Nano-encapsulation is a platform which offers a promising application for control release and the delivery of drugs in pharmaceuticals and antioxidant/ antimicrobial in food systems. Poly (lactic-co-glycolide acid) (PLGA) is a biodegradable and biocompatible co-polymer of lactic acid and glycolic acid which is used for synthesizing food based polymeric nanoparticles (NP). The aim of this study was to evaluate the morphological and physicochemical properties and the controlled release of bioactive components derived from Aloe vera gel loaded PLGA NP. The results shows the mean hydrodynamic diameter of the unloaded NP is 103 nm which is significantly ( p < 0.01) smaller than the loaded freeze dried powered gel (FDG) (147 nm) and liquid gel (LG) (221 nm) and the particle size distribution given by the Poly-dispersity Index were 0.2, 0.2 and 0.3, respectively. The zeta potential for unloaded, FDG and LG NP were ±60, ±28 and ±22 mV, respectively, hence were electrokinetically stable NP. No significant ( p > 0.05) inhibition of the antioxidant potential was observed with loaded NP. The entrapment efficiency for the FDG synthesized was 87%, and the burst effect was observed after 4 h as a result of the encapsulation effect. The release kinetics of bioactive is govern by the combination of mass diffusion and capillary action.
The importance of reactive oxygen species (ROS) and free radicals has attracted increasing attention over the past decade due to their impact on human health. The ROS include free radicals such as superoxide anion radicals (
Renolds & Dweck [
The bioaccessibility of these bioactive compounds has been limited due to the harsh conditions encountered during food processing. Processing parameters such as temperature, light, pH severely degrade these bioactive components, furthermore conditions (pH, enzymes) in the gastrointestinal tracts or during storage when exposed to light and oxygen [
A mean Poly (Lactic co-Glycolide Acid) (PLGA) NP diameter (148 nm) loaded with (antioxidant thermoquinone) NP was reported by Nallamuthu, Parthasarathi, & Khanum [
The particle size, morphology and zeta potentials of loaded NP are significant properties that control the entrapment efficiency (EE) and the time releases of the bioactive compound. Nanoparticles synthesize with polymers such as Poly (Lactic co-Glycolide Acid) were reported to have a high EE due to its molecular structure that is associated to its active sites [
Poly (lactic-co-glycolide) acid (1:1, MW: 10,000 - 15,000 Da), DMAB (Dimethylamine borane), ethyl acetate and HPLC grade water were purchased from Fisher Scientific. Aloe vera gel (100%) was collected from the Aloe vera plant grown at the Alabama A & M University’s greenhouse.
The harvested leaves were properly washed with portable water and sliced open to extract the gel using an ethanol sterilized surgical knife. The extract was blended using a coffee grinder and pour into a freeze drying glass canisters. The gel samples in the freeze frying glass canisters were frozen overnight at −20˚C. The frozen samples were freeze dried using a freeze dryer (Labconco FreeZone-6, Kansas City, Mo) at −52˚C and 0.808 mbar for 48 h.
Nanoparticles were formed using the ultra-sonication solvent evaporation technique. The organic phase was formulated by dissolving PLGA in ethyl acetate along with about 44 mg of the samples (freeze dried Aloe veragel and liquid Aloe vera gel) at a ratio of 1:9 relative to PLGA. The samples were vortexed to dissolve the PLGA. The organic phase was added drop-wise to the aqueous 0.5% (w/v) DMAB solution with water (HPLC grade) and continually stirring with a Magnetic stirrer. Once all the compound/polymer mixture was added the emulsion was sonicated (Sonicator 3000, Ultrasonic liquid processor, Church Hill, CT) at 75 W for 15 min. Then the organic solvent, ethyl acetate, present in the emulsion was evaporated using Rotary vacuum evaporator (RE 301, Yamato Scientific Co. LTD, Tokyo, Japan) at 40˚C. The same procedure was repeated for synthesizing the unloaded (control) without the gel. The NP were collected by centrifugation at 10,000 g for 20 min at 4˚C. Finally, recovered NP was resuspended in 2 mL cryoprotectent solution (2% sucrose) and freeze dried using the same methods as described above.
The freeze dried NP was suspended in distilled water at the concentration of 10 mg/mL and 2 mL of each sample was pipetted into a quartz cuvette and the particle size distribution (PSD) and the polydispersity index (PDI) were measured using the dynamic laser scattering Malvern Zetasizer Nano series (Nano ZS90, Malvern Instruments Ltd., Worcestershire, UK) according to the method developed by Kassama et al. [
About 1 mL of each sample was pipetted into a zeta potential capillary cell which was placed in the Dynamic Light Scattering Malvern Zetasizer Nano series (Nano ZS90, Malvern Instruments Ltd., Worcestershire, UK) according to the method developed by Kassama et al. [
The morphology of the nanoparticle was determined by a transmission electron microscope (TEM) at University of Auburn, Auburn, AL at an accelerating voltage of 60 kV. About 10 µL of aqueous suspension of particles were placed on 300 mesh copper grids and stained with a 2% (w/v) phosphotungstic acid in dH2O to provide a contrast under magnification. The suspension was allowed to dry before viewing at 40,000 to 100,000× magnification.
The entrapment efficiency was determined as a function of the decreased DPPH concentration over time. The NP were dispersed in the 95% acetonitrile solution for a period of 72 h with periodic mixing to allow bioactive compounds to be diffused in the solution. The solutions were centrifuged at 3000 g to separate from PLGA. About 1 mL of the supernatant collected was mixed with 3 mL of 0.1 mM DPPH solution. The 95% acetonitrile gel solution (1 mL) was taken as the control. The reaction kinetics of the DPPH solution recovered from NP and control gel solutions determined by using the microplate spectrophotometer (Spectra-Max 250, Molecular Device Corp., Sunnyvale, CA) at 517 nm for 24 hours. The same procedure was repeated for the Aloe vera liquid samples. The entrapment efficiency was calculated as shown in Equation (1):
Release kinetics methodology was developed based on the modified version of the DPPH antioxidant potential method developed by Braca et al. [
where A is the absorbance at time t, A0 is the absorbance at time 0, Aα is the final absorbance and K is the release rate constant of the bioactive component released in the medium.
The modified 2,2-diphenyl-1-picrylhydrazyl(DPPH) assay used in this study was based on the method proposed by Nallamuthu et al. [
where A0 is the absorbance of the control, and A1 is the absorbance of treatment or standard sample.
The freeze dried and liquid Aloe vera gel were used with PLGA to synthesize Aloe vera freeze-dried powder (FDG) and liquid-gel (LG) loaded NP. The results of the physicochemical measurements of the synthesized FDG and LG loaded NP are shown in
No significant differences (p > 0.05) of the materials synthesized either in the PLGA unloaded NP and FDG loaded NP was observed on the PDI values of 0.2 (
Treatments | Hydrodynamic Diameter (nm) | TEM Diameter (nm) | Zeta Potential (mV) | Polydispersity index | EE (%) |
---|---|---|---|---|---|
PLGA Unloaded NP | 102.9 ± 0.35a | 60 | −60 | 0.2 | - |
Freeze Dried Aloe Vera gel Loaded PLGA NP | 146.2 ± 1.06b | 100 | −28 | 0.2 | 86.3 |
Liquid Aloe Vera gel Loaded PLGA NP | 221.4 ± 0.86c | 200 | −21.9 | 0.3 | 67.0 |
The NP dispersed in solution are usually energetic and under constant motion, and the repulsive forces between adjacent charges particles is one of the determinant factors to particle stability. Hence, the electrokinetic potential is a measure of the charges to establish stability of the NP. The results of the colloidal dispersion measured for this study varies within ±60 mV as shown in Figures 2(a)-(c), hence significantly different (p < 0.05) amongst the different treatments.
The mean zeta potential values were −60 mV, −28 mV & −21 mV for the unloaded, FDG and LG NP, respectively as shown in Figures 2(a)-(c). The zeta potential value for unloaded NP were higher than the loaded NP, this is largely due the influence of the bioactive compounds attached to the polymer charge, hence the adsorption of the bioactive compounds on the surface of the nano-carriers. The zeta potential values reported in this study shows strong electrostatic repulsive forces, hence will prevent particle aggregation, which ultimately suggests a highly stable emulsion. Pool et al. [
The size and structural morphology of the unloaded, FDG and LGNP are important characteristic features in the control release of encapsulated compound, hence the image by TEM was carried to determine their formation as shown in Figures 3(a)-(c).
present in the nano-emulsion. The actual particle diameters measures with TEM are shown in
The entrapment efficiency (EE) was determined based on the method described above and the values were calculated using Equation (1). The EE of the FDG synthesized is significantly different (p < 0.05) from the LG loaded NP, hence the values determined were 87% and 67%, respectively. These values are much higher than 48% and 39% for PLGA50-CBE and PLGA65-CBE, receptively reported by Hill et al. [
The controlled release kinetic profile of bioactive component derived from Aloe vera gel encapsulated with PLGA was studied as a function of decrease bioactive as shown in
Time (min) | Freeze dried Aloe vera gel | Freeze dried Aloe vera gel Loaded PLGA NP | ||
---|---|---|---|---|
Rate Constant (min−1) | R2 | Rate Constant (min−1) | R2 | |
Ph-I: 0 - 240 | 3.6 × 10−3 | 0.88 | 1.2 × 10−3 | 0.93 |
Ph-II: 240 - 300 | - | - | 6.5 × 10−3 | 0.99 |
Ph-III: 300 - 540 | - | - | 1.5 × 10−3 | 0.97 |
leading to burst effect [
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to determine the effect of the antioxidant potentials on nanoencapsulation of PLGA NP, hence the inhibition profile is shown in
assay was based on the reduction of hydrogen donating antioxidant due to the formation of diphenyl-1-picrylhydrazyl. The DPPH free radical scavenging power for the FDG loaded PLGA NP was 51% as shown in
Freeze dried powdered Aloe vera gel and Aloe vera gel liquid nanoparticles were synthesized with PLGA by using the solvent evaporation technique. The results by all indications show significant effect (p < 0.05) of Nano-encapsulation on the physicochemical properties of the nanoparticles. The mean hydrodynamic diameter of the unloaded PLGA NP is 103 nm which is significantly (p < 0.05) smaller than the loaded Aloe vera FDG (147 nm) and LG (221 nm) and the measured electrokinetic values indicates stable nanoparticle. No significant (p > 0.05) inhibition of the antioxidant potential was observed with encapsulated NP. The entrapment efficiency for the FDG synthesized was 87%, hence the burst effect was observed after 4 h as a result of the encapsulation effect. The release kinetics of bioactive is govern by the combination of mass diffusion and capillary action.
The authors’ wishes to acknowledge the financial support of the USDA National Institute of Food and Agriculture, [USDA-NIFA Capacity Building Grant Project Title: Nanotechnology Application in the Food Engineering Curriculum. Accession number 230755]. Likewise the AAMU experimental station for committing resources in support of the research project.
Kassama, L.S. and Misir, J. (2017) Physicochemical Properties and Control Release of Aloe Vera (Aloe bar- badensis Miller) Bioactive Loaded Poly (La- ctic Co-Glycolide Acid) Synthesized Nano- particles. Advances in Chemical Engineer- ing and Science, 7, 333-348. https://doi.org/10.4236/aces.2017.74025