Mesoporous Hydroxyapatite as Olanzapine Carrier Provides a Long

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Mesoporous Hydroxyapatite as Olanzapine Carrier Provides a LongActing Effect in Antidepression Treatment Yan-Jye Shyong,† Mao-Hsien Wang,‡ Hsiang-Chien Tseng,§ Chen Cheng,† Kuo-Chi Chang,*,∥ and Feng-Huei Lin*,†,⊥ †

Institute of Biomedical Engineering, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan Department of Anesthesia, En Chu Kon Hospital, Sanshia District, New Taipei City 23702, Taiwan ROC § Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei 11101, Taiwan ROC ∥ Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No. 1, Sec. 3, Chung-Hsiao East Road, Taipei 10608, Taiwan ⊥ Institute of Biomedical Engineering and Nanomedicine, National Health Research Institute, No. 35, Keyan Road, Zhunan, Miaoli County 35053, Taiwan ‡

S Supporting Information *

ABSTRACT: An antidepressant carrier was designed to maintain over 2 weeks of constant medication release. The carrier was injected into muscle, where cellular activity was employed to achieve the goal of constant release. Mesoporous hydroxyapatite (mesoHAP) was synthesized into an adequate size by a coprecipitation method; it then went through a series of hydrophobic surface modifications for olanzapine (OLZ) loading by physical absorption to produce mesoHAP−OLZ. Because of its hydrophobic nature, OLZ was not effectively released from mesoHAP−OLZ in an aqueous environment. However, once engulfed by macrophages, the lysosome/endosome hybrid ruptured due to alterations in osmotic pressure, resulting in the release of OLZ into the cytoplasm. OLZ was then exocytosed to the extracellular space due to a high calcium ion (Ca2+) concentration and finally reached the blood circulation. Our findings provide a useful treatment strategy to achieve long-term drug release with a single intramuscular (IM) injection, helping to solve the problem of nonadherent medication intake that often occurs in antidepressant therapy.

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INTRODUCTION Depression describes a transient mood state experienced by almost everyone at some time in their life; it is often associated with clinical or behavioral syndromes.1 Depression has become increasingly common in adults; by the year 2020, the World Health Organization (WHO) has estimated that depression will be the second major cause of disability in the world next to HIV.2 Depression can be characterized into five stages based on seriousness, extension, and features. In the first stage, minor depression is often prodromal to major depressive disorder (MDD), and anxiety, irritable mood, anhedonia, and sleep disorders may occur. In the second stage, major depressive episodes occur; it is still frequently considered to be a benign disorder, amenable to full remission upon appropriate © XXXX American Chemical Society

pharmacological treatment. In the third stage, residual symptoms may occur after the completion of drug or psychotherapeutic treatment; this stage has been associated with poor outcomes and may progress to symptoms of relapse. In stage 4, recurrence is a common and serious problem of depressive illness. In stage 5, chronic major depressive episodes occur for at least 2 years without breaks; additionally, remission and relapse in symptoms can occur periodically over time.3 There is no definite way to prevent depression with currently available technology. Minor depression may be cured by psychology, religion, or self-control, but medication is still the Received: May 8, 2015

A

DOI: 10.1021/acs.jmedchem.5b00714 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. MesoHAP−OLZ particle preparation and in vivo delivery via intramuscular (IM) injection. The cartoons describe the sequences of mesoHAP−OLZ particle synthesis. Also, the method of IM injection of mesoHAP−OLZ particles is indicated and so are the multiple steps and mechanisms involved for the OLZ delivery to the blood circulation system.

acting injectable design has been reported using a polylactic-coglycolic acid (PLGA) microsphere to enclose OLZ.10 However, its release profile was not able to meet the standard clinical requirements; during the designed two-week release, the medication level fluctuated from over- to under-medication. Other non-PLGA polymer-based microspheres include polyphosphazene, polyethylene imine carbonate, polyphosphate, polyanhydrides, polyorthoester copolymers, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, etc.11 However, all microsphere systems generally involve water-in-oil or oil-inwater microemulsion methods; surfactant may need to be added into the system to prepare microspheres in which to enclose olanzapine. The surfactant is sometimes difficult to remove and causes toxicity unless a complex removal process is performed, which is very costly and inefficient. In the current study, we developed a long-acting drug administration strategy to allow more than 2 weeks of constant drug release by IM injection. This design employs cellular activity to achieve the constant release of medication, as described below. Hydroxyapatite (HAP) is used as the carrier for OLZ because HAP has been used as a drug carrier for decades.12,13 However, its mechanism of release is largely based on diffusion across different concentration gradients14 and it causes a huge initial burst, resulting in the nonconstant release of medication. To solve this problem, mesoporous HAP

best way to treat the last three stages. Early medication is the key to preventing the progression of early depression to the more severe stages. Many antidepressants have been developed to battle depression, such as selective serotonin reuptake inhibitors (SSRIs), serotonin−norepinephrine reuptake inhibitors (SNRIs), tricyclic antidepressants (TCAs), and monoamine oxidase inhibitors (MAOIs).4 All these antidepressants possess similar side effects such as nausea, diarrhea, anxiety, and sleeping disorder.5 Olanzapine (OLZ), a second-generation antidepressant generally used for the treatment of depression and schizophrenia, appears to be more effective on the associated symptoms and shows fewer side effects compared with other antidepressants.6−8 Most developed antidepressants have had good clinical outcomes during trials; however, nonadherence to antidepressant therapy has become a major obstacle to effectively treating depression. Approximately 60% of primary care patients fail to take their antidepressant medications before the completion of the recommended 6 months of therapy due to memory loss and/or rhembasmus.9 A promising solution to this problem could be long-acting injectable depot formulations of antidepressants to replace conventional oral intake. A patient could receive one long-acting injection during the hospital stay or clinic visit that would last until the next outpatient appointment, without any mediation in between. This longB



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(mesoHAP) was synthesized with an adequate grain size, pore size, and porosity by a coprecipitation method; it then underwent a series of hydrophobic surface modifications for OLZ loading by physical absorption (so-called mesoHAP− OLZ). The prepared mesoHAP−OLZ was tested in animals using IM injection. Our hypothesis was that OLZ would not be simply released out of mesoHAP in an aqueous environment because of its hydrophobic nature; instead, OLZ could be released while host defense cells take up the mesoHAP−OLZ particles to form the early endosome and merge with the acidic lysosome thereafter. The prepared particles would be completely dissolved in the lysosome, resulting in high concentrations of Ca2+ and PO43− ions; this increase of osmotic pressure will break down lysosome to release OLZ into the cytoplasm. The excess Ca2+ ions from the dissolved mesoHAP will also trigger exocytosis to push out OLZ into extracellular matrix, eventually reaching the blood circulation.15 The developed particles are gradually taken up by defense cells in the injection site to achieve constant release and maintain the OLZ concentration at a therapeutic level for several weeks.16,17 The entire process is illustrated in Figure 1. In this study, coprecipitation was used to synthesize HAP particles with a mesoporous structure.18,19 After synthesis, the crystal structure of the developed particles was confirmed and identified by X-ray diffractometer (XRD). Additionally, scanning electron microscopy (SEM) and electron dispersive spectrophotometry (EDS) were used to determine the morphology and chemical composition of the synthesized particles, respectively. The Zeta-sizer was used to measure the particle size to determine the suitable size for endocytosis by host defense cells. Surface area, pore size distribution, and porosity of the mesoHAP particle were measured by Brunauer, Emmett, and Teller (BET) and a mercury porosimeter. MesoHAP particles then further underwent a series of surface modification processes by stearic acid to increase their hydrophobicity. OLZ was then adsorbed into the surface of the particle. Fourier transform infrared spectroscopy (FTIR) was used to determine the functional groups of mesoHAP before and after surface modification. Thermogravimetric analysis (TGA) was used to determine the quantity of OLZ loading to the mesoHAP. Cytotoxicity and cell viability were evaluated by lactase dehydrogenase (LDH) and water-soluble tetrazolium (WST-1) tests, respectively. The processes of mesoHAP−OLZ particles internalized by defense cells were examined under a transmission electron microscope (TEM). The release profile of the developed system was monitored in neutral and acid conditions to mimic the mesoHAP−OLZ in physiological and lysosome environments, respectively. The bioactivity of OLZ release from the defense cells was examined in vitro using coculture of mesoHAP−OLZ and macrophages. To test our hypothesis in vivo, the animal study was employed to further verify whether the OLZ concentration in blood could maintain a therapeutic level more than 2 weeks after IM injection of mesoHAP−OLZ particles into Wistar rats.

Figure 2. X-ray diffraction pattern of mesoHAP. All the characteristic peaks were matched to the standard pattern of HAP as JCPD card of 09-0432.

(002), (211), and (300) from the HAP crystal lattice, respectively. Dividing the area of the major peaks by the total area, the synthesized mesoHAP showed high crystallinity without any second phase in approximately 77% of samples. The morphology of mesoHAP was examined under high-power field emission SEM, as shown in Figure 3a. The rod-like grains aggregated into particles to form a mesoporous structure with uniform pore size, adequate porosity, and homogeneous distribution. The chemical composition of the synthesized mesoHAP was analyzed by SEM-accessorized EDS; the ratios of calcium and phosphorus in the synthesized particles by weight were 8.36% and 3.24%, respectively, as shown in Figure 3b and summarized in Table 1. The data on the size distribution of mesoHAP analyzed by the Zeta-sizer are presented in Figure 4. The results indicated that the particle size was in the range of 900−2800 nm, a suitable size range for macrophage to engulf.20 Table 2 shows that the BET surface area and porosity of mesoHAP; the BET surface area was 28.8 m2/g, the Langmuir surface area was 39.5 m2/g, and the porosity was 50.1%. In comparison to the surface area of commercialized HAP powder in the range of 10−15 m2/g,21 the surface area of the synthesized mesoHAP particles was much larger than that of the commercialized HAP powder; this could effectively increase the loading capacity of OLZ. The pore size distribution of the synthesized mesoHAP measured by BET was in the range of 40−100 nm based on the absorption and desorption of Barrett, Joyner, and Halenda (BJH), as shown in Figure 5a,b, respectively. The FTIR pattern of mesoHAP shown in Figure 6a indicated that the absorption bands at 3630, 1103, 1036, 604, and 567 cm−1 were assigned to the O−H stretching vibrations mode, the PO43− bending vibrations mode, and the PO43− stretching vibrations mode, respectively, as those in standard HAP. The spectrum of stearic acid indicated that the absorption bands on 3340, 3280, and 1720 cm−1 corresponded to the C−H stretching vibrations mode and CO stretching vibrations mode (Figure 6b). After the mesoHAP surface was modified by stearic acid, the FTIR spectrum showed characteristic absorption bands contributed by both mesoHAP and stearic acid (Figure 6c).

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RESULTS Characterization of the Materials. The synthesized mesoHAP crystal structure was identified using an X-ray diffractometer (XRD). The XRD profile of synthesized mesoHAP particles (Figure 2) indicated that all the characteristic peaks matched with the standard pattern of HAP as a JCPD card of 09-0432. The three major peaks at 25.7°, 31.6°, and 32.8° corresponded to the diffracted beam of the planes C



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Table 2. BET Surface Area and Porosity of mesoHAP Where the BET Surface Area Was 28.8 m2/g, Langmuir Surface Area Was 39.5 m2/g, and the Porosity Was 50.1% instrument name

Table 1. Chemical Composition of mesoHAP from EDS Analysis by Weight Ratio and Atomic Ratio wt %

atomic %

C O P Ca totals

61.51 26.89 3.24 8.36 100.00

71.98 23.62 1.47 2.93

BET surface area: 28.8 ± 0.03 m2/g correlation coefficient: 0.9999985 langmuir surface area: 39.5 ± 1.15 m2/g correlation coefficient: 0.998731

mercury porosimeter

porosity: 50.1%

OLZ Loading. Thermogravimetric analysis (TGA) was used to evaluate the efficiency of OLZ loading. The TGA curve of mesoHAP indicated very minor weight loss in the temperature range from room temperature to 600 °C. The evaporation temperature (boiling point) for the stearic acid was 383 °C.22 The OLZ TGA curve indicated that the first weight loss was approximately 100 °C, possibly the boiling of surface water, and that the second weight loss occurred at the temperature of 233.8 °C, the flash point of OLZ (Figure 7a). In contrast, the mesoHAP−OLZ TGA curve showed three weight losses at 100, 233, and 383 °C, corresponding to the evaporation of water, OLZ, and stearic acid, respectively. The first loss was approximately 1.2% in weight. The second weight loss was approximately 23.5%, and the third weight loss was 1.7% (Figure 7b). On the basis of the results, OLZ was effectively loaded into the mesoHAP with a weight ratio as high as 23.5%. Cell Viability and Cytotoxicity. To test the cell viability, the WST-1 test was performed in 3T3 cells treated with mesoHAP−OLZ and examined on day 1 and day 3. The results showed no significant differences among the different groups with mesoHAP−OLZ concentrations ranging from 0 to 0.2 mg/mL, indicating that mesoHAP−OLZ treatment did not affect cell viability (Figure 8a). Additionally, the potential cell cytotoxicity after mesoHAP−OLZ treatment was examined using LDH tests on 3T3 cells (Figure 8b); similar to the findings of the WST-1 tests, mesoHAP−OLZ treatment did not increase cytotoxicity at the concentration range from 0 to 0.2 mg/mL. Cellular Uptake of mesoHAP−OLZ and the OLZ Release Profile. The UV−vis absorption spectrum of OLZ exhibited two absorption bands at 226 and 272.5 nm, as shown in Figure 9a. However, 226 nm was used as the working peak because it showed greater maximum absorbance than 272.5 nm.23 Figure 9b shows the OLZ release profile of mesoHAP− OLZ in a normal physiological environment at pH 7.4. It showed a 15% initial burst on the first day, which did not

Figure 3. Morphology of mesoHAP examined under SEM. (a) Rodlike grains aggregated into particles to form a mesoporous structure with uniform pore size, adequate porosity, and homogeneous distribution. (b) Chemical composition of the synthesized mesoHAP was semiquantified by energy dispersive spectrophotometer (EDS), where calcium and phosphorus could be traced in the synthesized particles.

element

result

BET

Figure 4. Size distribution of mesoHAP particles analyzed from the Zeta-sizer. The data indicated that the particle size was in the range of 900−2800 nm. D



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Figure 5. Pore size distribution of mesoHAP particles. The pore sizes of mesoHAP particles were measured by BET, and their ranges were from 40− 100 nm based on the (a) absorption and (b) desorption of BJH.

Figure 6. FTIR spectrophotography. (a) mesoHAP, (b) stearic acid, and (c) surface modified mesoHAP; absorption bands identified on 3630, 1036−1103, and 567−604 cm−1 were matched to the standard HAP. Absorption bands on 3340, 3280, and 1720 cm−1 were matched to the steric acid.

change significantly in the release profile thereafter, as almost no OLZ was released in the aqueous environment. Figure 9c shows the OLZ release profile of mesoHAP−OLZ in the acid solution with pH 3−5 to mimic the conditions of mesoHAP− OLZ in the endosome/lysosome hybrid. OLZ was 82%, 41%, and 20% released from acid-dissolved mesoHAP−OLZ within in the first day in pH = 3, pH = 4, and pH = 5, respectively. OLZ was 100% released for at most 6 days. Figure 9d shows the OLZ release profile of mesoHAP−OLZ cocultured with RAW-264.7 (commercialized macrophage cell line). Approximately 75% of OLZ was constantly released from mesoHAP− OLZ within 2 days due to the cellular activity. This study suggested that the OLZ in mesoHAP−OLZ could only be released when combined with phagocytic activity and was not released from the normal physiological environment.

The TEM images of RAW-264.7 cultured with mesoHAP− OLZ particles after 24 h are presented in Figure 10, demonstrating the interface of RAW-264.7 cells and mesoHAP−OLZ particles before endocytosis. Figure 10b shows that RAW-264.7 cells took up mesoHAP−OLZ particles and enclosed them in the endosome. An enlarged image of the cell membrane is shown in Figure 10c, and the indentation of cell membrane during endocytosis could be observed. The image showed that mesoHAP−OLZ particles were transported to lysosomes, as shown in Figure 10d. MesoHAP−OLZ particles were digested into the endosome/lysosome hybrid, and then the hybrid inflated and ruptured due to the change of osmotic pressure, indicated by red arrow. The process of defense cell uptake of the mesoHAP−OLZ particles shown in Figure 10 is consistent with our hypothesis that OLZ could be released by the cellular activities shown in Figure 1. E



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Figure 7. Flash points of different compounds examined by TGA. (a) The flash point of OLZ was analyzed by TGA. The first weight loss at 100 °C was water evaporation. The second loss at 233 °C was OLZ decomposition temperature. (b) The TGA curve of mesoHAP−OLZ showed that the weight loss of 23.5% was at 233 °C, that would be in terms of the percentage of OLZ loaded in mesoHAP.

OLZ Release Study in Vivo. The OLZ release profile in vivo is presented in Figure 11, and the in vivo release profile of OLZ from the mesoHAP−OLZ lasted for more than 2 weeks. The release was faster in the first few hours, which may have been due to the initial burst. After macrophages accumulated and were activated, constant release was achieved afterward, and OLZ was depleted at the third week of the experimental period. The minimum effective dose of OLZ was indicated by the red line in the figure.

contains many nonpolar side chains such as leucine, alanine, and valine. The nonpolar side chains undergo steric separation to inhibit HAP crystal growth so that mesoHAP could be synthesized into nanoparticles with high surface area and high porosity (Table 2). Albumin not only acts as a foaming agent to create adequate pore size and porosity but also acts as a dispersion agent to disperse the nuclei in the solution to inhibit crystal overgrowth and allow the synthesis of nanoscale mesoparticles.25−27 The precipitation of mesoHAP consists of two steps as follows: nucleation and crystal growth. Nucleation is a process whereby molecules in solution randomly assemble to form aggregates that generally cannot reach a critical mass to become a stable nucleus for crystal growth. Crystal growth, on the other hand, can only occur when the size of the nucleus is over the critical size. When a solution contains more solute than that at equilibrium, the solution is supersaturated. The polar side chains on the albumin attract Ca2+ or PO43− to the molecule’s neighbor to locally increase solute concentration for nucleation and crystal growth. The parameter that describes the degree of supersaturation of a solution is relative supersaturation, which is expressed as

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DISCUSSION In this study, we successfully synthesized mesoHAP particles using coprecipitation method with albumin as the foaming agent. The size of the synthesized particles varied but was adequate for the activation of the macrophage endocytosis pathway. We also increased the hydrophobicity of the particle surface by adding stearic acid to mesoHAP to further facilitate OLZ loading. The synthesized mesoHAP−OLZ particles were tested for cytotoxicity and examined for drug release profiling in both in vitro and in vivo environments. Our results demonstrated that the synthesized mesoHAP−OLZ particles were able to achieve the long-term delivery of OLZ, which primarily involved the activation of macrophage endocytosis pathways. Factors Affecting mesoHAP Particle Synthesis. In this study, egg whites were added into the system as a foaming agent during mesoHAP synthesis.24 Albumin in egg whites is one of the major proteins in the blood serum, with long amino acids that contain positively and negatively charged side groups such as glutamic acid, aspartic acid, lysine, and arginine. These polar side groups could effectively bind to the Ca2+ or PO43− to develop into the nucleation site to precipitate HAP crystals from the solution. Albumin is a macromolecule that also

(Q − S ) ÷ S

(1)

where Q is the concentration of the solution and S is the concentration at equilibrium.28 The more solutes in solution, the greater the level of supersaturation. It has been shown that relative supersaturation affects the rate of nucleation more than the rate of crystal growth as nucleation proceeds faster than crystal growth in a highly supersaturated solution. Therefore, very tiny particles are formed in a suspension, resulting in small final sizes of the precipitate. Precipitation of mesoHAP involves both Ca2+ and PO43+ simultaneously; therefore, they both need to be concentrated to facilitate a high rate of nucleation.29 As a F



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suggest that the hydrophilic surface of mesoHAP may be rendered hydrophobic by stearic acid. Stearic acid is an 18carbon fatty acid without double bonds in the main hydrocarbon chain; it is an amphiphilic molecule with a hydrophilic carboxyl group and a hydrophobic C−H tail. The hydrophilic surface of mesoHAP would interact and bind to the carboxyl group on the stearic acid, exposing the hydrophobic C−H tail of the stearic acid on the particle surface and making the surface hydrophobic. The spectrum of the surface-modified mesoHAP possesses additional absorption bands at 3340, 3280, and 1720 cm−1 (Figure 6c). These adsorption bands corresponded to the C−H stretching vibration mode and the CO stretching vibration mode from stearic acid.32 The stearic acid-modified mesoHAP with a hydrophobic surface increases the binding of hydrophobic OLZ due to hydrophobic interactions. As indicated in the TGA curve of mesoHAP particles, a relatively minimal weight loss occurred at temperatures over 600 °C. The TGA curves for OLZ and mesoHAP−OLZ both showed a single and minimal weight loss at approximately 100 °C that was attributed to water evaporation. The second substantial loss at the temperature of 233.8 °C was the flash point of OLZ. The third minimal weight loss at the temperature of 383 °C was the evaporation temperature for stearic acid due to the surface modification, as shown in Figure 7b. The efficiency of OLZ entrapment of the mesoHAP−OLZ was calculated using the following formula:33 entrapment efficiency(%) = (the amount of OLZ in mesoHAP

Figure 8. Cell viability and cytotoxicity tests for mesoHAP−OLZ. (a) Water-soluble tetrazolium (WST-1) test in 3T3 cells was performed using different concentrations of mesoHAP−OLZ at day 1 and day 3 (n = 3). The data suggest no significant effect of affecting cell viability by mesoHAP−OLZ. (b) Lactase dehydrogenase (LDH) test was performed in 3T3 cells using different concentrations of mesoHAP− OLZ at day 1 and day 3 (n = 3). The data suggest that mesoHAP− OLZ did not increase cytotoxicity.

/total OLZ in the system) × 100%

(2)

The weight loss of OLZ in mesoHAP−OLZ was 23.5% (Figure 7b), in which 23.5 mg of OLZ was loaded for every 100 mg of mesoHAP. The total OLZ in the system was 30 mg. On the basis of the previous formula, the OLZ entrapment efficiency of mesoHAP−OLZ was approximately 78%. Factors Affecting OLZ Release from mesoHAP−OLZ Particles. Because of its hydrophobicity, hydrophobic OLZ is generally not released in a water-rich environment and tightly binds to surface-modified mesoHAP. Although a 15% initial burst occurred on the first day in vitro in PBS due to physical adsorption, OLZ was not further released from the mesoHAP− OLZ particles because hydrophobic interactions provided strong binding between OLZ and surface-modified mesoHAP (Figure 9b).34 One source of the initial burst was the rapid release of drugs from the material surface, accounting for up to 80% of the total drug loading. This poses a serious threat of toxicity, especially in the first few days of treatment. In the treatment of depression, an overdose of antidepressants due to the initial burst could be harmful to the nervous system and heart and, more seriously, lead to suicide. Many methods have been developed to reduce the initial burst during treatment. One key solution is to increase the hydrophobic interaction between the drug and the material, as the higher the interaction, the higher the efficiency of drug loading and lower the amount of the initial burst.35−37 Most antidepressants, including OLZ, have a very high hydrophobicity because the target region of these drugs is usually inside the brain; therefore, these drugs are designed to pass through the blood− brain barrier (BBB). Only hydrophobic and small-molecule drugs are able to go through BBB.38 In our study, mesoHAP is relatively hydrophilic compared with OLZ; therefore, without

result, a mixture of solutions with high concentrations of both phosphorus and calcium promotes the formation of nanosized mesoHAP particles (approximately 40 nm). Factors Affecting mesoHAP Particle Size and Morphology. In previous studies, most of the synthesized HAP exhibited a needle-like shape with the c-axis along the long axis of the needle grain. In the current study, the grain axis was shortened to form a rod-like structure (Figure 3a) due to the effect of steric separation by albumin macromolecules limiting growth along the long axis (002). The unstable brushite phase, as well as the other amorphous phases, transforms into a more stable mesoHAP in alkaline conditions.30 Several experimental parameters were found to affect the surface area, particle size, and morphology of mesoHAP particles, including pH level, foaming agent, aging temperature, and aging time.31 By adding albumin as the foaming agent and adjusting the pH to 8.5 at 85 °C for 22 h, the resulting particles from this solution showed a much larger BET surface area than those of commercialized HAP powder (Table 2), which could effectively increase the loading capacity of OLZ. The mesoHAP particles synthesized in the current study also fit into the range of the particle size and pore size (Figures 4 and 5) suitable for the engulfment of macrophages. Hydrophobic Modification of mesoHAP Particles and OLZ Loading. The data from the FTIR spectrum analysis G



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Figure 9. (a) The UV/vis absorption curve of OLZ shown a working peak at 226 nm. (b−d) OLZ releasing profile of mesoHAP−OLZ. (b) At pH 7.4, OLZ was released about 15% from mesoHAP−OLZ in the first day, and no further OLZ was released from mesoHAP−OLZ thereafter. (c) When in pH 3−5, mesoHAP−OLZ was dissolved and OLZ was released in about 82%, 41%, and 20%, respectively, within the first day. (d) Cocultured with RAW-264.7, 75% of OLZ was constant released within 2 days, presumably due to the cellular activity.

Figure 10. Transmission electron microscope (TEM) images of RAW-264.7 cultured with mesoHAP−OLZ particles. (a) The interface of RAW264.7 cell and mesoHAP−OLZ particles before cellular uptake. (b) RAW-264.7 cells start to uptake mesoHAP−OLZ particles and enclose them in the endosome. (c) The indentation of cell membrane during endocytosis. (d) MesoHAP−OLZ particles transported to lysosome, where inflated and ruptured of lysosome/endosome hybrid due to the change of osmotic pressure was indicated by red arrow.

dissolved in acidic solution.40 We demonstrated in Figure 9c that the mesoHAP−OLZ was able to dissolve in an acidic solution with a pH from 3 to 5, and OLZ was completely released within 6 days. Additionally, this condition could be mimicked in the acidic environment of lysosome/endosome hybrids, suggesting that the key mechanism of OLZ release from mesoHAP−OLZ particles under pathophysiological

surface modifications, a high initial burst will occur at the beginning of drug treatment. By mixing stearic acid with mesoHAP at 90 °C, stearic acid will melt and form a thin layer on the surface of mesoHAP, resulting in a large increase in hydrophobicity.39 After surface modification, the initial burst of mesoHAP−OLZ was reduced to only 15% in the first 24 h, and no further release occurred in a water-rich environment. However, OLZ was released when mesoHAP−OLZ was H



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CONCLUSION In this study, mesoHAP particles were synthesized to serve as the OLZ vehicle; the particles were characterized to determine their crystal structure and particle size, and the surface was further modified to increase particle hydrophobicity. The mesoHAP particles were also examined for their cytotoxicity, as well as their drug release profile and mechanism in both in vitro and in vivo environments. Our findings suggest that the long-term constant release of OLZ was not simply accomplished by diffusion; instead, a pathophysiological process implicated in the macrophage−endocytosis pathway was activated during the period of long-lasting drug delivery. The design and concept of achieving long-acting and constant delivery of medicine in vivo provide a valuable treatment strategy to resolve the problem of nonadherent medication intake that frequently occurs in antidepressant therapy. Additionally, the same strategy will be beneficial to help patients who require multiple daily injections of their medication.

Figure 11. Release profile in vivo of OLZ from the mesoHAP−OLZ for over 2 weeks. Maintained constant release was achieved to the third week of the experimental period until the depletion of OLZ.

conditions would be its combination with phagocytic activity (Figure 9d). mesoHAP−OLZ Delivery and OLZ Release in Vivo. On the basis of the above suggested mechanism, mesoHAP−OLZ particles were delivered by IM injection. They were engulfed by macrophages in an in vivo environment, where they form phagosomes or endosomes and later combine with lysosomes. Under the acidic lysosome/endosome hybrid condition, mesoHAP−OLZ was dissolved, resulting in an abrupt increase in the concentration of Ca2+ or PO43− in the hybrid. Sequentially, this caused the rupture of the lysosome/ endosome due to increased osmotic pressure. In multicellular organisms, exocytosis is Ca2+-dependent and is involved in signal transduction between cells/neurons. Exocytosis is performed by all cells, allowing the release of components to the extracellular matrix or the delivery of newly synthesized membrane proteins to be incorporated into the plasma membrane. Ca2+ ions are the key factors that regulate the process of exocytosis, and the rise in intracellular Ca2+ ions, such as that caused by the opening of the voltage-dependent Ca2+channels, triggers exocytosis.41 In our study design, Ca2+ ions did not originate from the influx of Ca2+ channels but from the Ca2+ ions embedded in the mesoHAP particles released when the particles were dissolved by the lysosome/endosome hybrid. Once the lysosome/endosome hybrid ruptured due to increased osmotic pressure, OLZ was released into the cytoplasm, and the excess Ca2+ from the dissolved mesoHAP particles triggered exocytosis and sent OLZ to the extracellular space, entering the circulatory system and reaching the target cells. This process is summarized in Figure 1, with evidence shown in a series of TEM pictures in Figure 10. To further validate the efficiency of our long-acting drug release design in vivo, we further conducted experiments in an animal model (Figure 11). The data suggest that OLZ release from mesoHAP−OLZ was not only based on the surface diffusion across different concentration gradients, as shown in other studies.42 The simple diffusion mechanism leads to a high initial burst and is not able to maintain OLZ at a constant blood level for a long time. Employing the advantages of our current design, various sizes of mesoHAP−OLZ particles were synthesized, resulting in different rates of endocytosis by macrophages; presumably the larger the particles, the more time needed for macrophages to take them in. Because of the differences in the rate of endocytosis, constant, long-acting delivery was achieved for over 2 weeks, until all the mesoHAP− OLZ particles taken up by macrophages and OLZ were depleted from the circulatory system.

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EXPERIMENTAL SECTION

Reagents and Cells. Calcium hydroxide (Ca(OH)2, SigmaAldrich, St. Louis, USA) and phosphoric acid (H3PO4, Sigma-Aldrich, St. Louis, USA) were used as sources of Ca2+ and PO43− to synthesize mesoHAP, respectively. Olanzapine was purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). Sodium hydroxide (NaOH, Merck, Darmstadt, Germany) and hydrochloric acid (HCl, SigmaAldrich, St. Louis, USA) were used to adjust the pH value during the preparation of materials. Stearic acid (Sigma-Aldrich St. Louis, USA) was used for the surface modification of mesoHAP. Methanol from Merck Chemicals (Darmstadt, Germany) was used for washing. 3T3 and RAW-264.7 cells were purchased from the Bio-Resource Collection and Research Center, FIRDI, Hsin-Chu City, Taiwan; 3T3 cells served as target cells for the evaluation of cytotoxicity and viability, and RAW-264.7, a type of mouse macrophage, was used as a cell model to observe how the defense cells internalize the developed particles in vitro, as examined by TEM. Synthesis of Mesoporous HAP (mesoHAP). Coprecipitation was used to synthesize mesoHAP.43 The reaction is described as follows:

10Ca(OH)2 + 6H3PO4 → Ca10(PO4 )6 (OH)2 + 18H 2O

(3)

Calcium hydroxide [Ca(OH)2] 3.86 g was dissolved in 100 mL of ddH2O and kept at 80−85 °C in a water bath during the reaction period. A stoichiometric amount (Ca/P molar ratio = 1.67) of 0.3 M phosphoric acid (H3PO4) was added dropwise to the previous 0.5 M Ca(OH)2 with a rate of approximately 3 mL/min, followed by the addition of 15 g of egg whites as a foaming agent to create an adequate pore size and porosity in mesoHAP. NaOH was added to adjust the pH value to 8.5. The mixture was stirred for 2 h and then aged for 20 h at a temperature of 85 °C. After aging, the precipitated powder was collected and washed three times by methanol and finally by deionized water. The powder was calcined at 800 °C to remove albumin from egg whites to obtain the mesoHAP particles. Particles were collected and stored for later surface modification. Supporting Information Table S1 shows the experiments that failed during mesoHAP preparation, resulting in different calcium phosphates prepared under different conditions. S Surface Modification of mesoHAP for Hydrophobicity and OLZ Loading (mesoHAP−OLZ). Stearic acid 0.05 g was vigorously stirred in 100 mL of deionized water and heated to 90 °C. Approximately 2.5 g of mesoHAP powder was added and stirred for 12 h at a temperature of 90 °C. The mixture was centrifuged at 3000 rpm for 10 min, washed with deionized water 3 times, and lyophilized in a freeze-drier. Approximately 100 mg of OLZ was added to 10 mL of deionized water and mixed with 250 mg of surface-modified mesoHAP particles. I



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RAW-264.7 was seeded on a T25 flask with a density of 2.5 × 106/ flask for culturing with 30 mg of mesoHAP−OLZ particles to demonstrate that OLZ is released from mesoHAP−OLZ in a relatively short period of time in endosome/lysosome hybrids. Because of the interference of culture medium on UV−vis, RAW-264.7 cells were cultured in 30 mL of PBS along with mesoHAP−OLZ for 3 days. PBS samples were collected, centrifuged, and further filtered with a Cornig 30K Spin-X concentrator to remove any interference with sequential analysis by UV−vis. The filtered PBS sample was analyzed with a UV− vis spectrophotometer (JASCO V-670) at a wavelength of 226 nm. This experiment was designed to test the hypothesis that OLZ would not be released from mesoHAP in normal aqueous conditions unless combined with cellular activity and the phagocytic process. Phagocytosis of Particles Taken Up by RAW-264.7 Cells. RAW-264.7 macrophages were seeded on a 24-well plate at a density of 105 cells/well and cultured for 3 days. The cells were then treated with mesoHAP−OLZ (0.24 mg/mL) for 24 h. After mesoHAP−OLZ treatment, cells were washed thoroughly with chilled PBS, pelleted by centrifugation, and fixed with 2.5% glutaraldehyde overnight and postfixed in 1% osmium tetroxide solution for 1−2 h. They were then rinsed with 0.2 M PBS buffer 3 times and dehydrated in ethanol (35%, 50%, 70%, 85%, 90%, 95%, and 100%). Cell cryotomy was performed with the help of the School of Veterinary Medicine of National Taiwan University, and images were recorded by TEM (Jeol, JEM-1200EX II, Tokyo, Japan) operated at 100 kV to observe how the particles were internalized by macrophages. Study of in Vivo OLZ Release. Wistar rats were purchased from BioLasco Taiwan Co., Ltd. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. All rats were administered 300 mg/kg of mesoHAP−OLZ by IM injection into the dorsum.46 Blood samples were collected after sacrifice at 0.5, 2, 4, 7, 14, and 21 days (n = 3 for each data point). Blood plasma was obtained after the samples were centrifuged at 1200 rcf for 10 min. Blood plasma was further filtered with a Corning 30K Spin-X concentrator to remove plasma protein from the blood sample to reduce the interference of sequential analysis by UV−vis. The filtered blood sample was analyzed with a UV−vis spectrophotometer (JASCO V-670) at a wavelength of 226 nm.

The vacuum system with a rotary pump was used to help OLZ travel into the pores of mesoHAP.44 The particles were collected by centrifugation at 3000 rpm for 10 min, washed with deionized water, and lyophilized in a freeze-drier. Material Characterization and Analysis. The synthesized mesoHAP was identified using an X-ray diffractometer (Rigaku Geigerflex, Tokyo, Japan) for crystal structure identification. The XRD pattern was obtained at 30 kV and 20 mA, with the diffraction angle set within the range of 20−60° at a scan rate of 1°/min.45 The morphology of the synthesized particles was observed under SEM (Philips XL30, Amsterdam, Netherlands) with an accelerating voltage of 15 kV. Particles were mounted on the sample stage of SEM and coated with a platinum film by sputtering PVD. The platinum film was used to increase the imaging resolution and prevent undesired charge accumulation. The chemical composition was analyzed by an SEM-equipped energy dispersive spectrophotometer (EDS). The particle sizes of mesoHAP were detected underwater by a Zetasizer (Malvern, Worcestershire, UK) operated at 10−70 °C (±0.1 °C). The detected range of particle sizes was 2 nm-8 μm. The specific surface area and pore size distribution of mesoHAP was determined by BET (Micromeritics ASAP2010, Georgia, USA) using nitrogen gas adsorption−desorption isotherms. The porosity of mesoHAP was also determined by a mercury porosimeter (Micromeritics Autopore 9520, Georgia, USA). The functional groups of mesoHAP, stearic acid and surfacemodified mesoHAP were characterized by FTIR (JASCO 410, Tokyo, Japan); the test sample was mixed with KBr at a ratio of 1:9 and pressed into a disk with a pressure of 10 MPa. All the spectra were collected in the wavenumber range of 400−4000 cm−1 at a 400 nm/ min scanning rate. Efficiency of OLZ Loading. The thermogravimetric analysis of OLZ and mesoHAP−OLZ was performed with TGA-DTA (TA Instrument SDT2960, New Castle, Germany) under a nitrogen atmosphere from 30 to 600 °C with a heating rate of 10 °C/min. Weight loss in the thermal analysis could be classified in terms of the amount of OLZ loaded on surface-modified mesoHAP. The Evaluation of Cell Viability and Cytotoxicity. The cell viability in response to the synthesized mesoHAP−OLZ was evaluated by WST-1 assay (BioVision, Milpitas, USA) under the guidelines of ISO-10993. Approximately 1 mg of synthesized particles was immersed in 10 mL of DMEM (Sigma-Aldrich, St. Louis, USA) supplemented with 10% fetal bovine serum (FBS, Gemini Bioproducts, Calabasas, CA) and 10% biotin for 24 h. The synthesized particles were centrifuged and discarded; the solution was collected as an extraction solution for later cell culture. 3T3 cells were seeded on the 96-well plate with a cell density of 104/mL/well. The extraction solution and fresh medium were mixed at a ratio of 1:1 and added to the culture well. The results of WST-1 assay were measured by an ELISA reader at a wavelength of 450 nm. The higher the OD value measured by ELISA, the higher the cell viability. The cytotoxicity of the synthesized particles was evaluated by LDH assay (Promega, Madison, USA). The particles were treated with the same immersion process as described in the previous section to prepare the extraction solution. The 3T3 cells were seeded on a 96well plate with the same seeding density as in the WST-1 assay. The results of the LDH assay were measured at a wavelength of 490 nm. The higher the OD value, the higher the percentage of cell death. By comparing the OD values between the control and the extraction group, we could evaluate the cell toxicity of mesoHAP−OLZ. In Vitro OLZ Release Profile. The evaluation of the release profile of mesoHAP−OLZ was conducted in saline with a pH value of 7.4 and 3.0−5.0 to mimic the conditions of physiological environment and the lysosome/endosome hybrid, respectively. OLZ released from mesoHAP was measured by a UV−vis spectrometer (JASCO V-670, Tokyo, Japan) at a wavelength of 226 nm. The accumulated percentage of OLZ released from mesoHAP−OLZ was recorded for up to one month. The calibration curve of OLZ was also performed by UV−vis in the range from 0.0125 to 0.2 mg/mL (Supporting Information Figure S1), and the adsorption/desorption isotherms of OLZ under different conditions are shown in Supporting Information Table S2.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00714. Calibration curve of OLZ, adsorption/desorption isotherms of OLZ under different conditions, table of failed experiment during mesoHAP preparation (PDF) HAP X-ray CIF data (deposited with the Cambridge Crystallographic Data Centre (CCDC)) (CIF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*For K.-C.C. phone, 886-2-28910819; fax, 886-2-27317117; Email, [email protected]. *For F.-H.L.: phone, 886-2-23912641; fax, 886-2-23940049; Email, [email protected]. Author Contributions

Yan-Jye Shyong and Kuo-Chi Chang designed and performed experiments, analyzed data and wrote the paper. Mao-Hsien Wang, and Hsiang-Chien Tseng assist in the animal study. Chen Cheng assistsed in information collection. K.-C.C. and F.H.L. contributed equally to this work. Notes

The authors declare no competing financial interest. J



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ACKNOWLEDGMENTS We show our gratitude to the Animal Center of National Taiwan University for their professional assistance in the animal study. We also appreciate the contributions of the National Science Council, Taiwan, and National Health Research Institute, Taiwan, for their financial support of current research and make this research possible.

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ABBREVIATIONS USED IM, intramuscular injection; MesoHAP, mesoporous hydroxyapatite; OLZ, olanzapine; MDD, major depressive disorder; PLGA, poly lactic-co-glycolic acid; RAW-264.7, mouse leukemic monocyte macrophage cell line; 3T3, standard fibroblast cell line

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Mesoporous Hydroxyapatite as Olanzapine Carrier Provides a Long

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