Risperidone-Loaded PLGA–Lipid Particles with Improved Release Kinetics: Manufacturing and Detailed Characterization by Electron Microscopy and Nano-CT

For parenteral controlled drug release, the desired zero order release profile with no lag time is often difficult to achieve. To overcome the undesired lag time of the current commercial risperidone controlled release formulation, we developed PLGA–lipid microcapsules (MCs) and PLGA–lipid microgels (MGs). The lipid phase was composed of middle chain triglycerides (MCT) or isopropylmyristate (IPM). Hydroxystearic acid was used as an oleogelator. The three-dimensional inner structure of Risperidone-loaded MCs and MGs was assessed by using the invasive method of electron microscopy with focused ion beam cutting (FIB-SEM) and the noninvasive method of high-resolution nanoscale X-ray computed tomography (nano-CT). FIB-SEM and nano-CT measurements revealed the presence of highly dispersed spherical structures around two micrometres in size. Drug release kinetics did strongly depend on the used lipid phase and the presence or absence of hydroxystearic acid. We achieved a nearly zero order release without a lag time over 60 days with the MC-MCT formulation. In conclusion, the developed lipid-PLGA microparticles are attractive alternatives to pure PLGA-based particles. The advantages include improved release profiles, which can be easily tuned by the lipid composition.


Introduction
The use of parenteral controlled release drug delivery systems (CR-DDS) is a highly attractive and rational way to improve drug therapy. Potential benefits include decrease of administration frequency, increase of bioavailability and less side effects. Polylactic acid (PLA) and poly(lactic-co-glycolic acid The properties and the release kinetics of the micro-DDS will depend on the general structure and the physicochemical state of the drug and the excipients. Possible structures of (drug-free) micro-DDS are shown in Figure 1. PLGA microparticles are expected to form a homogeneous polymer matrix (structure I). Microcapsules composed of PLGA and oil could show structures with a single oily core and a PLGA shell (structure II) or multiple oily droplets encapsulated in PLGA (structure III). The addition of the oil gelator will increase the viscosity, but again, the oily phase could exist as a single core (structure IV) or as multiple cores (structure V). The drug could be dissolved in the polymer and the oil and/or exist as an own solid (crystalline or amorphous) phase. A rational development of the DDS requires an appropriate characterization. For particle sizing of micro-DDS, static light scattering is the standard method. We decided to use X-ray diffraction for the detection of crystalline structures in our DDS. In addition, we wanted to explore the microstructure inside the DDS. For this purpose, we used two complementary imaging techniques, which were focus ion beam scanning electron microscopy (FIB-SEM) and Zernike phase-contrast X-ray nano-computed tomography (nano-CT). By means of focused ion-beam preparation, we could cut the particles in situ and we were able to visualize the morphology of the microstructure at the location of the cut. In addition, we performed nano-CT experiments. Due to the high penetration depth of X-rays, nano-CT provided highresolution three-dimensional images of the DDS and we were able to visualize their internal structures at any virtual slice without the need for cutting or slicing them.
In summary, the main goals of this work were: (i) development of risperidone loaded PLGA-lipid microcapsules (MCs) and PLGA-lipid microgels (MGs) with an optimized release profile (no lag time, constant release rate) (ii) characterization of the three-dimensional inner structure of risperidone-loaded MCs and MGs by using focused ion beam (FIB) preparation and three-dimensional X-ray imaging (nano-CT).

Materials
Expansorb  PLGA microparticles are expected to form a homogeneous polymer matrix (structure I). Microcapsules composed of PLGA and oil could show structures with a single oily core and a PLGA shell (structure II) or multiple oily droplets encapsulated in PLGA (structure III). The addition of the oil gelator will increase the viscosity, but again, the oily phase could exist as a single core (structure IV) or as multiple cores (structure V). The drug could be dissolved in the polymer and the oil and/or exist as an own solid (crystalline or amorphous) phase. A rational development of the DDS requires an appropriate characterization. For particle sizing of micro-DDS, static light scattering is the standard method. We decided to use X-ray diffraction for the detection of crystalline structures in our DDS. In addition, we wanted to explore the microstructure inside the DDS. For this purpose, we used two complementary imaging techniques, which were focus ion beam scanning electron microscopy (FIB-SEM) and Zernike phase-contrast X-ray nano-computed tomography (nano-CT). By means of focused ion-beam preparation, we could cut the particles in situ and we were able to visualize the morphology of the microstructure at the location of the cut. In addition, we performed nano-CT experiments. Due to the high penetration depth of X-rays, nano-CT provided high-resolution three-dimensional images of the DDS and we were able to visualize their internal structures at any virtual slice without the need for cutting or slicing them.
In summary, the main goals of this work were: (i) development of risperidone loaded PLGA-lipid microcapsules (MCs) and PLGA-lipid microgels (MGs) with an optimized release profile (no lag time, constant release rate) (ii) characterization of the three-dimensional inner structure of risperidone-loaded MCs and MGs by using focused ion beam (FIB) preparation and three-dimensional X-ray imaging (nano-CT).

Materials
Expansorb Risperidone-loaded microcapsules were prepared using an oil-in-water (o/w) emulsion solvent extraction/evaporation technique.
For this purpose, 100 mg PLGA 75-7E, 100 mg of the oil phase (MCT or IPM), and 50 mg of Risperidone were weighted into a 4 mL glass vial and dissolved into the final volume of 2 mL dichloromethane by vortexing. The organic solutions were drawn up into a 2.5 mL GASTIGHT ® syringe (Hamilton Germany GmbH, Planegg, Martinsried, Germany) and injected with a flow rate 5.2 µL/min by a syringe pump (Pump 11 Elite, Harvard Apparatus, Holliston, MA, USA) directly into 300 mL of aqueous PVA 4-88 (Merck, Darmstadt, Germany) solution 0.25% (w/v) stirred with a Rotating Paddle, USP at 240 rpm at room temperature. The aqueous phase contains double distilled and filtrated water with 0.25% polyvinyl alcohol PVA 4-88 (Merck, Darmstadt, Germany). When particle formation was completed, the suspensions were transferred into a 500 mL round bottom flask and the remaining DCM were removed under vacuum 40 mbar for 20 min. Thereafter, sedimented microcapsules were transferred by a glass pipette into a 15 mL tube and the supernatants were removed carefully. Then, the microcapsules were washed thrice with sterile filtrated double distilled water, filled up to the final volume of 1 mL and lyophilized.

Lyophilization
For freeze-drying, the samples were frozen rapidly with liquid nitrogen at −196 • C and lyophilised on a Christ Alpha 2-4 freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) in combination with a Vacuubrand RC 6 vacuum pump (Vacuubrand GmbH, Wertheim, Germany) for 24 h. The chamber was evacuated to 0.05 mbar, corresponding to −48 • C on the sublimation curve of ice. The prepared microcapsules were stored at −20 • C until further use.

Static Light Scattering Measurements
Routine measurements of the size distribution were carried out by laser diffractometry on a Mastersizer 2000 (Malvern Instruments, Malvern, United Kingdom) combined with a Hydro 2000 S wet dispersion unit. The samples were measured at a laser obscuration of 5%, corresponding to 11%-15% obscuration of the blue laser, in purified water. A series of five runs was evaluated by the Mastersizer 2000 software version 5.60, using the Mie theory, assuming spherical particles with a refractive index of 1.44, absorption of 0.001, and a refractive index of 1.33 for the dispersant as optical properties.

Powder XRD
XRD analysis was performed on a Bruker D8-Advance diffractometer, equipped with a one-dimensional silicon strip detector (LynxEye) operating with Cu Kα radiation. Diffraction was measured from 2θ = 5 • to 70 • with a step size of 0.01 • and a counting time of 1 s.

High-Performance Liquid Chromatography (HPLC)
A HPLC Agilent 1100 Series (Agilent, Santa Clara, CA, USA) was used. Separations were carried out using a BDS-Hypersil ® C18 5 µm 250 × 4.6 column (Thermo Fisher Scientific, Waltham, MA, USA) and a mobile phase of 75% (v/v) methanol/25% (v/v) water, 1% (v/v) tetramethylammoniumhydroxid pentahydrate solution 25% w/v. Runs were carried out at 0.5 mL/min over 16 min and the absorption at 280 nm was recorded. The retention time of risperidone was 7.5 min. Injection volumes of 20 µL were used for determination of drug loading. Risperidone-loaded microcapsules were weighed and diluted in methanol to get a final concentration of 0.5mg/mL. All the samples were sonicated for 10 min to accelerate the dissolving of MC. Thereafter all samples were diluted 0.05 mg/mL and filtered (Minisart SRP 4, 0.45 µm syringe filter, Sartorius Stedim Biotech GmbH, Göttingen, Deutschland). The concentration of risperidone was determined via HPLC. Drug loading and EE were calculated as following equations from literature: Drug loading = (weight of drug entrapped/weight of MC used) × 100%; EE = (experimental drug loading/theoretically drug) × 100% [23].

In Vitro Drug Release Assay
The in vitro release of risperidone from MCs was carried out in a heated bath shaker (Memmert GmbH + Co. KG, Schwabach, Germany) at 37 • C and constant shaking at 50 rpm. A total of 6 mg of formulation I-IV and 3 mg of Risperdal Consta ® were suspended in 1 mL of 10 mmol phosphate buffer saline (PBS) at pH 7.4 and transferred into a Spectra Por Float-A_Lyzer ® G2 1000kD. The float-A Lyzer was placed into a 100 mL measuring cylinder and filled up with PBS to the final volume of 60 mL so that the float-A Lyzer is submerged in PBS. To maintain sink conditions, the buffer solution was drawn off half (half change) when the risperidone concentration reaches 7%-8% of the solubility in PBS. Drug quantity was determined by UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). All measurements were performed in triplicate and the measurement points were represented as the mean ± SD.

Scanning Electron Microscopy and Focused Ion Beam Preparation
High resolution investigations of the microparticle morphology were done by scanning electron microscopy (SEM) using a Quanta 3D FEG (field emission gun) instrument from FEI Company, (Hillsboro, OH, USA). The microparticles were carefully placed on SEM sample holders using carbon tape and subsequently coated with a thin layer of platinum using a magnetron sputtering system (HVD, Dresden, Germany) to achieve a conducting surface. The SEM images were obtained under high vacuum conditions using an acceleration voltage of 5 keV, a working distance of 6-10 mm and electron beam currents of 12 pA determined with an Everhart-Thornley detector.
The microscope is designed as a dual beam (electron and ion beam) which gives the possibility to perform FIB preparations in the same device. Here, the gallium ion beam and the electron beam operate independently of each other. The point of coincidence of the two beams is located at a working distance of 10 mm. The angle between both beams is 52 • . To allow vertical cutting with the FIB, the sample was tilted by this angle. Thus observation of the cross-sections with the electron beam was also done at an angle of 52 • . After screening the samples by conventional SEM, the particle of interest was selected for cross-sectional preparation (target preparation). As the first step in cross-section preparation at predefined areas of interest coarse material was ablated with gallium ions accelerated at 30 kV with ion currents of 15 nA. After this coarse milling step lower beam currents of Afterwards, 1-0.5 nA were used to polish the cross sections. The samples were not cooled during FIB milling. The patterning conditions used conform to the standard patterning conditions for silicon materials. The SEM observation can be done during ion milling or after subsequent milling steps.
2.2.9. Nano-CT X-ray imaging experiments were performed at the Institute of Physics, Martin Luther University Halle-Wittenberg, in a Carl Zeiss Xradia 810 Ultra (Cr source, 5.4 keV, for instrument details see [24]). Phase-contrast imaging mode was used with a phase-ring positioned near the back focal plane of the zone plate. In each experiment, a total of 901 projections with a field-of-view (FOV) of 65 µm were obtained over 180 • , with an exposure time of 15 s per projection, a detector binning of 2, and voxel size of 128 nm. Image reconstruction was performed by filtered back-projection algorithm using the software integrated into the Xradia 810 Ultra. For each sample, two or three imaging experiments were performed, moving the sample vertically to enable the visualization of one entire microsphere in one single direction (from now on named Z-direction). For each sample's datasets, the brightness and contrast were adjusted in ImageJ ® and the datasets were then stitched together using the Merge module of the Thermo Fisher Scientific Avizo ® software (version 9.2, Thermo Fisher Scientific, Hillsboro, OR, USA). Median denoising and nonlocal mean filter were applied to the datasets. The 3D renderings presented here were created using either the commercial software arivis Vision4D ® (version 2.12.6, arivis AG, Rostock, Germany) or Avizo ® . For the dispersed internal spherical structures (ISS) evaluation, a volume located in the center of the 3D image was extracted consisting of 325 × 325 × Z voxels. Z varies among the samples, as it is almost equal to the number of pixels of the microsphere diameter, but is smaller than it to prevent voxels that do not consist of sample to be added to the calculation. The results obtained here represent the ratio of ISS compared to the total extracted volume of the sample (PLGA+ISS). For binarization of the datasets into PLGA and ISS, automatic thresholding with IsoData criterion was used, followed by island removal for the removal of small clusters of voxels (<15). A surface was generated from the segmented dataset in order to smooth sharp voxel edges and the specific surface area (SSA) values obtained represent the ratio of the constructed surface area (SA) to the volume (V) for each sample.

Preparation of MCs and MGs
For the preparation of MCs and MGs, the conventional oil-in-water (o/w) emulsion solvent evaporation method was used [2,25]. All components were dissolved in dichloromethane. The components of the four different formulations are listed in the Table 1. In general, the composition followed a 2:2:1 mass ratio of PLGA/Lipid/Risperidone. Therefore, the theoretical drug load was 20% [m/m]. High encapsulation efficiencies (>92%) were achieved, as seen in Table 1. To get reproducible particle sizes, it was important to keep all size-influencing parameters constant, e.g., the o-phase volume and solvent, the concentration and type of polymer, the volume of the continuous phase and the type and concentration of stabilizer, the temperature, and the stirring speed [26]. After lyophilization the MCs did not agglomerate and were resuspendable in water.

Particle Size Measurements
The particle size and morphology of MC are key factors which potentially affect EE, drug release rate, and the biodistribution [27,28]. The optimal size range for microparticles with controlled release characteristics is considered to be 10-200 µm. Particles in the lower micron range will show a faster drug release, and they are also more easily phagocytosed by immune cells [29]. Sizes larger than 200 µm are difficult to inject and will show more inhomogeneous degradation [30]. The particle size distribution is shown in Figure 2.
Pharmaceutics 2019, 11, x 7 of 18 µ m are difficult to inject and will show more inhomogeneous degradation [30]. The particle size distribution is shown in Figure 2.  Table 2. It can be concluded, therefore, that the incorporation of hydroxystearic acid leads to slightly smaller particles, probably due to the impact on the surface tension during particle formation. The determined D(0.5) of the market product is 89.1 µ m, which is in line with a size range between 25 and 150 µ m reported in the literature [31]. The particle size distribution of the commercial product is considerably broader compared to our formulations (Span Risperdal Consta ® 1.165 vs. span values between 0.612 and 0.637), as seen in Table  2.   Table 2. It can be concluded, therefore, that the incorporation of hydroxystearic acid leads to slightly smaller particles, probably due to the impact on the surface tension during particle formation. The determined D (0.5) of the market product is 89.1 µm, which is in line with a size range between 25 and 150 µm reported in the literature [31]. The particle size distribution of the commercial product is considerably broader compared to our formulations (Span Risperdal Consta ® 1.165 vs. span values between 0.612 and 0.637), as seen in Table 2.  Figure 3. The market product Risperdal Consta ® shows a high heterogeneity of particle size, shape, and surface structures. Surprisingly, structures showing partial engulfment, as seen in Figure 3d, and collapsed structures are visible, as seen in Figure 3c. Larger magnification of the surface indicated the presence of anisotropic material, which are most likely drug crystals, as seen in Figure 3e,f.  Figure 3. The market product Risperdal Consta ® shows a high heterogeneity of particle size, shape, and surface structures. Surprisingly, structures showing partial engulfment, as seen in Figure 3d, and collapsed structures are visible, as seen in Figure 3c. Larger magnification of the surface indicated the presence of anisotropic material, which are most likely drug crystals, as seen in Figure 3e,f. The microcapsules and microgels do not show the irregular structures of the commercial product. Most of MCT microcapsules are spherical, some show an elongated shape, which probably results from the coalescence of two particles, as seen in Figure 4a, white stars. The surface of the MC-MCT microcapsules shows a pattern similar to golf balls, as seen in Figure 4b,c. Very rarely, small anisotropic structures are found on the particle surface, as seen in Figure 4c, top middle, which most likely represent risperidone crystals. Cutting of the particles by focused ion beam (FIB) provides insights into the internal structure, as seen in Figure 4b and d. A multicore structure with dimensions in the lower micron range becomes visible. Within the internal structures, anisotropic shapes are visible which most likely present precipitated drug crystals, as seen in Figure 4d. The microcapsules and microgels do not show the irregular structures of the commercial product. Most of MCT microcapsules are spherical, some show an elongated shape, which probably results from the coalescence of two particles, as seen in Figure 4a, white stars. The surface of the MC-MCT microcapsules shows a pattern similar to golf balls, as seen in Figure 4b,c. Very rarely, small anisotropic structures are found on the particle surface, as seen in Figure 4c, top middle, which most likely represent risperidone crystals. Cutting of the particles by focused ion beam (FIB) provides insights into the internal structure, as seen in Figure 4b,d. A multicore structure with dimensions in the lower micron range becomes visible. Within the internal structures, anisotropic shapes are visible which most likely present precipitated drug crystals, as seen in Figure 4d.
The electron microscopic pictures of the corresponding microgel MG-MCT shows spherical particles with a structured surface that appears smoother compared to the MC-MCT surface ( Figure 5). In contrast to the MC-MCT microcapsules, we could not detect elongated shapes, as indicated by white stars in Figure 4a, which emerge from the coalescence of two particles. The pictures obtained by FIB cutting of the particles show internal multicore structures with dimensions in the lower micron range, as seen in Figure 5b,d.
IPM-loaded microcapsules (MC-IPM) have a spherical shape, as seen in Figure 6a. The surface appears similar to the MC-MCT capsules. Very rarely, small crystals can be detected on the particle surface, as seen in Figure 6c. After FIB cutting, multicore structures in the lower micron range become visible, as seen in Figure 6b,d.
The electron microscopy investigation on MG-IPM microgels shows the presence of spherical particles, as seen in Figure 7a. Higher magnifications of the particle surface show surface dips ("golf ball structure") similar to the MG-MCT microgels. In addition, elongated "hair-like" structures are visible, as seen in Figure 7c,d, which are not detectable in the other samples. Most likely, they are mainly composed of hydroxystearic acid, which is known to induce gel formation by elongated structures. Again, FIB cutting was used to explore the interior of the particles. Multicore microdomains became visible, as seen in Figure 7b, similar to the MG-MCT microgel particles with dimensions in the lower micron range.   Table  1). Intact particles are presented at lower (a) and (c) higher magnification. Focused ion beam (FIB) cut particles are shown at lower magnification in 4b and after deeper FIB milling and higher magnification in 4d. The white stars in Figure 4a indicate anisotropic particles, which were most likely formed by coalescence of two particles.
The electron microscopic pictures of the corresponding microgel MG-MCT shows spherical particles with a structured surface that appears smoother compared to the MC-MCT surface ( Figure  5). In contrast to the MC-MCT microcapsules, we could not detect elongated shapes, as indicated by white stars in Figure 4a, which emerge from the coalescence of two particles. The pictures obtained by FIB cutting of the particles show internal multicore structures with dimensions in the lower micron range, as seen in Figure 5b,d.  Table 1). Intact particles are presented at lower (a,c) higher magnification. Focused ion beam (FIB) cut particles are shown at lower magnification in (b) and after deeper FIB milling and higher magnification in (d). The white stars in (a) indicate anisotropic particles, which were most likely formed by coalescence of two particles.  Table  1). Intact particles are presented at lower (a) and (c) higher magnification. Focused ion beam (FIB) cut particles are shown at lower magnification in 4b and after deeper FIB milling and higher magnification in 4d. The white stars in Figure 4a indicate anisotropic particles, which were most likely formed by coalescence of two particles.
The electron microscopic pictures of the corresponding microgel MG-MCT shows spherical particles with a structured surface that appears smoother compared to the MC-MCT surface ( Figure  5). In contrast to the MC-MCT microcapsules, we could not detect elongated shapes, as indicated by white stars in Figure 4a, which emerge from the coalescence of two particles. The pictures obtained by FIB cutting of the particles show internal multicore structures with dimensions in the lower micron range, as seen in Figure 5b,d.  Table 1). Intact particles are presented at lower (a,c) higher magnification. FIB cut particles are shown at lower magnification in (b) and at higher magnification at (d).
particles are presented at lower (a) and (c) higher magnification. FIB cut particles are shown at lower magnification in (b) and at higher magnification at (d).
IPM-loaded microcapsules (MC-IPM) have a spherical shape, as seen in Figure 6a. The surface appears similar to the MC-MCT capsules. Very rarely, small crystals can be detected on the particle surface, as seen in Figure 6c. After FIB cutting, multicore structures in the lower micron range become visible, as seen in Figure 6b,d.  Table 1). Intact particles are presented at lower (a) and (c) higher magnification. FIB cut particles are shown at lower magnification in (b) and at higher magnification at (d).
The electron microscopy investigation on MG-IPM microgels shows the presence of spherical particles, as seen in Figure 7a. Higher magnifications of the particle surface show surface dips ("golf ball structure") similar to the MG-MCT microgels. In addition, elongated "hair-like" structures are visible, as seen in Figure 7c,d, which are not detectable in the other samples. Most likely, they are mainly composed of hydroxystearic acid, which is known to induce gel formation by elongated structures. Again, FIB cutting was used to explore the interior of the particles. Multicore microdomains became visible, as seen in Figure 7b, similar to the MG-MCT microgel particles with dimensions in the lower micron range.  Table 1). Intact particles are presented at lower (a,c) higher magnification. FIB cut particles are shown at lower magnification in (b) and at higher magnification at (d).   Table  1). Intact particles are presented at lower (a,b) and higher (c,d) magnifications. A FIB cut particle is shown in 7b. Elongated, "worm like" structures (most likely precipitated hydroxyl-stearic acid) are visible in 7c and 7d.
The crystallinity of the formulations was evaluated by X-ray diffraction. The diffractograms of PLGA, the pure drug risperidone, the commercial product, and the developed microcapsules and microgels are shown in Figure 8. It is known that the drug can exist in different polymorphs [32][33][34]. As expected, the polymer PLGA does not show sharp peaks, but only broad lines, indicating an amorphous state. The pure drug shows multiple sharp peaks. The X-ray diffractograms of the commercial product Risperdal Consta ® and the developed formulations show a similar pattern, but  Table 1). Intact particles are presented at lower (a,b) and higher (c,d) magnifications. A FIB cut particle is shown in (b). Elongated, "worm like" structures (most likely precipitated hydroxyl-stearic acid) are visible in (c,d).
The crystallinity of the formulations was evaluated by X-ray diffraction. The diffractograms of PLGA, the pure drug risperidone, the commercial product, and the developed microcapsules and microgels are shown in Figure 8. It is known that the drug can exist in different polymorphs [32][33][34]. As expected, the polymer PLGA does not show sharp peaks, but only broad lines, indicating an amorphous state. The pure drug shows multiple sharp peaks. The X-ray diffractograms of the commercial product Risperdal Consta ® and the developed formulations show a similar pattern, but they are different from the pure drug. From the measurements, it can be concluded that risperidone exists in all formulations at least partially in the crystalline state. The crystalline state in the formulation is different from the crystal structure of the parent drug, but very similar to the crystal form of risperidone, which has been observed after micronization with supercritical fluids [33].

X-Ray Nano-imaging (nano-CT)
X-ray imaging comprises a class of nondestructive techniques that are used to visualize the internal structure of different materials. In conventional X-ray imaging, differences in the X-ray absorption cross-section of different materials results in the image contrast [35,36]. Therefore, if the different parts composing one sample show only small differences in the X-ray attenuation, a poor image contrast will be obtained in conventional absorption-based X-ray imaging. This is specially the case for samples composed of elements with low electron density, making the characterization of the structures and visualization of the sample features a very hard task. Alternatively, phase-contrast imaging methods enable the visualization of features of weakly absorbing objects [35,37,38]. X-ray phase-contrast imaging is based on a different physical principle, and the contrast of the image is generated by the detection of the changes in the phase of the wavefront [36,39]. In this work, we imaged the different microspheres using Zernike phase-contrast imaging in a benchtop nano-CT scanner (Xradia Ultra 810). Phase-contrast nano-CT was used to characterize the inner structure of the commercial product Risperdal Consta ® (Figure 9) and the developed microcapsules and microgels ( Figure 10). Supplementary Videos S1-S5 show the inner 3D structure of the microparticles. The diameter and volume distributions of the internal structures are presented in Supplementary Figures S1 and S2.

X-Ray Nano-Imaging (Nano-CT)
X-ray imaging comprises a class of nondestructive techniques that are used to visualize the internal structure of different materials. In conventional X-ray imaging, differences in the X-ray absorption cross-section of different materials results in the image contrast [35,36]. Therefore, if the different parts composing one sample show only small differences in the X-ray attenuation, a poor image contrast will be obtained in conventional absorption-based X-ray imaging. This is specially the case for samples composed of elements with low electron density, making the characterization of the structures and visualization of the sample features a very hard task. Alternatively, phase-contrast imaging methods enable the visualization of features of weakly absorbing objects [35,37,38]. X-ray phase-contrast imaging is based on a different physical principle, and the contrast of the image is generated by the detection of the changes in the phase of the wavefront [36,39]. In this work, we imaged the different microspheres using Zernike phase-contrast imaging in a benchtop nano-CT scanner (Xradia Ultra 810). Phase-contrast nano-CT was used to characterize the inner structure of the commercial product Risperdal Consta ® (Figure 9) and the developed microcapsules and microgels ( Figure 10). Videos Supplementary Videos S1-S5 show the inner 3D structure of the microparticles. The diameter and volume distributions of the internal structures are presented in Supplementary Figures S1 and S2.  All microgels and microcapsules show internal spherical structures (ISS) in the submicrometer range, with sphericity values close to unit. However, the volume percentage of the ISS in the microcapsules is between 35% and 46% and is, therefore, much higher than the volume percentage of the ISS phase in the Risperdal Consta ® particles. The ISS phase can be attributed to the dispersed lipid phase for the microgels and microcapsules. In contrast, the ISS consists of risperidone only for All microgels and microcapsules show internal spherical structures (ISS) in the submicrometer range, with sphericity values close to unit. However, the volume percentage of the ISS in the microcapsules is between 35% and 46% and is, therefore, much higher than the volume percentage of the ISS phase in the Risperdal Consta ® particles. The ISS phase can be attributed to the dispersed lipid phase for the microgels and microcapsules. In contrast, the ISS consists of risperidone only for the Risperdal Consta ® system. A quantitative treatment of the 3D images indicates that the ISS have diameter around 2 µm for lipid-based systems and around 800 nm for the Risperdal Consta ® product, as seen in Table 3. The ISS diameter and volume distributions are shown in Figures S1 and S2. Compared to other imaging techniques, Zernike phase-contrast nano-CT has the advantage of producing high-resolution three-dimensional images of low-absorbing samples, which enables the estimation of the values shown here. As with most high-resolution imaging techniques, nano-CT has the disadvantage of producing images limited to a small field of view, thus, to obtain the images of one single microparticle, it was necessary to image the sample two or three times and stitch the datasets. One of the benefits of phase-contrast nano-CT for imaging low-absorbing samples is that it does not require any special sample preparation procedure or induce any sample damage within the imaged area, in contrast with FIB-SEM. Overall, the results obtained by FIB-SEM correlate well with those obtained by nano-CT, and the same structures were observed. Both methods indicate the presence of a high percentage of internal structures (ISS) with sizes around two micrometers. Therefore, the microcapsules and microgels represent structures III and V of Figure 1, respectively.

Drug Release
The in vitro release of risperidone from the commercial product in the developed formulations is shown in Figure 11. For all formulations, a low percentage of the drug (<10%) is initially released. Most likely, drug molecules on the surface (which were observed by electron microscopy) are released very quickly. After the initial release, there is almost no release detectable for two weeks for Risperdal Consta ® . Thereafter, the release accelerates. The developed formulations show different release profiles. The fastest release was observed for the MG-IPM microgel. The slowest release rate observed for the lipid formulations was for the MC-IPM microcapsules system. Therefore, it can be concluded that the oleogelator hydroxystearic acid had a strong impact on the release kinetics. The impact of the oleogelator hydroxystearic acid is also visible for the MCT systems, but to a smaller extent. Again, the microparticles with the incorporated oleogel (MG-MCT) release faster compared to the HS-free microcapsules (MC-MCT). The desired release profile was obtained with the MC-MCT microparticles: an almost linear release profile over two months with no lag time has been achieved.
observed for the lipid formulations was for the MC-IPM microcapsules system. Therefore, it can be concluded that the oleogelator hydroxystearic acid had a strong impact on the release kinetics. The impact of the oleogelator hydroxystearic acid is also visible for the MCT systems, but to a smaller extent. Again, the microparticles with the incorporated oleogel (MG-MCT) release faster compared to the HS-free microcapsules (MC-MCT). The desired release profile was obtained with the MC-MCT microparticles: an almost linear release profile over two months with no lag time has been achieved.

Conclusions
The results of our study show that lipid-PLGA hybrid systems are interesting alternatives to pure PLGA microparticles. The saturated liquid lipids MCT and IPM were incorporated in the form of microcapsules and microgels. Hydroxystearic acid (HS) was used as an oleogelator. The particle

Conclusions
The results of our study show that lipid-PLGA hybrid systems are interesting alternatives to pure PLGA microparticles. The saturated liquid lipids MCT and IPM were incorporated in the form of microcapsules and microgels. Hydroxystearic acid (HS) was used as an oleogelator. The particle structure was investigated by FIB-SEM and nano-CT imaging. Both methods support the existence of a highly dispersed internal phase with spherical particle sizes of around 2 micrometers. The noninvasive nano-CT imaging enabled the production of 3D representations of the microparticles' structures in high-resolution images. After image processing and segmentation, it was possible to estimate the amount, size distribution, and sphericity of the internal structures. The presence of hydroxystearic acid accelerated the drug release. The drug release kinetics can be tuned by the lipid composition. An almost ideal release profile for risperidone was achieved with PLGA-MCT microcapsules (MC-MCT). In contrast to Risperdal Consta ® , no lag time and an almost linear release over 60 days was observed. Therefore, the developed systems are suitable candidates to overcome the need for double medication (injected microparticles and tablets), which is currently needed due to the release lag time of Risperdal Consta ® .