A pre-formulation study of tetracaine loaded in optimized nanostructured lipid carriers

This study reports the development of three optimized NLC pre-formulations capable of encapsulating TTC at high doses (4%). The NLC were prepared with mixtures of solid and liquid lipids (CP-TRANS, CP-DK and MM-DK), plus P68 as a stabilizer. The choice of lipids and their concentrations was based on previous reports^8,16. Differently than a previous report in the literature in which TTC was incorporated in NLC aimed for topical application³¹ the formulation in here is to be used by different routes of administration.

Experimental design

In order to analyze the factors that influence the different NLC formulations, three 2³ factorial designs with triplicate at the central point were carried out. Using three independent variables: (a) surfactant concentration (% P68), (b) total lipid concentration (% TL), and (c) solid:liquid lipid ratio (expressed as % SL), eleven NLC formulations (of nine different compositions) containing 4% TTC were prepared in each of the 2³ factorial design. Table S1 shows the composition of these systems and corresponding experimental responses (size, PDI, ZP), determined by DLS.

The mean particle size of the NLC ranged from 250 to 330 nm. Table S2 shows that in the formulations composed of CP-TRANS/TTC, two variables significantly affected the particle size: % P68, % SL and their interactions (p < 0.05), as exemplified in Fig. 1A. For CP-DK/TTC and MM-DK/TTC only % P68 (Fig. 1D,G and Table S2) significantly influenced particle size. The positive effect of the amount of solid lipid (% SL) on the size of CP-TRANS/TTC nanoparticles is a consequence of the higher viscosity of the lipid phase, reducing the effectiveness of particle breaking (homogenization and sonication) processes^32,33 and will also be discussed further (SANS and Raman Imaging data). For all the 3 NLC types the surfactant concentration (% P68) had a negative effect on the particle size, which means that the increase in the concentration of P68 decreases the particle size, due to the decrease of interfacial tension between the nanoparticles and the external phase³³.

The low PDI values (0.1 > PDI > 0.25) in Table S1 confirmed the homogenous distribution of particle sizes for CP-TRANS/TTC, CP-DK/TTC and MM-DK/TTC. Figure 1B,E,H and Table S2 revealed that only P68 had a significant (negative) effect, decreasing PDI in the 3 NLC formulations, a result that confirms that surfactants play a major role in determining the size distribution of the nanoparticles.

ZP values in the three types of formulations were in the range of − 20 to − 40 mV (Table S1) and P68 was the only variable that significantly affected ZP values in a negative way: the higher the P68 concentration the lower, in modulus, the surface electric potential of the NLC (Table S2, Fig. 1C,F,I). The negative ZP values are attributable to the polarization of surfactant P68, followed by adsorption of water molecules on the polarized surfaces of NLC^{32,33,34,35,37}.

Desirability functions were used to determine the preferred formulation, following the criteria: lower particle sizes, minimum PDI and maximum ZP values. According to that, the following systems were selected: CP-TRANS/TTC = 20% TL, 70:30% SL, and 4.4% P68, CP-DK/TTC = 18% TL, 70:30% SL, and 4.4% P68 and MM-DK/TTC = 17% TL, 72:28% SL, and 5% P68. The subsequent experiments were conducted only with these optimized formulations.

DLS and NTA results

Table 2 shows the average size, PDI, ZP and nanoparticle concentration (NC) values for the optimized formulations revealed by Factorial Design, and their respective controls. The three types of NLC formulations displayed sub-micron diameters (~ 200 nm) and monodisperse size distribution (0.1 < PDI < 0.20) with proper electrical charge repulsion between the particles (ZP > |17| mV) to ensure good shelf-stability^{36,37,38,39,40}. Visual analysis confirmed the homogeneous appearance of the fresh samples, with suspensions of whitish coloring, liquid consistency and no evidence of aggregates.

NTA is an alternative methodology to DLS for in vitro characterization of nanostructured colloidal systems⁹. NTA measurements allowed determination of the nanoparticles concentration (NC) in the optimized formulations (Table 2), an analytical parameter used in nanotoxicity, pharmacokinetic and stability (rupture, aggregation) studies of nanoparticles^41,42. The slightly lower NC values of CP-TRANS/TTC, CP-DK/TTC, MM-DK/TTC reflect their higher sizes, in comparison to the controls without tetracaine.

Most importantly, we have used the NC values and %EE to estimate the number of molecules of each excipient per nanoparticle⁸, as shown in Table S3. These numbers revealed a significant number of TTC molecules inside each particle (6–8 × 10⁵), corresponding to TTC:total lipid molar ratios of 0.14–0.17 that justify the increased diameter of TTC-containing nanoparticles.

Tetracaine encapsulation efficiency and drug loading

The three types of NLC formulations developed in this study showed very good capacity to carry TTC, with %EE values ranging from 63.7 to 68.1% and %DL > 11 (Table 2), reflecting the strong partition of the non-ionized form of TTC in the lipid milieu⁴³. Indeed, the encapsulation efficiency determined for TTC in these nanoparticles was in the range of those reported (> 55%) for other hydrophobic local anesthetics such as dibucaine, bupivacaine and ropivacaine^16,44,45 and higher (< 37%) than those observed in NLC with more hydrophilic agents such as lidocaine and prilocaine⁸. The drug loading capacity is another parameter that expresses the upload capacity of DDS (Eq. 2). The DL values of the optimized formulations for TTC were 11.1% with CP-TRANS/TTC and 11.6% with CP-DK/TTC and MM-DK/TTC, above those reported (%DL < 10) for other local anesthetics in NLC^16,46,47. %DL values were also in good agreement with the TTC:TL ratios (Table S3).

Transmission electron microscopy

TEM images showed that the nanoparticles, in despite of the different lipid matrices, had spherical morphology with a well-delimited surface (Fig. 2). In addition, encapsulation of TTC did not affect the integrity of the nanoparticles (Fig. 2A,C,E vs. B,D,F).

Infrared analysis (ATR-FTIR)

ATR-FTIR analyses were performed to investigate possible interactions between the anesthetic and the lipid matrix of NLC. Figure 3A shows the spectra of NLC excipients and TTC, while the spectra of the optimized NLC formulations and their controls are given in Fig. 3B. TTC spectrum showed absorption bands at 3370 and 1532 cm⁻¹ attributed to N–H groups and C–N stretching vibrations from the aromatic amine group, respectively, while the bands at 2952 and 2861 cm⁻¹ are due to asymmetric CH₃ stretching and symmetrical CH₂ stretching vibrations, respectively. Other bands at 1683 cm⁻¹ corresponding to the C=O stretching vibration of ester and 1600 cm⁻¹ due to C=C of the aromatic ring were detected, as well as those at 1168 and 1118 cm⁻¹ which refer to antisymmetric and symmetric stretching of C–O–C, respectively^5,48.

Control NLC (CP-TRANS, CP-DK and MM-DK) showed bands related to their major components (the solid lipids CP and MM) at 2917 cm⁻¹ and 2849–2850 cm⁻¹ corresponding, respectively, to ν_aC–H and ν_s C–H vibration modes of CH₂. Other bands related to ester bonds were observed in 1733 cm⁻¹ (ν C=O); 1463 cm⁻¹ and 1342–1343 cm⁻¹ (δ C–H in CH₂)^15,49. Finally, characteristic bands of P68 molecule were observed at 963 cm⁻¹ to 1108 cm⁻¹ and attributed to the symmetrical structure of C–O and the asymmetric stretching vibrations of C–O in the ether groups of –OCH₂CH₂ residues, repeated throughout the structure of P68⁵⁰.

ATR-FTIR spectra of NLC loading TTC showed similarities to those of the control NLC spectra (without TTC), indicating that incorporation of the anesthetic did not affect the overall arrangement of the NLC excipients in the nanoparticle⁸. Among all the NLC spectra, only those of formulations containing tetracaine exhibited typical bands of pure TTC at 1278 cm⁻¹ (C–N stretching) and 1683 cm⁻¹ (C=O stretching) which were shifted to 1281–1285 cm⁻¹ and 1603–1606 cm⁻¹, respectively. Such displacements confirm the insertion of TTC into the NLC, probably due to interactions between the amine groups of TTC and available groups of NLC matrices, as described before⁴⁹.

Differential scanning calorimetry (DSC) analysis

Figure 4A shows the thermograms obtained with the optimized NLC formulations, their major (solid lipid) excipients or TTC. The peak belonging to TTC (36.3 °C) could not be seen in any of the three optimized formulations, indicating insertion of the anesthetic inside the nanoparticles⁵¹. In agreement with the literature, endothermic peaks corresponding to the melting of CP and MM were observed at 54.7 and 42.6 °C, respectively^15,52. Incorporation of the liquid lipids slightly changed the transition of cetyl palmitate to higher (CP-TRANS) or lower temperatures (CP-DK) or, in the case of myristyl myristate, to lower temperatures (MM-DK) plus the appearance of another transition at higher temperature (53 °C). The decrease in the transition of the solid lipid, observed with CP-DK and MM-DK, are expected since the liquid lipid causes a reduction in crystallinity of the solid lipid⁵¹. As for the shift in the transition of cetyl palmitate to higher temperatures (56.5 °C in the case of CP-TRANS) it indicates an increase in the crystallinity index of the solid lipid inside the NLC, probably because of miscibility problems with TRANS (as will be discussed latter, in the Raman imaging results). This shift was also observed in the presence of tetracaine (56.8 °C for CP-TRANS/TTC).

X-ray diffraction (XRD) analysis

X-ray diffraction experiments provided information regarding the crystalline structure of NLC and TTC. Figure 4B shows the diffractograms of the optimized NLC formulations, their controls, major excipients (solid lipids, CP and MM) and TTC. The diffractogram of TTC showed three typical peaks at 2θ = 8.66°, 12.90° and 17.23°, and other peaks of lower intensity, confirming the crystalline nature of the anesthetic⁵³. These narrow peaks were not detected in the diffractograms of the optimized formulations, indicating that the anesthetic was solubilized in the lipid matrix of the NLC. In addition, diffractograms of control NLC (CP-TRANS, CP-DK and MM-DK) were different from those of the pure solid lipids (CP and MM), showing peaks of lower intensities (2θ = 6.94°, 21.72°, 24.02° for CP, and 2θ = 7.96°, 21.78°, 23.97° for MM, respectively). These data indicate the lower crystallinity of the NLC lipid matrix in relation to the pure lipids (CP, MM), thus reflecting a less ordered structure that results from the presence of liquid lipids in the core of the nanoparticles.

The diffraction patterns of TTC-containing particles CP-DK/TTC, MM-DK/TTC and their respective controls (CP-DK and MM-DK) were similar, confirming that addition of TTC did not change the overall organization of these nanoparticles, in agreement with TEM (Fig. 2) and DSC (Fig. 4A) data. In the case of CP-TRANS/TTC sample there is a narrowing in the more intense CP peaks at 21.72° and 24.02° promoted by TTC, indicating that the anesthetic increases the crystallinity of cetyl palmitate, in agreement with DSC results.

Small angle neutron scattering

SANS measurements were performed in order to get further information on the structural organization of the optimized NLC, with and without tetracaine. The samples were prepared in D₂O to reach a significant contrast between the solvent and the nanoparticles. First, all the NLC systems exhibit negligible changes when measurements were conducted at 25 °C and at 37 °C (as shown in Figure S1 for CP-DK/TTC and MM-DK/TTC). SANS data then revealed several systematic tendencies in the internal arrangement of the nanoparticles (Fig. 5). For those prepared with cetyl palmitate and Transcutol (CP-TRANS/TTC, CP-TRANS) correlation peaks in the SANS curves indicated the existence of lamellar structures inside the NLC (Fig. 5A), in agreement with previous reports in the literature, obtained with Electron Paramagnetic Resonance⁴⁵ and molecular Dynamics⁵⁴. Indeed, among the blends of solid and lipid lipids tested, cetyl palmitate and TRANS have the largest difference in polarity, and their SL:LL molar ratio (0.66) was the smallest among the three optimized formulations (Table S3). Because of that, the lamellar structure revealed by SANS results from the reorganization of CP molecules in the lipid NLC core, avoiding the contact with TRANS molecules (see “Discussion” below). Interestingly, and in agreement with that, the Design of Experiments study revealed that only for the CP-TRANS formulation (Fig. 1A) the amount of solid lipid (CP) played a significant effect, determining increased particles size.

Moreover, inclusion of tetracaine in the CP-TRANS nanoparticles induced a variation in the observed lamellar structure, since the 1 0-plane spacing (d10) diminished from 259 Å in CP-TRANS, to 230 Å in CP-TRANS/TTC. Such reduction in the lamellar interplanar distances (d10) suggests that tetracaine interacts with the CP molecules, decreasing the thickness or the lamellae by promoting lateral expansion, a phenomenon already observed for TTC in monolayers and bilayers^55,56. TTC really causes dynamic rearrangements in lamellar phases, as recently demonstrated by Hu et al. in dioleylphosphatidylcholine supported bilayers, increasing the lipid chain mobility and even inducing the formation of curved tubular structures prior to membrane disruption, at high TTC:lipid ratios⁵⁷.

No such correlation peaks were observed in the SANS profile of the NLC prepared with Dhaykol 6040 as the liquid lipid (Fig. 5B,C). But to gain a further insight about the DK-based NLC systems, we modeled the SANS data using Eq. (3), as shown in Figure S1 (“Supporting information”). The analysis of the correlation length parameter for the CP-DK and MM-DK samples showed that the latter had a bigger correlation length ((xi_{{MM{ – }DK}} = 78.68) Å) than the former ((xi_{{CP{ – }DK}} = 44.69) Å) suggesting that MM-DK nanoparticles had greater hydrophobic clusters (nanoclusters formed by the hydrophobic interactions between solid and liquid lipids)⁵², in comparison to CP-DK.

In the representations at Fig. 5D–F we depicted the different organizations proposed for the solid and liquid lipids inside the NLC, revealing the hydrophobic clusters observed for CP-DK and MM-DK, but not CP-TRANS. The size of the hydrophobic clusters did not change in the presence of TTC, nor with temperature (Figure S1).

Physicochemical stability studies

A long-term stability study was conducted with the optimized NLC formulations and their controls, by monitoring particle size, PDI, ZP, pH and visual aspects such as color and homogeneity for 365 days at 25 °C. The pH of the formulations remained in the range of 8.0–8.5. CP-DK/TTC and MM-DK/TTC and their respective controls did not show any significant variation by visual inspection or in any of the analyzed parameters over time (Fig. 6), reflecting the physical stability of these formulations (i.e. maintenance of nanoparticles structure).

On the other hand, CP-TRANS/TTC and CP-TRANS showed a significant increase in nanoparticle size and polydispersity (254.7 nm and 0.29, respectively, for CP-TRANS/TTC after 120 days) with ZP values tending to zero (p < 0.05) during storage (Fig. 6). Visual analysis confirmed the instability of this formulation, with phase separation starting after 30 days that prevented analyses after 120 days. Therefore, CP-TRANS NLC was found unstable over time, with changes in particle size, polydispersity and ZP values compatible with particle aggregation^37,21.

Miscibility of lipid excipients measured by Raman mapping

In an attempt to get more information on the stability of the formulations, Raman imaging analyses were used to evaluate the miscibility of their lipid components. Figure S2 shows Raman spectra of solid and liquid lipids, and their mixtures. These spectra were very similar to prior reports in literature and also previous works from our group^16,22. As expected, solid lipids had narrower bands than liquid lipids because of their more ordered molecular structures. Assignment of the main bands of each excipient is given in Table S4.

In this case, univariate methods (i.e. single wavenumber) could not be used to treat the data due to the high spectral overlap (Figure S2). Therefore, and since the spectrum of each individual component was available, the multivariate CLS method was employed, allowing the use of all spectra information to generate the chemical images. Figure 7 shows chemical imaging and histograms obtained for the three pairs of solid and liquid lipids of the optimized NLC. Each pair of SL/LL is shown on the left and right sides, respectively of Fig. 7A–C. The predicted mean scores and their ranges for each component in the pixels are given in the histograms. The scores are in the same scale, so they can be directly compared to evaluate lipid miscibility. SD_hist values of 13.6, 3.2 and 6.1 were found for the lipid mixtures CP-TRANS, CP-DK and MM-DK, respectively. Pixels with 0 or 100 score values would indicate full immiscibility between excipients, but they were not observed in any of the mixtures. CP-TRANS sample showed very wide histograms, with two maxima (Fig. 7A). Such behavior is a clear indication of aggregation, with CP concentrated in the right side (red in the chemical map) and TRANS condensed in the left side (green in the chemical map). The histograms of CP-DK were the narrowest ones (Fig. 7B), and MM-DK showed an intermediate behavior (Fig. 7C). It should be noted that unlike CP-TRANS, the last two systems showed a single distribution, with only one maximum for each (SL and LL) excipient. According to the Raman image analyses, the degree of miscibility in three mixtures decreased in the order: CP-DK > MM-DK >  > CP-TRANS, corroborating the SANS results (Fig. 5). These results also explain the instability of CP-TRANS under storage, as revealed by DLS (Fig. 6), and confirm the applicability of Raman mapping for the selection of excipients in pharmaceutical studies^22,23. So, for stability reasons, only the formulations containing DK as the liquid lipid were used in subsequent steps.

In vitro release kinetics

The in vitro release of TTC, in solution and encapsulated in CP-DK/TTC and MM-DK/TTC formulation (Fig. 8), was measured during 48 h at 37 °C. TTC in solution reached equilibrium (100% release) in 6 h. The NLC formulations displayed slower TTC release: 40.9% and 93.0%, respectively for CP-DK/TTC and MM-DK/TTC, after 48 h. The release curves were treated with several kinetic models, and the better fit (r² = 0.9991, and 0.9978, for CP-DK/TTC and MM-DK/TTC, respectively) was found with the Korsmeyer–Peppas model (Eq. 4). The determined values of n were 0.47 (CP-DK/TTC) and 0.49 (MM + DK/TTC). According to the Korsmeyer-Peppas model, n values from 0.43 to 1 indicate an anomalous, non-Fickian transport³⁰. This means that probably two mechanisms drove the release of TTC: an initial “burst” release due to the non-encapsulated TTC (ca. 35%, see Table 2), and a sustained release regimen related to the fraction of TTC loaded by the nanoparticles. The prolonged release of TTC encapsulated in CP-DK/TTC correlates well with the higher degree of miscibility in the lipid core of these nanoparticles in comparison to MM-CK/TTC, as revealed by Raman Imaging (Fig. 7) and SANS (Fig. 5) data. This modeling confirmed the NLC ability to extend the release of local anesthetics, as previously observed¹⁶.

In vitro cytotoxicity tests

Finally, we evaluated the cytotoxicity of the optimized (CP-DK/TTC and MM-DK/TTC) formulations through the MTT test (Fig. 9), in cultures of murine Balb/c 3T3 fibroblasts. After 24 h of treatment, the IC₅₀ of free TTC was 0.6 mM, in good agreement with the literature²⁴. Slightly higher IC₅₀ values were detected with CP-DK/TTC and MM-DK/TTC formulations (0.7 mM and 0.9 mM, respectively), showing that the NLC formulations decrease the intrinsic cytotoxicity of TTC, probably due to the sustained drug release over time. Similar observations were reported for TTC encapsulated in NLC composed of glyceryl monostearate, oleic acid and Tween 80³¹. Control formulations (CP-DK and MM-DK) were also tested, and they showed no (MM-DK) or low (CP-DK) effect over cell viability at the concentrations tested, indicating the safety of the nanocarrier systems.