INTRODUCTION
Cockle shells derived from bivalve mollusks or Anadara granosa are a waste product of Malaysian aquaculture industry (Othman et al., 2013). The quest to reduce waste and looking inwards for alternative uses of this abundant natural reserve of calcium carbonate, the main chemical constituent of cockle shells, has resulted in its wide application in the field of engineering, pharmacology, and medicine (Razalia et al., 2016). Biocompatibility of calcium carbonate has been described and this makes it a perfect candidate for nano drug delivery (Kamba et al., 2013). Direct bactericidal activity of nanoparticles which results in improved therapeutic index and efficacy has prompted the loading of antibiotics into calcium carbonate nanoparticles with the aim of better treatment while reducing costs (Isa et al., 2016; Saidykhan et al., 2016).
Oxytetracycline (OTC) is a very common antibiotic widely used in veterinary medicine for the treatment of bacterial infections caused by both Gram-positive and Gram-negative organisms; however, the emergence of resistant strains has questioned its therapeutic effects (Larbi-Bouamrane et al., 2016). Employing the use of nanoparticles to deliver antibiotics in the treatment of bacterial infections has yielded good results due to the unique properties of nanoparticles such as ultra-small size, large surface to mass area, and bacterial membrane adhering properties (Adhikari et al., 2013). More so, the successful synthesis of nanoparticles loaded drug with right physicochemical properties for effective in vitro and in vivo interactions rely on the methods of synthesis. Different methods of synthesizing nanoparticles have been described and all revolve mainly around a top- or bottom-down approach. For the top-down approach, the fabrication of nanoparticles usually starts from the large-sized raw material and is broken down to the level of desired size of nanoparticles as the end product or from a bottom-up approach where individual elements to make diverse fine nanoparticles are synthesized from scratch, while the bottom down approach entails the synthesis of these nanoparticles from individual elements (Dhand et al., 2015; Priyadarshana et al., 2015). Three processes are basically used for the synthesis, namely, physical, biological, and mechanical process. In the physical method, heat, gas, and vapor are applied to the precursors of the different nanoparticles through processes like gas condensation, vacuum deposition and vaporization, laser pyrolysis, melt mixing, and high energy ball rolling mill (Rawat et al., 2016). The biological process involves the synthesis of nanoparticles from some microorganisms or plants while the chemical process involves nanoparticles fabrication via polyol synthesis, microemulsion technique, sol gel, and chemical vapor (Dhand et al., 2015).
Using the right method of synthesis, we, therefore, hypothesize that oxytetracycline will retain its physicochemical and pharmacological properties when loaded into cockle shell-derived calcium carbonate aragonite nanoparticles (CS-CaCO3NP). In this study, we synthesized (CS-CaCO3NP) by a top-down approach, loaded it with oxytetracycline, and characterized it for use against livestock resistant bacteria species.
MATERIALS AND METHODS
Synthesis of CaCO3 nanoparticles from cockle shell powder
Cockle shell was washed and milled mechanically to get micron-sized cockle shell powder as described by Danmaigoro et al. (2017). The prepared micron size cockle shell powder was further processed; thus, 2 g of 75 µm micron-sized powder of CS-CaCO3 powder was weighed with an electronic balance (Biobase, BA 2204 B) and put in a flat bottom flask with a magnetic stirring bar within it. 50 ml of deionized water was added to the powder. The resultant solution was stirred on a systematic multi hot plate stirring machine (Systematic Multi-Hotplate Stirrers 6 Positions, WiseStir® Korean) set at 1,000 rpm (27oC for 2 hours). After 5 minutes of agitation, 0.5 ml of dodecyl dimethyl betaine (BS-12) Sigma-Aldrich (Steinheim, Germany) was added gradually into the solution and covered with aluminum foil. After 2 hours of stirring, the solution was filtered with filter paper of diameter 18.0 cm and the surfactant rinsed off through the filter paper with double deionized water, leaving the sediments which are the nanoparticles on the filter paper. This was then allowed to dry for 3 days in the oven at 50oC. The dried newly synthesized nanoparticle was placed in a cylindrical jar of 8 cm in diameter (with a 7 cm flat iron fixed perpendicular to it) containing 15 ceramic balls and placed on a roller mill at 200 rpm for three consecutive days overnight (54 hours). The resulting fine CS-CaCO3 nanoparticle was then packed in glass bottles and stored in the oven at 50oC till further analysis.
Loading of oxytetracycline into synthesized CS-CaCO3 nanoparticles
The loading of oxytetracycline was done by mixing the free oxytetracycline and the right concentration of synthesized CS-CaCO3NP with the aid of lab multistirrer (Systematic Multi-Hotplate Stirrers 6 Positions, WiseStir® Korean) overnight at room temperature set at 200 ± 1 rpm. After overnight mechanical agitation, the suspensions of the OTC-CS-CaCO3NP formulation was centrifuged at 20,000 rpm for 15 minutes. Supernatant from formulation was kept in a separate tube, while the pellet (OTC-CS-CaCO3NP) was washed, dried, and analyzed for morphological characteristics.
Morphological characterization of synthesized CS-CaCO3NP and OTC-CS-CaCO3N
The average Zeta size, charge, and polydispersity index (PDI) of the synthesized CS-CaCO3NP and OTC-CS-CaCO3NP was done by dynamic light scattering technique using a Zetasizer Nano ZS, Malvern Instruments (Malvern Version 7.02, Malvern Instruments Ltd. UK). Briefly, 0.1 mg of freshly prepared CS-CaCO3NP and OTC-CS-CaCO3NP was dissolved in a test tube containing 10 ml of de-ionized water and was sonicated (Power Sonic 505®) for 20 minutes. 2 ml of the supernatant was gently aspirated into a syringe, fitted with a TRP® Spritzen-/syringe filter 0.45 µm and then fixed to the cuvette which was subsequently loaded into the machine. Measurements were taken in triplicates at a light scattering angle of 90oC at 25oC.
The shape and particle size distribution of the synthesized CS-CaCO3NP and OTC-CS-CaCO3NP was determined using transmission electron microscopy (TEM) (IUPAC) while the surface morphology was done by field emission scanning electron microscopy (FESEM) equipped with an energy-dispersive X-ray spectroscopy unit. The speciï¬c surface area of CS-CaCO3NP was determined by the Brunauer–Emmett–Teller (BET) technique via a 3-flex surface characterization analyzer (Micromeritrics, Instrument Corporation, USA) using a total CS-CaCO3NP weight of 0.2 g. X-ray powder diffractometer (Shimadzu XRD-6000 powder diffractometer) using CuKα (λ = 1.540562 Å) at 40 kV and 30 mA was used to investigate the crystallinity of CS-CaCO3NP, OTC-CS-CaCO3NP, and OTC alone. The crystallinity phase of the samples was done at different diffraction angles ranging from 2° to 60°, set at 0.02°/seconds in 2ÆŸ at 37°C (Saidykhan et al., 2016). Functional group endings of the CS-CaCO3NP, OTC-CS-CaCO3NP, and OTC alone was determined using Fourier-transform infrared spectroscopy (Model 100 series, Perkin Elmer) at a range of 4,000 to 280 cm−1 with a resolution of 2 cm−1 and an average scan of 64 times.
Determination of OTC loading content and encapsulation efficiency
Six formulations of OTC-CS-CaCO3NP loading were prepared. The first set comprised of three increasing doses of free OTC against the same three doses of CS-CaCO3NP to make a 1:1, 2:1, and 3:1 ratio of OTC:CS-CaCO3NP. The second set consists of three increasing doses of CS-CaCO3NP with the same three doses of free OTC in 1:2, 1:3, and 1:4 OTC:CS-CaCO3NP ratio, respectively. The absorbance of the supernatants at 353 nm using ultraviolet-visible (UV-Vis) spectrophotometer (Shimadzu UV 1800) was measured and the concentration of the unloaded free OTC in all the supernatants was then determined using the equation generated from standard calibration curve for OTC at 353 nm (Fig. 1).
After which the loading content (LC) and encapsulation efficiency (EE) was calculated using Equations (1) and (2), respectively. The analyses of LC and EE was expressed as the mean percentage ± standard deviation (SD) based on two formulations that yielded similar results:
LC (%) = Wt –Wf/Wnp ×100 1
EE (%) = Wt–Wf/Wt × 100 2
where Wt is the total weight of drug fed, Wf is the weight of non-encapsulated free drug, and Wnp is the weight of nanoparticles. The results of drug loading were generated from the average of two independent experiments (Saidykhan et al., 2016).
In vitro drug release study of the OTC form OTC-CS-CaCO3NP
The profile of free OTC released from OTC-CS-CaCO3NP was determined using the method of Chakraborty et al. (2012) with modifications. A total of 10 mg of free OTC and OTC-CS-CaCO3NP was placed in a dialysis tube of 22 mm × 32 feet dry diameter (Thermo Scientific USA) and suspended in a glass jar containing 200 mL deionized water of four different pH (pH 4, 6, 7.4, and 8). The glass jar was covered and placed on a lab multistirrer (WiseCube® WIS-10; Wisd Laboratory Instruments, Witeg, Germany) at 37°C ± 0.5°C at 120 ± 1 rpm for 4 days (96 hours). At calculated time spans of 1 to 8 hours and then 24, 48, 72, and 96 hours, respectively, 1,000 μl of the solution was withdrawn and replaced by an equal amount of fresh deionized water of the equivalent pH to maintain a sink condition. The concentration of OTC released was determined at 353 nm (wavelength) using an UV-Vis spectrophotometer. The absorbance was interpreted according to an OTC standard calibration curve. The release study was conducted until there was an apparent total release of OTC. The percentage of OTC released was calculated based on the amount of OTC released at a given time to the amount of OTC loaded in OTC-CS-CaCO3NP expressed mathematically below. The results obtained was plotted on a graph of cumulative percentage of drug release versus time intervals.
 | Figure 1. OTC standard curve at 353 nm. [Click here to view] |
Amount of OTC released ( mg ) = (Concentration derived from standard curve mcg × volume of dissolution medium) / 1000
Percentage OTC released = Amount of OTC released (mg) / Total amount of OTC in OTC−CS−CaCO3NP) × 100
OTC-CS-CaCO3NP) ×100
Cumulative percentage OTC released = P(t–1) + Pt
where Pt = Percentage OTC released at time t;P(t–1) = percentage OTC released at time 1.
Statistical analysis
All data obtained were expressed as mean ± SD except for the elemental percentages which were expressed as mean ± standard mean of error (SEM).
RESULTS AND DISCUSSION
Zeta size, zeta potential, and polydispersity index (PDI)
The characterization of nanoparticles based on physicochemical properties such as size, shape, charge, and dispersity are important steps to understand it pharmacokinetics and biodistribution in biological medium (Murdock et al., 2008). These physicochemical properties are the basic determinants of effective drug loading, controlled release, pharmacokinetics, efficacy, and toxicity, as well as bacterial cell internalization. Table 1 and Figures 2–5 show the zeta size, zeta potential, and PDI of CS-CaCO3NP and OTC-CS-CaCO3NP. The zeta size of CS-CaCO3NP range from 93 to 98 nm while higher size (268–280 nm) was observed for OTC-CS-CaCO3NP. The average zeta size (95.96 nm) of the synthesized CS-CaCO3NP is within the limit (<100 nm) for which CaCO3NP sizes <100 can be classified as nanometer-level particle, and this size usually enables the dispersion of drugs loaded within them (Pan et al., 2018). Higher zeta size of 142 nm for CaCO3NP has been reported (Danmaigoro et al. 2017). The inclusion of BS-12 and longer period of milling ball impact on the power contributed to the lower zeta size and PDI observed. Higher size has always been associated with the agglomeration of the nanoparticles; however, the use of BS-12, a zwitterionic surfactant which acts as a capping agent, improved the dispersion of the CaCO3NP, hence resulting in lower zeta size. The presence of a metal at a 90° angle in the ceramic jar increases the kinetic energy of the ceramic balls on one another as they strike the metal and further break the bonds in the CS-CaCO3NP in the controlled energy ball milling mechanical process also contributed to further grind the CS-CaCO3NP in the controlled energy ball milling mechanical process also contributed to further spreading and reducing the size of the nanoparticles generated from the cockle shell powder (Dhand et al., 2015). Low PDI values have been reported for CS-CaCO3NP from different studies (Isa et al., 2016; Saidykhan et al., 2016) and it is an indicator of the spread of size distribution of the nanoparticles (Faustino-Vega et al., 2009; Masarudin et al., 2015).
 | Table 1. Effect of dynamic light scattering on CS-CaCO3NP and OTC-CS-CaCO3NP. [Click here to view] |
 | Figure 2. Zeta size distribution of CS-CaCO3NP. [Click here to view] |
The zeta potential of CS-CaCO3NP and OTC-CS-CaCO3NP fall within the range (−25 mV and +25 mV) of aggregation for nanoparticles due to the weak Van Der Waal nanoparticle forces existing between the nanoparticles. The loading of oxytetracycline further increased the zeta charge almost close to the higher limit (Fig. 5) which requires greater force in bringing two particles together. A higher negative potential of OTC-CS-CaCO3NP is suggestive of adsorption of negatively charged OTC on the internal surfaces of the CS-CaCO3NP making the OTC-CS-CaCO3NP more nano stable and remain longer in circulation with a reduced degree of agglomeration (Das et al., 2011; Faustino-Vega et al., 2009). The negative surface charge of CS-CaCO3NP is also responsible for its interaction in vivo as negative nanoparticles have a prolonged systemic time compared to positive ones with very high clearance time (Arvizo et al., 2011).
CS-CaCO3NP and OTC-CS-CaCO3NP morphology
The overall morphology of the synthesized nanoparticles was assessed using TEM and FESEM. The former providing information on particles size while the latter give shape and appearance (Liu et al., 2010). The TEM revealed the average size of 29.90 ± 6.3nm within the range of 17.6–41 nm for CS-CaCO3NP (Figs. 6 and 7) while OTC-CS-CaCO3NP showed a mean size of 62.4 ± 20.68 nm within the range of 23.02–81.50 nm (Figs. 8 and 9). Results of the FESEM analysis revealed a consistent spherical shape with a porous surface and solid or dense appearance for CS-CaCO3NP and OTC-CS-CaCO3NP, respectively (Figs. 10 and 11). The shape, size, and overall morphology of the CS-CaCO3NP as seen on TEM and FESEM are important characteristics for effective loading of OTC into it by physical adsorption mechanism (Fu et al., 2017). The solid-dense appearance of OTC-CS-CaCO3NP indicates the presence of OTC within the nanoparticles. When comparing the zeta size with TEM size, it can deduce that both followed a similar pattern of low and high sizes for CS-CaCO3NP and OTC-CS-CaCO3NP, respectively. Although there is a marked difference in the actual sizes quoted by both techniques. The Zeta size measures the apparent size as the particles move in fluid (Brownian motion), thus measuring not only the particle but concentric layers of fluid around it (i.e., hydrodynamic diameter) resulting in exaggerated size values. On the other hand, in TEM which is another method for measuring size, the air drying of CS-CaCO3NP and OTC-CS-CaCO3NP on the gold palladium after sonicating in distilled water during sample preparation resulted in a reduction in the size of the nanoparticles (Motwani et al., 2008).
 | Figure 3. Zeta size distribution of OTC-CSCaCO3NP. [Click here to view] |
 | Figure 4. Zeta potential distribution of CS-CaCO3NP. [Click here to view] |
 | Figure 5. Zeta potential distribution of CS-CaCO3NP. [Click here to view] |
 | Figure 6. Photomicrograph of cockle shell derived calcium carbonate aragonite nanoparticle CS-CaCO3NP) on transmission electron microscope showing the nanoparticle sizes ranging from 17.6 to 41 nm. [Click here to view] |
 | Figure 7. Average diameter, size, and distribution of CS-CaCO3NP on TEM. [Click here to view] |
 | Figure 8. Photomicrograph of oxytetracycline loaded cockle shell derived calcium carbonate aragonite nanoparticle (OTC-CS-CaCO3NP) on transmission electron microscope showing an average nanoparticle size of 53.73 nm. [Click here to view] |
Elemental analysis of CS-CaCO3NP and OTC-CS-CaCO3NP (Tables 2 and 3) showed high amounts of oxygen, carbon, and calcium in decreasing order. This is in line with the chemical formula (CaCO3) with oxygen being the highest. For CS-CaCO3NP, carbon, oxygen, and calcium together account for 95.3% of the total elemental composition while the other elements (Mg, Na, Al, Si, and K) make up the remaining 4.7%. Similar findings were reported for C, O, and Ca by Kamba et al. (2013) and Islam et al. (2012). However, for OCT-CS-CaCO3NP, the total elemental percentage for C, O, and Ca together is 91% while phosphorus alone is 5.6% and other elements (Na, Al, Si, K, Cl, and Cu) make the remaining 3.4%. The molecular formula of oxytetracycline hydrochloride is C22H25CIN2O9. Loading of OTC into CS-CaCO3NP increased the percentage of carbon atoms while decreasing the amounts of oxygen and calcium present in CS-CaCO3NP. The concentration of elements that makes up CaCO3 is still high at 91% of the total amount of the elements that make up the nanoparticle loaded with drug loading with OTC did not change the composition of CS-CaCO3NP.
 | Figure 10. Field emission micrograph (FESEM) of spherically shaped CS-CaCO3NP. [Click here to view] |
 | Figure 11. Field emission micrograph (FESEM) of spherically shaped OTC-CS-CaCO3NP. nanoparticle with the solid (dense) appearance. [Click here to view] |
 | Figure 9. Average diameter, size, and distribution of OTC-CS-CaCO3NP on TEM. [Click here to view] |
 | Table 2. Elemental analysis of CS-CaCO3NP. [Click here to view] |
FTIR and XRD analysis of CS-CaCO3NP, OTC-CS-CaCO3NP, and OTC
The characteristic vibration of the different functional groups present in CS-CaCO3NP and OTC were explained by FTIR analysis (Fig. 12). CS-CaCO3NP showed its most prominent peak at 1,452 cm−1. This peak vibration corresponds to the alkyl group of CS-CaCO3NP (Islam et al., 2012; 2013) and is due to the C=O stretching vibration in the alkyl group. CO32- peak that is characteristic of aragonite CS-CaCO3NP were seen at 1,072.29, 854.77, and 707.58 cm−1, respectively. The spectra of free OTC showed characteristic absorption bands at 1,010.64–1,622.76 cm−1 which is attributed to the C=O and C=C bonds of the aromatic structure of OTC. The vibration at 1,622.76 cm−1 is attributed to the C=N-H groups while that seen with peak at 3,317.91 is due to the N-H and N-C absorption bands of the amine group. The spectral bands of OTC-CS-CaCO3NP which were attributed to the OTC are seen at 1,248.87–1,609 cm−1, whereas bands located at 851, 938.43, 1,464, and 1,609.71 cm−1 are those of amides while those at 1,020.03 and 1,248.87 are those resulting due to stretching of aromatic C==C. The bands seen between 1,200 and 1,600 cm−1 in the free OTC are also present within the OTC-CS-CaCO3NP bands. The characteristic CO32- of aragonite CS-CaCO3NP between 860.16 meaning that the OTC did not react significantly with the CS-CaCO3NP following loading suggesting dispersion of OTC molecules within the OTC-CS-CaCO3NP (Larbi-Bouamrane et al., 2016).
The XRD diffractograms were done at 2ÆŸ peak position to identify fingerprints of the crystal phase of samples (Ni and Ratner, 2008). The XRD result pattern (Fig. 13) for CS-CaCO3NP showed three strong peaks at 26.2°, 45.8°, and 33.1° at 2 ÆŸ degree while that of OTC-CS-CaCO3NP also showed three strong peaks at 31.8°, 32.4°, and 9.3°. These XRD diffractograms match with the International Centre for Diffraction Data database for aragonite crystals (JCPDS 00-141-1475). The appearance of two strong peaks of OTC-CS-CaCO3NP very close to 33.10 is evidence that loading OTC into CS-CaCO3NP did not affect the crystallinity of the CS-CaCO3NP nanoparticle (Kamba et al., 2013; Danmaigoro et al., 2017). The first peak (9.50) of free OTC corresponding to one of the strongest peaks in OTC-CS-CaCO3NP is an indication that the drug is unaffected by loading into the nanoparticle.
BET surface area, average pore diameter, and total pore volume of CaCO3NP
The curve derived from the linear isotherm plot of CaCO3NP (Fig. 14) is convex to the P/Po axis throughout its range which qualifies it to be classified as the reversible Type III isotherm based on the IUPAC classification of adsorption isotherms (IUPAC, 1980). Classifying CaCO3NP based on pore size, the average pore diameter (185 nm) is greater than 50 nm; hence, the pore size of the CaCO3NP is biocompatible. The pore size is important for the adsorption of drugs into the nanoparticle because it enhances encapsulation efficiency and drug delivery (Xu et al., 2018). The large surface area of 8.4987 ± 0.0922 (m²/g) of the CaCO3NP compared to the pore volume 0.392931 (cm³/g) (Table 4) is an important unique characteristic of nanoparticles which allows for close interactions with other molecules compared to their bulked size counterparts. A large total pore volume of 0.392931cm³/g is referred to as the adsorptive space (IUPAC, 1980).
Loading capacity and encapsulation efficiency of OTC
The LC and EE of the loaded OTC-CS-CaCO3NP are shown in Table 5. Increasing concentrations of OTC resulted in increased loading capacity and encapsulation efficiency with maximal drug loss. Increasing concentrations of OTC resulted in increased LC and EE with maximal drug loss. This is because higher drug concentrations imply higher LC and EE but with increased loss of drugs outside the nanoparticle over the capacity than the nanoparticle can hold. While with increasing nanoparticle weight, there was decreasing LC with increased EE and minimal drug loss. This is because loading drugs into CS-CaCO3NP is a function of the weight of drugs fed into the nanoparticles in relation to the weight of the nanoparticles (Abd Ghafar et al., 2017; Muhamad and Selvakumaran, 2014). Incorporating drugs into CS-CaCO3NP depends on capillary action and not on the surface charge and solubility of the drug (Jaji et al., 2017). The negative charge of CS-CaCO3NP may be responsible for the adsorption of OTC due to the electrostatic interaction with the positively charged quaternary ammonium functional group of the OTC molecule (Harja and Ciobanu, 2018). Also, physical interaction like Van der Waals forces and hydrogen bonding between OTC and – COOH and – OH functional groups of CS-CaCO3NP may have contributed to the adsorption of OTC into CS-CaCO3NP (Deng et al., 2013). The pores on the CS-CaCO3NP leading to tortuous channels within the nanoparticles which the drug is carried into and loading of OTC into the CS-CaCO3NP overnight could be responsible for the high loading capacity as the drug had been absorbed into the calcium carbonate nanoparticle resulting expansion and subsequent increase in size and dark hollow centers seen in the OTC-CS-CaCO3NP on TEM.
 | Table 3. Elemental analysis of OTC-CS-CaCO3NP. [Click here to view] |
 | Figure 12. FTIR spectra of OTC, OTC-CS-CaCO3NP, and CaCO3NP. [Click here to view] |
 | Figure 13. XRD spectra peak of OTC, OTC-CS-CaCO3NP, and CaCO3NP. [Click here to view] |
 | Figure 14. Adsorption-desorption isotherm linear plot of CaCO3NP displaying the typical type III convex shape relative to the P/Po axis. [Click here to view] |
 | Table 4. BET surface area, average pore diameter, and total pore volume of CaCO3NP. [Click here to view] |
 | Table 5. Loading content and encapsulation efficiency of OTC-CaCO3NP ratios. [Click here to view] |
In vitro OTC release from OTC-CS-CaCO3NP
The essence of loading drugs into carriers is not only to get them delivered to target sites but also ensure the maximum efficient release of the drug from the carrier so as to maintain a given concentration of the drug within the system during the course of therapy (Narayan et al., 2012). Release study (Fig. 15) shows a fast release (99.4%) over 3 hours for free OTC at pH 4. The solubility of free OTC in deionized water makes it easier and faster to diffuse through the membrane to reach the medium from which the concentration was determined. However, for OTC-CS-CaCO3NP, there was an initial rapid “burst” release from 0 to 8 hours, with a gradual and steady release over the rest of the 96 hours of the release profiles for the pH 4, 6, 7.4, and 8 tested. The release was highest for pH 4 (98.2) and 6.0 (91.8%) when compared to pH 7.4 (87.7%) and 8.0 (82.4%). The release of OTC at different pH indicates that CS-CaCO3NP is an efficient carrier which can maintain the concentrations of antibiotic during infections as compared to the free antibiotic release (Pan et al., 2018). This high amount of OTC released from OTC-CS-CaCO3NP at pH 4 may be attributable to the high solubility of OTC in acidic media as mentioned by Larbi-Bouamrane et al. (2016).
.png) | Figure 15. In vitro oxytetracycline release profile from OTC-CS-CaCO3NP. [Click here to view] |
The slow release of OTC from OTC-CS-CaCO3NP implies that encapsulation of OTC into colloids creates a better microenvironment which results in slow release of OTC over a longer period when compared with free OTC release (Faustino-Vega et al., 2009). Furthermore, the initial burst release followed by the gradual steady release of OTC from OTC-CS-CaCO3NP suggest that OTC-CS-CaCO3NP is a core-shell structure (Xu et al., 2011), with the OTC adsorbed on the surface (Shell) of CS-CaCO3NP been released rapidly, while OTC loaded in the inner-core could only be released slowly from OTC-CS-CaCO3NP via the pores through dissolution and diffusion (Larbi-Bouamrane et al., 2016; Narayan et al., 2012). Earlier studies of drug release from CS-CaCO3NP showed slow decomposition and release rate at pH 7.4 and faster at pH > 6.5 (Dijaz et al., 2015).