Template-Free Synthesis and Control Drug Release of Calcium Carbonate-Hydroxylapatite Composite

We employed Gas expanded liquids (GXLs) technology to synthesize calcium carbon-ate-hydroxylapatite composite (CaCO3-HAp) nanoparticles and investigated the effects of reaction parameters on the properties of the composites as well as the performance of the composites as drug delivery agent using ibuprofen as a model drug in simulated gas- tric fluid. Separately, calcium carbonate and hydroxylapatite have been used extensively in pharmaceutical and drug delivery systems (DDS). However, both CaCO3 and HAp have individual draw-backs and limitations in applications. It has been reported that drugs and proteins embedded into the materials are usually loosely bound on the surface of the par- ticles (adsorbed); a condition which may lead to poor drug release. The results showed that the composite had good thermal stability and better surface area and pore (volume and size) properties. Studies on the performance of the composites as a drug delivery agent using ibuprofen as a model drug in simulated gastric fluid revealed that the composite promoted the dissolution of ibuprofen outstandingly. Whereas the low pressure loaded composite showed quicker relief potential (92% release) in 30 minutes with control release over desirable time frame (3h, 20 min), the high pressure loaded composite also exhibited quicker relief potential (80% release) in 30 minutes but with control release over remarkable time frame (6h, 40 min). The implication is that if a patient is administered with ibuprofen loaded into the synthesized composite at low pressure (10.0 MPa), the patient will experience quicker relief (92% release of ibuprofen) in 30 min and which will be sustained for almost 3.5 hours. Sim-ilarly, the high pressure (17.0 MPa) loaded ibuprofen will also provide a patient with quicker relief (80% release of ibuprofen) however, the relief in this case will be sustained for longer period; about 6.5 hours. Therefore, the high pressure loaded composite had tremendous effect on the control release of ibuprofen and can be used as effective drug delivery system (DDS) for drugs with similar characteristics.


INTRODUCTION
The anhydrous crystalline polymorph phases of calcium carbonate (CaCO3): vaterite, calcite and aragonite have received tremendous attention in the literature compared to their hydrated counterparts: monohydrocalcite and hexahydrocalcite (Domingo et al., 2006;Gomez-Villalba et al., 2012). CaCO3 is used for numerous chemical and industrial applications, and by living organisms (Domingo et al., 2006). Demand for CaCO3 will therefore continue to increase globally (Montes-Hernandez et al., 2010). Key factors driving this huge demand are the growing applications of nanometric CaCO3 in the fields of engineering, pharmaceuticals, medicine, as well as in health care, cosmetics and personal hygiene products (Harja et al, 2012). Industrially, CaCO3 is usually produced by the solid-solid-gas or solid-liquid-gas carbonation routes using Ca(OH)2 (Gomez-Villalba et al., 2012). Similarly, hydroxylapatite (HAp) has attracted much attention as the most suitable biocompatible candidate for use especially, in medical applications, coatings and prosthesis. It does not cause pyrogenic and inflammatory reactions; it is nontoxic, and does not form stringy tissues (Hui et al., 2010) when applied in tissue engineering. It has also been utilized in the ceramics and bioceramics industry, pharmaceuticals and as sorbents in environmental pollution control (Eduardo et al., 2012). For wider applications, control of its surface and pore properties, purity and morphology is most vital and many techniques have been explored to synthesize HAP with improved properties (Kim & Kumta, 2004;Sopyan, 2004). Meanwhile, substantial research has also been advanced towards the development of inorganic nanocrystals due to the potential applications. One of such important inorganic materials especially suited for application in the medical, pharmaceutical, drug delivery, tissue engineering, bone reconstruction and dental fields is calcium carbonate-hydroxylapatite (CC-HAp) composite (Goldberg et al., 2010). Separately, calcium carbonate and hydroxylapatite are perhaps, the most important and have been used extensively in pharmaceutical and drug delivery systems (DDS). For instance, CaCO3 has been employed for the imbedding and absorption of functional molecules and drugs (Marloes et al., 2010). Similarly, HAp has been used to encapsulate plasmid DNA, PEG and other drugs (Guoa et al., 2012). However, both CaCO3 and HAp have individual draw-backs and limitations in applications. It has been reported that drugs and proteins embedded into the materials are usually loosely bound on the surface of the particles (adsorbed); a condition which may lead to poor drug release (Ueno et al., 2005). As a result, several procedures aimed at effectively embedding drugs into inorganic solid matrices have been proposed (Ueno et al., 2005). Combining these versatile materials may alleviate https://journals.e-palli.com/home/index.php/ajmri Am. J. Multidis. Res. Innov. 1(2) 56-62, 2022 some of their individual draw-backs and lead to wider applications (Goldberg et al., 2010). The combination of porous carbonate hydroxyapatite (CHAp) and tri-calcium phosphate (TCP) has been applied for sustained release of anticancer drugs and antibiotics (Goh et al., 2013). A calcium phosphate (CaP)/ glycerylphosphorylcholine (GPC) and PEG (CaP/GPC-PEG) hybrid porous nanospheres were also used as drug carriers to alleviate multidrug resistance (MDR) (Sun et al., 2012). Liquids which have their volumes expanded as a result of been pressurized with a gas which has the ability to condense have equally received attention from the research world (Akien & Poliakoff, 2009). These systems, known as gas expanded liquids (GXLs) comprise of solvents or mixture of solvents which have been used in variety of engineering processes due to their pressuretunable properties (Akien & Poliakoff, 2009). Carbon dioxide expanded liquids (CXLs) are the most popularly utilized group among the GXLs due to its obvious advantages (Aaron et al., 2009). We applied the CO2-expanded carbonation (CXC) technique for direct and template-free synthesis of high surface area and pore volume CaCO3-HAp nanoparticle composite with superstructures (Ibrahim et al., 2014a) and investigated the performance of the composites as drug delivery agent using ibuprofen as a model drug in simulated gastric fluid.

Experimental Materials
Analytical reagent grade Ca(OH)2 with 95.0% containing ∼3% CaCO3 (Figure 1a) with 61 nm mean crystallite size (as measured by Jade), Phosphoric acid (95.0%) and ethanol (≥ 95.0%) were purchased from Sinopharm Reagent Co. Ltd, China and used as received whereas CO2 (99.99%) gas was purchased from Linde Gas, Xiamen Corporation Ltd (China). For comparison, CaCO3 (100%) (Ibrahim et al., 2013) and HAp (100%) (Ibrahim et al., 2015) were produced using previously reported techniques. Procedure Illustration of the experimental setup and the reaction procedure are shown in a previous publication (Ibrahim et al., 2013). Briefly, appropriate masses of Ca(OH)2 and phosphoric acid consisting of Ca/P molar ratio of 2 were measured. The Ca(OH)2 was transferred to the reaction vessel and the phosphoric acid was gently added to the vessel. To this mixture, 50mL of 4:1 ethanol−water ratio solution was added. The reaction vessel was sealed and heated to 60℃ with a water bath (DF-101T, Yuhua-China) under agitation (30 rpm). After thermal equilibrium, CO2 at 8.0MPa was fed faster (within 30s) into the reactor under continuous agitation to produce a CO2-expanded suspension for the onward reaction within set time. Products were recovered, washed severally with ethanol to remove traces of water and vacuum dried at 60℃ for 12h. Note that each batch experiment was repeated at least three times for reproducibility (Ibrahim et al., 2013;Ibrahim et al., 2014b).

Characterizations
Phase purity and composition of the products were characterized by X-ray diffraction (XRD); data were taken in 2θ° using a step size of 0.02° in 20s counting range between 5-60° with Cu Kα radiation (Rigaku Ultima IV). The morphology and structure of the particles produced were studied by scanning electron microscopy (SEM; Hitachi, S-4800). Functional group analysis was by Fourier transform infrared spectroscopy (FT-IR; Nicolet 330) within 400-4000 cm-1 using KBr with 4 cm-1 resolution whereas the surface area and pore properties of the powders were studied by Brunauer Emmett and Teller (BET; Micromeritics ASAP 2020, USA) analysis. The XRD patterns for commercial Ca(OH)2, CaCO3,    (Figure 1 a; d). This is a confirmation of the synthesis of CaCO3-HAP composite because the composite exhibited the characteristic peaks for both CaCO3 and HAP (Figure 1 b; d).

RESULTS AND DISCUSSIONS
Effect of temperature XRD patterns for the effect of temperature are shown in Figure 3. The reaction temperatures above the critical temperature of CO2 (T≥35℃) yielded complete conversions to pure CaCO3-HAP composites. The products of these reactions exhibited similarity in the XRD patterns (Figure 3a-d). However, the reaction temperatures below the critical temperature of CO2 led to emergence of new calcium phosphate compound: brushite (CaHPO4·(H2O)2) with diffraction peaks at 2θ = 11.7°, 18.0° and 20.9° (Figure 3, dotted inset). The XRD analysis revealed that the calcite content decreased with decreasing temperature from 60℃ to 40℃ whereas HAp content increased correspondingly with these temperatures. However, the calcite content increased with further decreasing temperature from 35℃ to 25℃ with corresponding decrease in HAp content. Even so, the 30℃ and 25℃ samples contained brushite ( Figure 3; Table S1). Therefore, the highest calcite content was realized at the lowest temperature condition (25℃) whereas the highest HAp content was at the moderate temperature condition (40℃). The BET analyses of the composites are also shown in Table S1. The specific surface area exhibited a contrary trend to the content of calcite. It increased with decreasing temperature from 60℃ to 40℃. However, it continues to decrease at 30℃ but increased at 30℃ and then decreased again albeit slightly at 25℃ (Table S1). Therefore, the surface area was dependent on the content of calcite and HAp or both. Specifically, the surface area was directly proportional to the content of HAp and inversely proportional to the calcite content (Table S1) from 60℃ to 35℃. The SEM images of the products from the temperature conditions (60℃, 50℃, 40℃, 30℃) are shown in Figure 4.
Besides the particle sizes, the structure and morphology of the particles were almost the same; mixture of rodlike, plate-like and flower-like structures. However, the size of the rod-like particles for the 40℃ and 30℃ were smaller compared to those obtained from the 60℃ and 50℃, perhaps, confirming their better surface properties (Table S1). Effect of pressure XRD patterns for effect of different pressures are shown in Figure 5. Almost all the pressures (5MPa, 8MPa, 10MPa and 12MPa) gave complete conversions (pure CaCO3-HAP composites) except for 4Mpa. There was about 3±0.2% (Table S2)      (calcite, HAp, monetite) were observed (XRD patterns in Figure 5). Rietveld analysis of the XRD patterns showed that the calcite content decreased at 4Mpa to 8Mpa but increased at 12Mpa. However, the HAp phase increased 4Mpa to 8Mpa but decreased finally at 12Mpa (Table S2). The BET specific surface and pore property analyses of the composites are also shown in Table S2. The surface area was inversely proportional to the calcite content at 4MPa to 8MPa but directly proportional to the HAp content at 8MPa to 12MPa. The surface area increased with decrease in the calcite phase but increase in the HAp content at 4MPa to 8MPa. However, the surface area decreased with increase in the calcite phase but decrease in the HAp content at 8MPa to 12MPa. https://journals.e-palli.com/home/index.php/ajmri Am. J. Multidis. Res. Innov. 1(2) 56-62, 2022 Effect of time XRD patterns for effect of different times are shown in Figure 7. All the times gave complete conversions of the starting material; Ca(OH)2 (Table S3) but not all the conditions yielded pure CaCO3-HAP composites. All the Ca(OH)2 was not transformed to the desired composite. There was brushite and monetite in <1 minute, monetite in 5 min and monetite in 10 min (Table S3). The SEM images for the products at the various times are shown in Figure 8. The structure and morphology of the particles appear to the same consisting of mixture of agglomerated spherical, rod-like, plate-like and flower-like structures.

Functional group analysis
Functional group analysis was also conducted on the CaCO3-HAP composite using FTIR between 400cm-1 to 4000cm-1 (Figure 9). All the functional groups (OH-, H2O, PO43-and CO32-) belonging to the composite were present and appeared at the usual wave lengths. Water (H2O) appeared at about 3433 cm-1 and 1640 cm- Figure 9. IR spectra of the CaCO3-HAP composites at different reaction times Figure 11. The XRD patterns for products before and after supercritical loading at low and high pressures; inset: ibuprofen loaded.  (Figure 9). In fact, XRD analysis had shown that CaCO3-HAP Composite was produced which is consistent with the results from the FTIR analysis. Thermal stability analysis Thermal stability analysis was conducted using SDT-Q600 analyzer in N2 atmosphere from room temperature (RT) to 1000℃ at a heating rate of 10℃/min and the results are shown in Figure 10. The total weight loss of the composite sample over the entire heating period (at RT to 1000℃) was about 20%, indicating good thermal stability.

Drug loading and dissolution
Solute-saturated supercritical loading procedure (Song et al, 2017;Ibrahim et al., 2019) was used to load ibuprofen into the composite sample produced at 40℃ and 8MPa in 30min. The composite contained 36.4% CaCO3 and 63.6% HAP with 133.60 m2/g specific surface area, 0.74 cm3/g pore volume and 10.60 nm pore diameter. XRD patterns of the samples before and after the supercritical loading experiments are shown in Figure 11 (inset is diffraction peaks for ibuprofen demonstrating effective loading at 2θ = 16.6 °, 17.7 ° and 20.2°). The diffraction peaks for ibuprofen was not clearly visible at lower pressure (10.0 MPa) loading perhaps, due to the minimal amount of loading (0.17g ibuprofen/g composite). At higher pressure (17.0MPa) loading, the diffraction peaks for ibuprofen was clearly visible attributed to substantial amount of ibuprofen loaded (0.28g ibuprofen/g composite) and/or deposited on the surface of the composite (Chen et al., 2013;Ibrahim et al., 2015). After the supercritical loading, 2.3 mg of the ibuprofencomposite samples loaded at 10.0 MPa and 17.0 MPa were dissolved in simulated gastric fluid (HCl-KCl buffer solution, pH = 1.4 ± 0.02) with a test apparatus (RCA-1A, Huanghai Medicine & Drug Testing Instruments co., Ltd., Shanghai, China) at 37 ± 0.5 °C with a paddle speed of 100 ± 1 rpm (Chen et al., 2013) for the dissolution test and the results are shown in Figure 12. The low pressure (10.0 MPa) loaded composite released 92% of the loaded ibuprofen within 30 min whereas the high pressure (17.0 MPa) loaded composite released 80% of the loaded ibuprofen within the same time period. However, the dissolution of pure ibuprofen sample was much lower; 18% of the loaded ibuprofen was released within 30 min. The high release of the loaded ibuprofen from the composites is due to the fact that the loaded ibuprofen particles were dispersed in the interior and the surface of the composite. This increased the contact surface with the buffer, thereby improving the dissolution rate. The low pressure (10.0 MPa) loaded composite gave faster dissolution compared to the high pressure (17.0 MPa) loaded composite up until about 150 min because of the less loading effect. Majority of the ibuprofen was dispersed and deposited uniformly on the surface of the low pressure composite leading to faster dissolution rate. Another important observation is that the low pressure loaded composite exhibited quick and faster release of the drug; all the ibuprofen was released within 200 min whereas the high pressure loaded composite exhibited slow and controlled release of the drug over 400 min. The implication of the results of the study is that if a patient was administered with a drug (for instance, ibuprofen) loaded into the synthesized composite (CaCO3-HAp) at low pressure (10.0 MPa), the patient will experience quicker relief (92% release of ibuprofen) in 30 min which will be sustained for almost 3.5 hours. The high pressure (17.0 MPa) loaded drug will also provide the patient with quicker relief (80% release of ibuprofen) however, the relief in this case will be sustained for longer period; about 6.5 hours. Therefore, the high pressure loaded composite had tremendous effect on the controlled release of ibuprofen and can be used as effective drug delivery system (DDS) for drugs with similar characteristics.

CONCLUSIONS
We employed Gas expanded liquids (GXLs) technology to synthesize calcium carbonate-hydroxylapatite composite nanoparticles and investigated the effects of reaction parameters on the properties of the composites. Based on the results we conclude that the composite had good thermal stability, better surface area and pore properties. The performance of the composites as a drug delivery agent using ibuprofen as a model drug in simulated gastric fluid was commendable. Whereas the low pressure loaded composite showed quicker relief potential (92% release) in 30 minutes with controlled release over desirable time frame (3h, 20 min), the high pressure loaded composite also exhibited quicker relief potential (80% release) in 30 minutes with controlled release over remarkably longer time frame (6h, 40 min). Therefore, the loaded composites had tremendous effect on the controlled release of ibuprofen and can be used as effective drug delivery system (DDS) for drugs with similar characteristics globally.