion (ME) method”, each leading to a different SLN size and composition. This method is a relatively easy technique and involves biocompatible conditions. Stearic acid has been used as lipid material of which the ratio is kept under 4% to prevent particle growth. Particle size and surface properties of the synthesized SLNs were controlled using various combinations of emulsifiers such as lithocholic acid, Pluronic F127, Tween 20, lecithin and butanol. Furthermore, the mean size of the particles was adjusted by changing the ME:water ratio in the dilution step which is independent from composition. It was found that most of the SLNs were in the colloidal size range (below 100 nm) and spherical in shape, which provides high surface area to exploit, as an alternative adsorptive drug carrier system. Also, the surface charge density values of SLNs were calculated by considering size and zeta potential values which then helps in understanding the surface potential of particles. MB was chosen as a model molecule and entrapped on the surface of SLNs after the preparation, to determine the loading capacities and release efficiencies. As such, SLNs have been successfully produced which have controllable drug entrapment efficiency and release capacity according to the chosen combination of emulsifiers and dilution ratio. Thus, this study contributes to the improvement of alternative drug delivery systems with biocompatible and stable newly formulated SLNs. Keywords: Solid lipid nanoparticle, Microemulsion, Drug delivery, Methylene Blue

1. Introduction Solid lipid nanoparticles (SLNs), prepared using various physiological lipids, emulsifiers and water, have been used since the 1990s as colloidal nanocarrier systems, either in combination with or as an alternative to liposomes, emulsions and polymeric nanoparticles [1,2,3]. Possessing adjustable release kinetic, determined by the method of preparation used, its level of biocompatibility, its stability and the degree to which active materials may be encapsulated within its structure, SLNs are ideal for use within the body and may be administered via different routes (parenteral, topical, ocular, etc.)

[4,5,6]. Another advantage is that, in

comparison to other systems, like the ones with liposomes for instance, SLNs have less storage and drug leakage issues. Moreover, they have good stability which they can maintain for up to 2-3 years [7,8]. To date, many hydrophobic and hydrophilic drugs, such as doxorubicin, paclitaxel, tobramycin, cyclosporine A, carmustine, pilocarpine, cortison etc. have been incorporated into SLN [9-15]. There are several methods that could be utilized to synthesize SLNs, including high pressure homogenization, high shear homogenization, etc [16]. However, if the incorporation of selected compounds is intended, particularly drugs, genes, proteins and other biomolecules, methods such as high pressure or high shear homogenization which create harsh environments for the material, need to be avoided. By using microemulsion method, the nanoparticle formulations of interest can be prepared in vials rapidly, reproducibly and cost effectively in a two-step process involving only mild operating temperatures. Moreover, the uniform nanoparticles produced become more biocompatible due to the elimination of organic solvent use during its preparation. Furthermore the solubilization properties of hot microemulsions enable SLNs to be loaded with various drugs and is particularly useful for drugs with poor water solubility [17-19]. To our day various lipids and emulsifiers have been utilized to investigate the effect of composition on the size and surface properties of SLNs [20-22]. A large variety of compounds can be utilized as a lipid material which is used here in a broad sense such as fatty acids, triglycerides, partial glycerides and steroids. It is known that the mean diameter of SLNs is directly affected by the type and ratio of lipid in the structure. Westesen et al have reported that the size of SLNs increase when prepared from lipids with higher melting points via the hot homogenization method [23]. In another study, the effect of lipid chain lengths on the particles produced was investigated and it was found that shorter chains make the particle more compact and reduce its size [24]. A general finding is that increasing the lipid content causes an increment in the size of particles and lowers the monodispersity [25]. Along with 1   

the choice of lipid, the type of emulsifier used also has a great impact on the SLNs fabricated. In a study reported by Cavalli et al., ionic and nonionic emulsifiers have been compared and it has been found that using ionic emulsifiers in the microemulsion method, SLNs can be synthesized in a considerably smaller size [26]. Also, in another study focused on manipulating the surface properties of SLNs, different emulsifier concentrations were examined and it is found that increasing the amount of nonionic emulsifier resulted in the reduction of the zeta potential value of the particle [27]. In that study, Müller-Goymann et al. particularly focused on creating an alternative drug delivery system that is based on the adsorption of the material onto the particle surface. The surface properties of the particle become a vital issue which needs consideration. This remarkable approach, where the drug is loaded after the SLNs have been synthesized, helps to preserve the cargo from the conditions to which it may be exposed during the course of preparation. As a general assessment for nanoparticles, small size is more desirable due to factors such as high surface area per mass, greater contact with surrounding materials and accessibility to target areas as drug delivery agents [28-30]. However, as the particle gets smaller it becomes harder to attain higher levels of efficiency in drug encapsulation and a sufficient release capacity. In order to overcome this problem, the active ingredients can be carried onto the surface of the particle via adsorption rather than incorporation into the structure. In this way, the drug can also be protected from a high stress environment during particle preparation. Up until today, a variety of biomolecules like BSA, HSA etc. have been successfully incorporated onto SLNs via adsorption [31-33]. Apart from simply being a means of carrying a material on the surface of a particle, protein adsorption on nanocarriers intravenously injected is an important subject, as such studies provide foresight into whether a controlled development of drug delivery agents will be possible [34]. Some proteins such as immunoglobulin induce the clearance of particles via macrophages and inhibit the transport of pharmaceutical ingredients to the target area, and others such as albumin promote a prolonged circulation time of the nanocarrier in the blood [35,36]. Thus, rather than merely aiming to develop a carrier particle, it is important to investigate the adsorptive properties of the colloidal carrier from this point of view. Methylene blue (MB), which is a heterocyclic aromatic compound, has many uses in a number of different applications, such as redox indicators in analytical chemistry [37], as dyes or stains in biology [38], as U.S. Food and Drug Administration approved drugs for the methemoglobinemia treatment [39] and as photosensitizers in photodynamic therapy (PDT) 2   

[40]. In addition to this, MB is also an inexpensive compound which is encourages its use as a model molecule to mimic the behavior of most positively charged and aromatic drug molecules. In the present study, 12 types of SLNs with different size and compositions were synthesized using the “microemulsion method”. The effect of operational parameters such as temperature, stirring type and pH on the formation of transparent ME was investigated. Stearic acid was selected as the lipid material and the ratio was adjusted so as to not exceed 4%. Lithocholic acid, Pluronic F127, Tween 20, lecithin and butanol were used as emulsifiers and coemulsifiers. Different combinations, concentrations and ratios of emulsifiers and coemulsifiers were tested in order to understand the effect of both the emulsifier (in terms of its ionic charge and chemical nature), and the co-emulsifier (and its ratio) on the size and zeta potential of particles. Various techniques were used in the characterization of the SLNs. Furthermore, a stability study for the nanoparticles was performed, under room temperature conditions and over a 3 month period, on the basis of their mean diameter and zeta potential values. MB was chosen as a model molecule and its interaction with SLNs was investigated in order to obtain information about the drug entrapment capacity of these nanoparticles, as well as to observe their interaction with molecules that carry potential as drug delivery agents.

2. Materials and methods 2.1 Materials Stearic acid, Tween 20, monosodium phosphate and disodium phosphate were purchased from Merck (Darmstadt, Germany). Pluronic F 127, lithocholic acid, soybean lecithin and butanol were obtained from Sigma-Aldrich (St. Louis, MO, USA). Methylene blue and sodium hydroxide were purchased from Fluka. These materials were obtained from the indicated sources and used without further purification. All solutions were formulated using UP (ultrapure) water (Millipore Direct-Q3 UV). 2.2 Preparation of SLN Microemulsion method was used to prepare SLN formulations. In the first step, briefly, stearic acid as the lipid matrix was weighed and heated to ~70oC. Various amounts of emulsifiers either Tween 20, Pluronic F 127, lithocholic acid and lecithin or sometimes combination of them were dissolved in UP water and heated to the same temperature. The hot 3   

aqueous phase was added to the lipid phase and magnetically stirred at 500 rpm for 10 min to form transparent and thermodynamically stable microemulsion (ME). In the second step, hot microemulsion was added into the water at 2-3oC at desired ratio via hypodermic needle. For these two steps, operational parameters such as temperature, stirring type and ME:water ratio have been examined and the rest of the study was completed by using these optimum conditions. When lithocholic acid was used as emulsifier, the pH of the solution is adjusted to pH 12 which is higher than the pKa value of lithocholic acid. Otherwise the pH of the solution is kept as approximately 7. SLN dispersions were freeze-dried to remove water and the powder form was used during the thermal analysis. Glucose (3%) was used in the lyophilization process as a cryoprotectant. 2.3 Measurement of particle size and zeta potential Dynamic light scattering (DLS, ALV-CGS-3 Compact Goniometer) was used to determine the hydrodynamic diameter of the SLNs and polydispersity indices (PDI). Samples were diluted to 1:100 with UP water and the measurements were done at an angle of 90o. Each measurement was performed for at least three different batches of SLN solution. To verify the particle size and to obtain information about the geometry of particles, SLNs were analyzed by using AFM (PSIA Corporation, XE-100E) with Cr-Au cantilevers (ACTA 10M) with a frequency of 0.37 Hz in non-contact mode. The SLN dispersions were diluted by using UP water and then dropped onto clean microscope slide followed by vacuum drying for 24 hours at 25°C. The zeta potential values were determined by applying ~75 mV potential difference with Zeta Meter System 3.0 (Zeta Meter Inc.). Quartz-teflon GT-2 cell, molybdenum anode and platinum cathode were used. To measure zeta potential, the samples were 1000-fold diluted with UP water. Each measurement was performed for at least three different batches and ten particles. The average values were calculated and used for the surface charge density calculations. 2.4 Differential scanning calorimetry (DSC) Thermograms of SLNs were obtained with a differential scanning calorimeter (Perkin Elmer PYRIS Diamond™). About 5 mg of each SLN sample was weighed into an aluminum pan and the thermal behavior determined in the range of 10–190oC at a heating rate of 5oC min−1. 4   

Melting point values were determined from the endothermic peak while enthalpies were obtained by integration of the endotherms using linear baselines. The crystallinity index (CI) was calculated by using the enthalpies according to Eq. (1) to determine the degree of crystallinity of SLNs [41].

CI % 

EnthalpySLN ( J / g ) .100% Enthalpybulk material ( J / g )

[1]

2.5 Preparation of MB-loaded SLNs

200 µL aliquots of nanoparticles with the concentration of 7.89x1010 particle/ml were incubated with 100 µL MB with a last concentration of 0.0026 mg/ml to yield 1.1 ml total solution together for 24 h at room temperature. After incubation (15, 60, 180, 360 and 1440 min), samples were centrifuged at 7000 rpm, 25oC (Microcentrifuge MPW-55). Loaded MB amount was thereafter calculated by determining the remaining MB via UV-vis spectroscopy according to the calibration graph that was prepared before. UV-vis spectroscopy measurements have performed (Thermo Scientific™, GENESYS 10S) with 1 ml quartz cell (λ=664 nm) and the entrapment efficiency (EE%) was calculated by using Eq. (2). EE % 

Total amount of drug  Free drug inthe supernatant .100 Total amount of drug

[2]

2.6 In vitro release of MB from SLNs 1.1 ml of MB-SLNs with a concentration of 7.89x1010 particle/ml was centrifuged with 7500 rpm and SLNs were re-dispersed in PBS at pH 7.4 with a volumetric ratio of 1:10. At selected time intervals (10, 30, 60, 240 and 1440 min), 1 mL of samples were taken and centrifuged to isolate the SLNs from supernatant solution of MB. The supernatant solution was then analysed by using UV-vis spectroscopy to get information about the released amount of MB. Thereafter the corresponding MB concentration has been calculated by using the calibration graph considering the initial amount of MB loaded and release capacity (RC%) has been calculated by using Eq (3).

5   

RC % 

Free drug inthe supernatant .100 Total amount of entrapped drug

[3]

2.7 Stability of SLNs Stability studies of SLN suspensions were performed after the samples were stored at room temperature for 3 months for particle size and zeta potential. After 3 months, the size of the particles was measured via DLS.

2.8 Statistical studies All measurements were performed for at least three batches. Results were analyzed and presented as mean ± SD. Statistical analysis was performed using the one-way method via ANOVA. Differences between means were accepted statistically significant if the p-values were less than 0.05.

3. Results and Discussion 3.1. Preparation and characterization of the SLNs The SLNs were prepared using microemulsion method in which the formation of a transparent and thermodynamically stable microemulsion is considered as a vital step [19]. There are some key operation parameters that play a major role in this method such as temperature, pH value and type of stirring, all of which may affect final particle size, polydispersity, stability and ultimate product quality [42,43]. In our study, SLNs were successfully prepared using based on the aforementioned method and the procedure involved the addition of the hot water phase into the lipid phase, which comprised stearic acid, at a temperature range of 70-75oC. The stearic acid content was kept to under 4% within the SLNs in order to prevent the growth of particles [25]. The SLNs were then formed by dispersing the hot microemulsion in a large volume of cold water (2–3°C) under magnetic stirring. At this point of the procedure the temperature gradient becomes a significant issue and necessitates a solidification procedure. The lipid crystallization can be facilitated by high temperature gradient which prevents lipid aggregation and also known to have an effect on particle size. To find the optimal value for the preparation of ME, 65-70oC temperature range was also used and revealed that after the dispersion of the lipid phase in the water phase, the transparency in the oil phase could not be attained. The result also included an increase at the micron scale of 6   

particle diameter. Therefore, the temperature range in the first step of the preparation was kept at 70-75oC throughout the rest of the study. Furthermore, to see the effect of additional energy in the formation of ME, different stirring techniques such as homogenization and ultrasonication, which inhibit transparency, were also explored. Gasco and coworkers reported that since the droplet structure has formed when the suitable conditions provided in the microemulsion without needing an additional energy [44]. For either “oil-in-water” or “water-in-oil” microemulsions, the presence of an emulsifier is required to lower the oil-water interfacial tension by way of adsorption onto the interface, leading to a more thermodynamically stable form of ME [45]. Even though stirring is used to achieve a homogeneous solution (dispersion), the results indicate that due to the chaotic environment caused by applying high energy, the emulsifiers are unable to minimize the positive energy change resulting from surface formation and thus ME formation is prevented. Among the other techniques, it is observed that magnetic stirring (~500 rpm) helps build contact between emulsifiers and the interface thereby assisting ME droplet formation and submicron-sized particle formation. Based on this information our study focuses on the type of emulsifier used and its effect on both the surface properties of SLNs as well as the size of particles. The emulsifiers and lipids used, along with the operational conditions selected, are presented in Table 1. Another important parameter is the pH of the solution during the formation of ME step, which must be adjusted according to the materials used. Lithocholic acid, a bile acid, has a nonionic character in the conditions that we have studied (~pH7) [46]. Its HLB number is approximately 2.5 and slightly soluble in water [47]. For the first batch, the solution pH was kept at 7 during ME preparation. However, a transparent microemulsion could not obtained under this condition. Therefore, while using lithocholic acid as emulsifier, the pH was adjusted to 12 using NaOH during the preparation of ME. With this, the solubility of lithocholic acid in the water was maximized and the carboxylic acid group in the molecular structure of lithocholic acid transformed largely into a carboxylate form (COO-Na+). Notwithstanding that, the ultimate pH of the SLN solution is being adjusted to ~7 in the dilution step.

The composition and physicochemical properties of the SLNs synthesized have been summarized in Table 1. It can be seen from Table 1 that the mean diameter, measured using 7   

DLS and AFM, was quite similar for all particles. One of the parameters investigated was the ME:water ratio in the dilution step [12,48]. To understand the influence of this parameter, 2 different ME:water ratios were used in the first 4 batches. When size of SLNP1 and SLNP2 was compared to each other, we found that when the ME:water ratio decreases from 1:10 to 1:20, the mean particle size decreases from 135±2 to 88±6 nm. The same phenomenon was also observed for SLNL1 and SLNL2 for the mean diameters decreased from 144±8 to 79±10 nm by changing the ME:water ratio from 1:10 to 1:20, respectively. Based on these results 1:20 ME:water ratio was found to be optimal and was used throughout the rest of the study. The choice of emulsifier and its concentration has a great impact on the size of SLNs [21,22]. It has been previously reported that the particle size of the SLNs prepared with ionic emulsifiers becomes smaller compared to those synthesized using nonionic emulsification [26]. Parallel to this is that the change in the size of SLNs upon changing the emulsifier is also significant. In this study, to examine the effect of ionic emulsifier on the size of particles, lithocholic acid and Pluronic F127 were selected as the ionic and nonionic emulsifier, respectively. The first was used to synthesize L-coded (SLNL1 and SLNL2) and the latter to produce P-coded (SLNP1 and SLNP2) nanoparticles. In the preparation of these particles, coemulsifiers were not used in order to see the actual effect of emulsifiers clearly. The results show that DSLNL1 < DSLNP1, as expected, and comply with the literature. However, when the mean diameters of SLNL2 and SLNP2 (144±1.3 and 135±2) are compared to each other, it is seen that the same phenomenon does not occur. This can be attributed to the increase in the amount of emulsifier within the solution. In general, high concentrations of emulsifier reduce surface tension and provide facilitated particle formation. When the nanoparticles are partitioning, there must be enough emulsifier molecules to achieve the coverage of new surfaces rapidly and prevent the formation of uncovered lipid surfaces which cause an agglomeration. Thereby, the size of the nanoparticles can be reduced. However, an excessive amount of emulsifier molecules can also cause an increase in micelle formation, degraded physical stability of SLNs, colloidal coalescence and lipid reorganization, and enlarged particles, even though the lipid:emulsifier ratio is constant [27]. It is deduced that the concentration of emulsifier is also a significant parameter, as well as the ionic character which must be optimized by tracking changes in particle size. According to literature, the introducing of a co-emulsifier into the structure decreases the size of the particle due to both the rearrangement of compounds and also the increase in storage stability compared to formulations involving only one emulsifier [25]. However, it is known 8   

that the exclusive use of rapid distribution surfactants as co-emulsifiers often results in toxicity (e.g. SDS) because of their ability to cover surfaces quickly. In this study, to investigate the effect of co-emulsifier, such surfactants were avoided; lecithin, Tween 20, and butanol were used. SLN6 and SLN7 were synthesized by adding Tween 20 as a co-emulsifier into the structure of SLNP1 and SLNL1, respectively. Although the ratio of emulsifier to the lipid phase was kept constant, AFM results given in Table 1 show that the addition of Tween 20 during SLN6 and SLN7 preparation results in a particle size reduction from 104±1.4 to 72±0.5 nm and from 80±2.2 to 55±1.1 nm, respectively. Lecithin is a frequently-used emulsifier in SLN fabrication [48,49]. From Table 1 it can be seen that the incorporation of lecithin increases the size of SLNs produced in this study (SLN5 and SLN8). Schubert et al. investigated the effect of lecithin percentage in the lipid matrix on the size of SLNs [7]. They found that the presence of lecithin facilitates the formation of a large oil/water interface. Thus, when lecithin content increased which helps the formation of additional interfacial area, the mean particle size decreases. In our study, the percentage of lecithin according to stearic acid (in SLN5 and SLN8) is 55%. The effect of high content of lecithin can be understood by comparing the mean diameters of SLN6 and SLN8, the only difference between of which is the presence of lecithin in the structure of SLN8. There is an associated respective increase in the size of particles which changes from 72±0.5 to 88±1.3 nm. Considering the effect of Tween 20 on the size of particles, it is deduced that the use of lecithin in SLN5 fabrication makes the largest particles compared to the others registering a diameter of 164±1.4 nm. This is linked to the high content of lecithin in the structure of the particle. When the amount of lecithin reaches a critical value (which corresponds to the point above 30% lecithin/lipid matrice), it is seen that the presence of excess lecithin does not help to decrease the size of particles energetically. It is deduced that, at these concentrations, lecithin multilayers may accumulate on the particle’s surfaces despite the fact that at low lecithin concentrations, just a monolayer of lecithin is formed as an attempt to stabilize the particle interface [7]. Thus, it is understood that the ratio of lecithin/lipid matrice has to be kept under 30% in order to see the reducing effect of lecithin as an emulsifier. According to literature, it is expected that using an alcohol as a co-emulsifier in the preparation of SLNs, makes the particle smaller [48]. However, during the formation of ME, which is the first and

most important step in SLN

preparation with this method, the

incorporation of butanol prevented the ME formation when added to the aqueous phase. Thereafter the same amount of butanol was added to the lipid phase in the same initial step of 9   

SLN9 and SLN10 preparation and transparent ME formation was achieved. When the mean diameter value of particles was compared to see the effect of butanol incorporation, it was found that the presence of butanol in the lipid phase of both SLN6 and SLNL1 promotes the growth of the particles. As seen in Table 1, there is an increase in the mean diameter of SLN6 from 72±0.5 to 78±0.3 nm, and from 80±2.2 nm to 85±1.2 nm for SLNL1, upon the addition of butanol (SLN9 and SLN10). It can be concluded, therefore, from these results, that when added to the lipid phase, butanol makes the lipid matrix looser and abruptly increases the diameter of particles. In order to accurately determine the size of SLNs, AFM analysis was performed. This technique is widely used to obtain size, shape and distribution information about nanoparticles. Despite the fact that SLNs have a solid matrix and high long term stability, when the lipid materials have been exposed to an electron beam for a long time, as when they are during the collection of visuals , the material may be damaged. As DLS measures the hydrodynamic radius of particles, which is the total size of the particle and hydration layer together, the hydrodynamic diameter will always be greater than that measured using microscopic techniques [50]. Thus, AFM analysis is rather suitable for SLNs. Furthermore, when the diameters presented in the Table 1 are compared to each other, the difference between DLS and AFM results can be seen clearly and as such supports findings in literature. The images presented in Figure 1 show that the SLNs synthesized in our study have a low polydispersity. Also, it is clearly seen that particles are almost spherical in shape. In Table 1, the diameters of SLNs that were obtained from the AFM images can be seen. It is known that when using AFM, some samples may be seen larger than their actual size because of the tip used and scanning [51]. Zeta-potential can serve as an important parameter in the predictions of long term stability. Itis very important to foresee the capability of formulations in electrostatic interaction based drug release applications Stearic acid, the lipid material we have used, has a pKa value of 4 and in the conditions that we have synthesized SLNs (~pH6), it is negatively charged. From Table 1, it can be seen that this ionic structure makes the nanoparticles negatively charged, too. Furthermore, the addition of an ionic emulsifier such as lithocholic acid resulted in the increase of zeta potential. Table 1 shows that the higher zeta potential values belonged to the SLNs that have lithocholic acid in their structure such as SLNL1, SLNL2 and SLN10 with 42.4±1.1, -47±1.3 and -44.1±2.1 mV, respectively. Additionally, using only a non-ionic

10   

emulsifier and the co-emulsifiers Pluronic F127, Tween 20 and butanol, lower zeta potential values for SLN6 and SLN9 with -10±0.3 and -13.7±0.5 mV, respectively, were obtained. Surface charge density (σ) has a key role in obtaining more realistic quantitative information about the surface properties of particles. Since the synthesized SLNs were used to study the entrapment at their surface, it is very important to characterize the surface of SLNs to correctly estimate interaction potential; particularly when electrostatic interaction is of concern. We calculated surface charge density values which are dependent on the zetapotential value, composition, geometry and size of the SLNs. This calculation was performed using the previously reported equations and assuming that the particles are spherical [52,53] (the equations used for this calculation are given in Supplementary Material). Table 1, reveals that when the particle size increases, the surface charge increases relative to it, too. Furthermore, the addition of an ionic emulsifier also leads to a high surface potential. SLNL1 and SLNL2 have the highest surface charge densities at 415±8.8 and 160±15.0 C/m2 (x1014), respectively. Also, at 20±4.0, 21.4±1.2, 21.7± 1.2, 18.8±0.6 and 19.3±0.3 C/m2 (x1014), SLN6, SLN8, SLN9, SLNP1 and SLNP2 have lowest surface charge densities, respectively. The melting point of a material depends on its chemical nature, such as chain lengths and the amount of free hydroxyl groups within its structure. Owing to the different melting points and melting enthalpies of different compounds, DSC can give information about the sample structure and interactions between the components. DSC analysis is a particularly powerful for providing insight into the drug encapsulation mechanisms of SLNs. In the present study, since the model molecule was entrapped on the surface of SLN, the crystallinity or the enthalpy of transition of SLNs did not affect the loading efficiency. Nevertheless, thermal analysis of freeze-dried SLNs is important for understanding whether the ingredients have been incorporated into the structure and the production of particles has been accomplished. Differential scanning calorimetry thermograms for SLNs are shown in Figure S1 (See Supplementary Material). Also, melting point temperature (Tonset and Tmax) and enthalpy values which are based on the amount (g) of stearic acid are listed in Table 2. There are four different polymorphs of stearic acid which are A, B, C and E [54]. Form A is triclinic, and B, C and E are monoclinic. By comparison among the other polymorphs, The C-form is thermodynamically the most stable one and has a melting point at ~70oC [55]. The peaks obtained show that there are both crystalline and amorphous phases in the structure of SLNs. It is known that low melting point emulsifiers carry the melting point of the 11   

structure to a lower temperature and vice versa. For this reason, using Tween 20 (liquid at room and body temperature) and lecithin makes the particle more amorphous and decreases the melting point (Table 2). When we look at the melting points for SLNP1, SLN5, SLN6, SLN8, SLN9, SLN11 and SLN12, we see that their common property is the presence of low melting point emulsifiers within their structure. Pluronic F127 is in solid form at room and body temperature and its melting point is 57oC [56]. Since its melting point is lower than that of stearic acid, it affects the crystallinity of the structure and reduces the melting temperature. Additionally, using lithocholic acid without any co-emulsifier (SLNL1), increases the melting enthalpy and thus the melting point of particle, making the particle the most crystalline at 32.3% crystallinity .

Furthermore, DSC thermograms help us understand the structural form of the SLNs that have been produced. It can be seen from Figure S1 that, even though there are different compounds within the body of SLNs, there is only one melting peak. This result shows that SLN preparation has been achieved as planned. An exception to this is SLNL1, SLNP1 and SLN11 for each of which there are two melting peaks at 82-78oC, 68-54oC and 67-51oC, respectively. For these particles, it can be said that these peaks belong to the SLN lipid matrix and emulsifier shell. The reason for this may be that as the formulations were different to the other formulations that were used, lithocholic acid and Pluronic F127 covered the surface of the particle rather than being introduced into the lipid matrix. Similar results have been seen in the literature where the surfactant shell and lipid core are melting at two different melting points by revealing two peaks [57,58]. Although, there is an individual melting peak attributed to Pluronic F127 (54oC) which is compatible with literature [56], the presence of Pluronic F127 clearly reduces the melting point (Tmax) of the particle to 68oC and 67oC for SLNP1 and SLN11, respectively. In the case for SLNL1, the melting point of lithocholic acid shell decreased to 82oC from 188oC, which is the melting point value of lithocholic acid alone. Moreover, the melting point (Tmax) of the lipid matrix increased to 78oC with the addition lithocholic acid which accounts for the introduction of lithocholic acid into the structure. Additionally, even though there are some deviations due to the emulsifiers in the structure, from the melting point values of lipid material in SLNs, it can be deduced that stearic acid crystallized into its C-form in the structure of particles.

12   

Investigation of the crystallinity of nanoparticles is necessary for the prediction of the drugloading capacity. In the present study, the crystallinity of the bulk stearic acid phase was expected to be 100% and the other CI% values were calculated according to this value. It is understood from the reduction of the enthalpy of bulk lipid that there is also a decrease in the crystallinity of the lipids after the formation of SLNs. However, despite located on the structure of a particle, lipids still possessed some degree of crystallinity (Table 2). As a general rule, the stability related modifications of the materials means more crystalline structures which usually exhibit lower drug entrapment efficiencies [59]. This phenomenon is especially important in cases where the active pharmaceutical ingredient is to be loaded into the particle. However, when concerned with adsorptive drug loading, surface charge density value becomes more important than the morphological state of SLN. It is seen from the results in Table 2 that the SLNs produced have crystallinity degrees within a broad range of, 32.3% to 1.0%. The highest crystallinity value belonged to SLNL1 due to the high melting point of lithocholic acid and it has behaved as a shell material despite diffusing its diffusion into the lipid matrix and its influence on its crystallinity. According to literature, the usage of excess emulsifier reduces the crystallinity. Moreover, some co-emulsifiers such as Tween 20 help increase the fluidity of the lipid matrix resulting in a more amorphous structure. Our results indicate that SLN5, SLN6, SLN8 and SLN9, all of which have Tween 20 in their structure as a co-emulsifier, have the lowest crystallinity values as expected. However, this is not the case for SLN7 even though there is Tween 20 in its structure. The high degree of crystallinity of the structure is therefore related to lithocholic acid. It can be said that using only lithocholic acid as emulsifier in SLN production, such as SLNL1, is not very favorable. Even though SLNL1 is a very suitable for use as a nanocarrier since its high surface charge density value enabling adsorption of materials, its high crystallinity impedes its drug loading capacity. However, using lithocholic acid in combination with different co-emulsifiers such as Pluronic F127, Tween 20 and butanol (SLN7, SLN10, SLN11 and SLN12), by-passes the issue of crystallinity and makes the ultimate structure more suitable to carrying a pharmaceutical ingredient. An alternative solution is to use Pluronic F127 alone or with a co-emulsifier such as Tween 20 (SLNP1 and SLN6) to produce drug carrying SLNs.

3.2 Stability

13   

The zeta potential is a key factor in order to obtain information about the stability of nanoparticles. As Table 3 demonstrates, in the process of time, zeta potential values of particles decreases. According to the literature, a system can be deemed as stable if the electrostatic repulsion prevails the attractive van der Waals forces [60]. It is known that, agglomeration of particles have formed due to the lack of the electrostatic repulsion between the particles. If the kinetic energy of the particles increases due to the high temperatures or light, the particles will collide and may overcome the energy barrier which can be resulted with the agglomeration [61]. Thereby, the zeta potential values of particles affected. Also, it has been proven that nanoparticles which possess ~25 mV zeta potential value can remain physically stable without any change in their size or zeta potential value. The stability study results reveal that at a 8.2, 9.5 and 16.0% reduction, the most dramatic decreases belong to SLN6, SLN8 and SLN9, respectively. Considering the respective low zeta potential values of these SLNs, it can be said that they are not as stable as the other SLNs. Also the zeta potential value of SLNP1, SLNP2, SLN5, SLN7, SLN10, SLN11 and SLN12 decrease to less than 5%. However, it is obvious from these values that after the months of storage at room temperature, the SLNs prepared have acceptable electrochemical and physical stability [62,63].

Likewise, in order to evaluate the stability of the SLNs fabricated, size is generally used as a characterization tool. In the present study, the mean diameter values were measured using DLS 3 months after preparation. From Table 3, we can see that SLN6 is the most unstable in terms of zeta potential with a 9.6% increment in its size, which is the highest percentage among SLNs. It can be said though, according to these results, that the particle size remains almost constant.

3.3 Determination of MB entrapment efficiencies of SLNs The MB entrapment efficiency of SLNs was determined with UV-vis spectroscopy. The MB adsorption behavior of SLNs can be seen in Figure 2. The incorporation of MB onto the surface of SLNs appears be enhanced when surface charge is increased and reduced when surface charge is decreased. This phenomenon indicates that adsorption occurs via an electrostatic interaction between the MB and the surface of SLN.

14   

When SLNP1 – SLNP2 and SLNL1 – SLNL2 are compared to each other in terms of their entrapment efficiency, it is seen that even though their compositions are the same, their entrapment efficiencies are different. This may be attributed to surface charge densities. SLNL2, the larger particle, has the higher surface charge density and EE% (92.2%). Figure 2, displays the low interaction of SLNP1, SLNP2, SLN6, SLN8 and SLN9 with MB which results in a low amount of adsorption onto their surfaces. SLNL1, SLN5, SLN7, SLN10, SLN11 and SLN12 behave like more effective carriers, in terms of adsorption, than SLNP1, SLNP2, SLN6, SLN8 and SLN9. Table S1 shows the relationship between the entrapment efficiency of SLNs and their corresponding surface charge density. It is seen that at 5.0% and 5.2%, respectively, SLNP1 and SLNP2 have the lowest entrapment efficiency values among SLNs. When these results are evaluated together with surface charge density values, and if drug loading via electrostatic interaction between particle and MB is considered to be completed, the entrapment efficiency values appear to be acceptable. Table S1 also shows the adsorbed MB amount corresponding to the entrapment efficiencies. It can be seen that this amount changes between ~230-3400 µg/mg which, according to literature, is considered to be a high adsorption capability [27].

Moreover, in order to confirm the influence of surface charge density on the entrapment efficiencies of SLNs, the relation is demonstrated in Figure 3. Linear regression of MB entrapment data with a correlation coefficient of R2=0.9933 suggested a linear correlation between the entrapment efficiency and the surface charge density values of SLNs which depicts the importance of surface charge density calculation. As an exception, it is seen that SLNL2 which has the highest surface charge density value, has not been included into the trendline. SLNL2 adsorbed 92.2% of the total MB in the medium and it can be deduced that the adsorbed MB amount can be increased with the increasing MB concentration. Due to that, the deviation of the correlation of this particle from linearity can be explained with the MB concentration which is allowed to be entrapped on the surface of SLNL2.

3.4 In vitro release of MB from SLNs In the present study, the in vitro release of MB from SLNs for 24 hours was investigated. The release profile (Figure 4) demonstrates that nearly ~90% of the MB is released within the first 15   

4 hours and the release continues gradually over time, reaching a maximum before 24 hours (See Supplementary Material for in vitro release full profile). As the electrostatic interaction between MB molecules and the surface of the SLNs during loading is the dominant factor, the strength of this interaction plays important role in the release profile as well. The particles SLNP1, SLNP2, SLN6, SLN7, SLN8 and SLN9, which are ranked at the bottom of MB loading, have higher release efficiency compared to the others (Figure 4). However, in terms of concentration of MB released, the 89.7% and release efficiency of SLNP2 and 92.8% of SLNP1 correspond to ~0.12 µg/ml, which is a rather low value. The low amount of initial MB load can be the reason for this result. When we investigated the release performance of SLN5 and SLN10, we found that they had moderate surface charge density values which enables efficient cargo load/unload interactions when required.

To conclude, the entrapment and release efficiencies of SLNs are inversely proportional to each other, as expected, and can be seen in Figure 5. Due to electrostatic interactions acting as the dominant factor keeping MB on the surface of SLNs, the surface charge of nanoparticles, which strongly depends on the zeta potential and size of SLNs, becomes the key parameter to consider.                       

4. Conclusion The composition and operational parameters involved in the preparation of SLNs via microemulsion method have a significant impact on the formation of ME as well as the size and surface properties of SLNs. In our study, 12 types of SLNs have been successfully synthesized using stearic acid as the lipid material. Different combinations of lithocholic acid, Pluronic F127, Tween 20, lecithin and butanol have been used as emulsifiers. The dilution ratio in the preparation of SLNs directly affects the size of the particles and the obtained mean particle diameter is considerably small for 1:20 ME:water ratio. Besides, by changing the content of the particle, the size and the zeta potential values of SLNs can be accordingly adjusted. Additionally usage of co-emulsifiers decreases the particle diameter by ~30 nm when the appropriate co-emulsifier amount is used. Moreover, it is proved that the zeta potential value of the particles can be increased by using an ionic emulsifier such as lithocholic acid. This feature may be exploited in order to adjust the surface properties and 16   

prepare a nanomaterial appropriate for the intended function or use. The entrapment and release studies conducted with methylene blue (MB) as the model molecule showed that surface charge densities of the particles are quite effective on the entrapment performance and the highest surface charge density leads the highest entrapment efficiency (i.e. 92.2%). Similarly, the release capacity of the SLNs can be adjusted by reducing the surface charge densities which cause a weak electrostatic interaction between particle surface and MB. Ultimately, with the development of SLNs with various size and surface properties we provide the opportunity of having the control over their drug entrapment/release performance. This ability as well as the biocompatible and stable character of these SLNs makes them promising alternatives in the controlled drug delivery field.

Acknowledgements This work is supported by the Scientific and Technical Research Council of Turkey (TUBITAK) through the grand of the project with Grant Number 215M759.

17   

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[55] F.G. Gandolfo, A. Bot, E. Flöter, Phase diagram of mixtures of stearic acid and stearyl alcohol, Thermochim. Acta 404 (2003) 9-17. [56] J. Dai, J. Kim, Photo and thermal properties of cinnamoyl Pluronic F-127, Polym. Int. 63 (2014) 501-506. [57] S. Kumar, J.K. Randhawa, Solid lipid nanoparticles of stearic acid for the drug delivery of paliperidone, RSC Adv. 5 (2015) 68743-68750. [58] A. Carbone, A. Campisi, T. Musumeci, G. Raciti, R. Bonfanti, G. Puglisi, FA-loaded lipid drug delivery systems: Preparation, characterization and biological studies, Eur. J. Pharm. Sci. 52 (2014) 12-20. [59] H. Svilenov, C. Tzachev, Solid Lipid Nanoparticles – A Promising Drug Delivery System, in: A. Seifealian, A. de Mel, D.M. Kalaskar (Eds.), Nanomedicine, London, 2014, pp. 187-237. [60] G. Lagaly, Energetische Wechselwirkungen in Dispersionen und Emulsionen, in: H. Asche, D. Essig, P.C. Schmidt (Eds.), Technologie von Salben, Suspensionen und Emulsionen, APV paperback series, vol. 10. Wissenschaftliche Verlagsgesellschaft, Stuttgart, 1984, pp. 32 – 61 [61] C. Freitas, R.H. Müller, Correlation between long-term stability of solid lipid nanoparticles (SLN) and crystallinity of the lipid phase, Eur. J. Pharm. Biopharm. 47 (1999) 125-132. [62] A. Radomska-Soukharev, Stability of lipid excipients in solid lipid nanoparticles, Adv. Drug Deliv. Rev. 59(6) (2007) 411-418. [63] K. Westesen, B. Siekmann, Investigation of the gel formation of phospholipid-stabilized solid lipid nanoparticles, Int. J. Pharm. 151(1) (1997) 35-45.

23   

Figure 1. AFM images of SLNs in “Topography” mode.

24   

Figure 2. Loading of MB onto the surface of SLNs with time

25   

Figure 3. Influence of surface charge density on entrapment efficiency±SD (error bars) of SLNs (Linear regression line and coefficient of determination value don’t involve SLNL2)

Linear fit, R² = 0,9933

Figure 4. In vitro release profile of MB from SLNs.

26   

 

Figure 5. Entrapment efficiency (EE%) and release capacity (RC%) of SLNs (after 24 hours) 

27   

Table 1. Composition and physicochemical properties of the synthesized SLNs (mean±SD, n=3)

PDI

Zeta Potential (mV)

Surface Charge Density (σ) (C/m2)x1014

Diameter (nm) Formulation

Composition

Percentage (%)

SLNP1

Stearic acid Pluronic F127 Stearic acid Pluronic F127 Stearic acid Lithocholic acid Stearic acid Lithocholic acid Stearic acid Lecithin Tween 20 Stearic acid Pluronic F127 Tween 20 Stearic acid Lithocholic acid Tween 20 Stearic acid Pluronic F127 Tween 20 Lecithin Stearic acid Pluronic F127 Tween 20 Butanol Stearic acid Lithocholic acid Butanol Stearic acid Pluronic F127 Lithocholic acid Stearic acid Pluronic F127 Lithocholic acid Butanol

3.3 2.5 3.3 2.5 3.3 2.5 3.3 2.5 3.3 4 1.7 3.3 0.8 1.7 3.3 0.8 1.7 3.3 0.8 1.7 4 3.3 0.8 1.7 0.8 3.3 2.5 2.7 3.3 2.5 2.5 3.3 2.5 2.5 2.7

SLNP2 SLNL1 SLNL2 SLN5 SLN6 SLN7

SLN8

SLN9

SLN10 SLN11

SLN12

DLS

AFM

104±1.4

88±6

0.12±0.04

-28.2±1.9

18.8±0.6

148±1.3

135±2

0.22±0.05

-28.1±1.3

19.3±0.3

80±2.2

79±10

0.14±0.03

-42.4±1.1

160±15.0

162±2.4

144±8

0.29±0.02

-47±1.3

415±8.8

164±1.4

148±5

0.13±0.02

-23.6±0.5

143±12.8

72±0.5

62±3

0.21±0.03

-10±0.3

20±4.0

55±1.1

54±5

0.11±0.03

-35.3±1.3

57±0.8

88±1.3

85±3

0.13±0.05

-18.4±0.8

21.4±1.2

78±0.3

94±4

0.27±0.02

-13.7-±0.5

21.7±1.2

85±1.2

83±8

0.26±0.03

-44.1±2.1

136±12.3

39±0.1

40±6

0.12±0.02

-28.3±0.3

126±4.5

68±1.1

56±5

0.28±0.04

-31±0.2

119±11.5

28   

Table 2. Melting point (MP), melting enthalpy (ΔH) and crystallinity index (CI%) values of SLNs

ΔH (J/g)

CI %

P1

MP (oC) Tonset Tmax 64 68-54

36.2

19.1

L1

77

82-78

61.3

32.3

5

53

60

2.7

1.5

6

44

47

1.9

1.0

7

79

81

14.3

7.5

8

65

68

2.8

1.5

9

61

63

4.3

2.3

10

78

80

27.1

14.3

11

65

67-51

3.3

1.8

12

63

65

1.4

1.0

SLN

Table 3. The results at month 3 of the stability study for SLNs performed at room temperature (mean ± SD, n=3)

SLN P1 P2 L1 L2 5 6 7 8 9 10 11 12

Zeta Potential (mV) After After preparation months -28.2±1.9 -27.1±0.8 -28.1±1.3 -27.2±0.2 -42.4±1.1 -41.9±0.5 -47±1.3 -46.7±1.1 -23.6±0.5 -21.9±1.4 -10±0.3 -8.4±0.1 -35.3±1.3 -33.7±0.8 -18.4±0.8 -16.9±1.7 -13.7-±0.5 -12.4±0.8 -44.1±2.1 -41.9±1.0 -28.3±0.3 -26.7±0.2 -31±0.2 -30.6±1.2

Particle Diameter (nm) 3 After After preparation months 104±1.4 110±1.3 148±1.3 152±1.2 80±2.2 84.6±1.4 162±2.4 164.7±1.3 164±1.4 170.7±1.2 72±0.5 79.6±0.2 55±1.1 58.2±0.4 88±1.3 92.8±1.1 78±0.3 80±0.3 85±1.2 90.2±0.4 39±0.1 41±1.4 68±1.1 70±1.4

3

29