Pollen-derived microcapsules for aspirin microencapsulation: in vitro release and physico-chemical studies

Aspirin, also known as acetylsalicylic acid (ASA), is one of the most crucial therapies needed and/or used in a basic health system. Using biocompatible materials to encapsulate ASA would improve its therapeutic efficacy and reduce its side effects via controlled release in physiological environments. Consequently, we explore in this study the feasibility of encapsulation of ASA into robust Lycopodium clavatum L. sporopollenin (LCS) microcapsules. After extracting sporopollenin from their natural micrometer-sized raw spores, the physico-chemical features of the extracted sporopollenin, pure ASA, and sporopollenin loaded with ASA were characterised using various methods, including optical microscopy, Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible (UV-vis.) spectroscopy, thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and X-ray diffraction (XRD). Additionally, we demonstrate the in vitro release profile of ASA in a triggered gastrointestinal environment utilizing kinetics analysis to investigate the mechanism of release. The LCS microcapsules were found to be excellent encapsulants for the crucial ASA drug and achieved controlled in vitro release, that would enable further investigations to rationally design versatile controlled delivery platforms.

Where C is the percent of drug remaining at time t, C 0 is the initial amount of drug in the solution, K 1 is the first-order rate constant and t is the time. Log cumulative % of drug remaining is plotted versus time, and -K 1 /2.303 is the slope of the obtained line. Higuchi model based on Fickian diffusion mechanism is expressed by the simplified Higuchi equation:

Q t = K H × t 1/2
Where K H is the Higuchi constant and t is the time. To determine the drug release kinetics, cumulative amount of the released drug is plotted against the square root of time. Korsmeyer-Peppas model to understand the dissolution mechanisms from the matrix is expressed by the equation:

Mt/M∞ = K kp t n
Mt/M∞ is a fraction of drug released at time t, log (Mt/M∞) = log K kp + n log t, Mt is the amount of drug released in time t, M∞ is the amount of drug released after time ∞, n is the diffusional exponent or drug release exponent, K kp is the Korsmeyer release rate constant. To study release kinetics a graph is plotted between log cumulative % drug release log (Mt/M∞) vs. (log t), n value is used to characterize different release mechanisms. 3

Scanning electron microscopy (SEM)
Scanning electron microscope analysis was acquired using JSM-5400 LV JEOL (Japan). Samples were coated with 20 nm gold using a gold sputter (JEOL JFC-1100E). An acceleration voltage of 15 kV was used during capturing the images at different magnifications to observe the surface morphology.

UV-vis spectrophotometry
The UV-vis spectrophotometric analyses were performed with a double beam Unicam Helios Alpha spectrophotometer (made in England) fitted with deuterium and tungsten lamps, using a scan range of 200 -400 nm. Quartz cuvettes with optical path length of 1 cm were used.

pH-measurements
The pH was measured using a pH-meter model HI 8014 from HANNA instruments (Portugal) with Adwa (AD 1230B) gel-filled pH electrode. The pH-meter was calibrated before use with standard buffer solutions of pH= 4, 7 and 10 at 25 °C .

Optical microscopy
Optical images were taken by an OPTIKA (B-293) microscope (Italy) fitted with Optikam B5 (modelꓼ 4083.B5) digital camera and the images were processed using Optika IS view software. Micrometer slide was used for software calibration by using the microscope at different magnifications to calculate scale bars of images in microns. Calibration was made at the same resolution and magnification used through imaging.

Combustion CHN elemental analysis
CHN elemental analysis was conducted using a calibrated VarioEL III CHN automatic elemental analyzer (Elementar, Germany). Prior to conducting elemental analysis, to ensure combustion efficiency, all samples were dried at 60 °C for a minimum of 1 h before being combusted in excess oxygen at high temperature to release compositional carbon %C, hydrogen %H and nitrogen %N. The percentage of protein in different samples was calculated using the obtained percentage of nitrogen and the total Kjeldahl Nitrogen (TKN) conversion factor of 6.25 in accordance to recommendations from the Association of Analytical Communities (AOAC) to transform weight percent of nitrogen into weight percent of protein using the equation: 5 % Protein = % Nitrogen × 6.25
Samples were ground with anhydrous potassium bromide (Spectrosol grade) with a ratio of 1/9 (w/w) to obtain disks. All spectra were obtained in the 400 -4000 cm -1 range as a result of scan against a background, employing OMNIC software.

X-ray Diffraction (XRD) and Thermogravimetric Analysis (TGA)analysis
XRD was measured using a JEOL JSX-60PA diffractometer equipped with Ni-filtered Cu Kα radiation, at a wavelength (λ) of 1.5418Å, 35 kV voltage, and a current of 30 mA. The diffraction data were recorded in the range of 4°-100° with 0.1° 2θ step size.
TGA analysis was carried out with a TGA-50H from (SHIMADZU-Japan), with a heating rate of 10 °C /min up to 600 °C in a flow of nitrogen at 20 mL/min.