Corresponding author: Krassimira Yoncheva (
The study was focused on the evaluation of two copolymers as micellar carriers for kaempferol delivery. The copolymers comprised identical hydrophilic blocks of poly(2-(dimethylamino)ethyl methacrylate and different hydrophobic blocks of either poly(ε-caprolactone) (PDMAEMA9-b-PCL70-b-PDMAEMA9) or poly(propylene oxide) (PDMAEMA13-b-PPO69-b-PDMAEMA13). The calculation of Flory-Huggins parameters and determination of encapsulation efficiency showed that PDMAEMA-b-PCL-b-PDMAEMA copolymer possessed higher capacity for kaempferol loading. The diameter of the micelles before and after lyophilization was not changed, suggesting that the micelles could be lyophilized and redispersed before administration. The in vitro release of kaempferol from PDMAEMA-b-PPO-b-PDMAEMA micelles was faster than the release from PDMAEMA-b-PCL-b-PDMAEMA micelles, probably due to the higher affinity of kaempferol to this copolymer. Further, the higher affinity resulted in a retention of antioxidant activity of kaempferol in the presence of DPPH and KO2 radicals. Thus, PDMAEMA-PCL-PDMAEMA was considered more appropriate carrier because of the higher encapsulation efficiency and preservation of antioxidant activity of the drug.
Copolymeric micelles are core-shell nanoaggregates formed by amphiphilic block copolymers that self-assembly in an aqueous medium above certain concentration known as critical micellar concentration. Copolymeric micelles are intensively investigated drug delivery carriers due to the high potential for efficient loading of hydrophobic active molecules in their core (
Antioxidants are an important class of active substances related to the treatment of many diseases associated with oxidative stress. However, many of these substances, especially those with a natural origin, are highly unstable in vitro or in vivo. For example, flavonoids could be degraded during processing or storage at inappropriate conditions, e.g. light or oxygen exposure (
The aim of the present study was to evaluate two amphiphilic copolymers as micellar carriers for kaempferol delivery. The copolymers comprised nearly the same shell-forming blocks of poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA) and different hydrophobic core-forming blocks (PCL or PPO). Thus, the work was focused on assessing the main physicochemical properties of kaempferol loaded micelles prepared from the two copolymers as well as their potential as antioxidant delivery systems.
Kaempferol, 1,4-dioxane, 2,2-diphenyl-1-picrylhydrazyl (DPPH), luminol and potassium superoxide were purchased from Sigma-Aldrich. The triblock copolymers PDMAEMA13-b-PPO69-b-PDMAEMA13 and PDMAEMA9-b-PCL70-b-PDMAEMA9 were previously synthesized as reported elsewhere (
Flory-Huggins parameter χsp was calculated applying the equation:
χsp = Vs (δs-δp)2/ RT
where Vs is the molar volume of the drug, δs and δp are the Schatchard-Hildebrand solubility parameters of the drug and polymer block forming the core, R is the gas constant and T is the Kelvin temperature (
Kaempferol loaded PDMAEMA-b-PCL-b-PDMAEMA and PDMAEMA-b-PPO-b-PDMAEMA micelles were prepared by the solvent evaporation method. Briefly, the selected copolymer (10 mg) and kaempferol (1.5 mg) were dissolved in 5 ml of 1,4-dioxane. After incubation for 30 min. (700 rpm), 2 ml of purified water was added dropwise to the organic phase. Next, the dioxane was evaporated under reduced pressure (Buchi-144, Switzerland) and the resulted micellar dispersions were filtered (0.22 µm) to separate the micelles from non-encapsulated drug. The filter was rinsed with ethanol and this drug fraction was collected to determine the drug loading efficiency. The aqueous micellar dispersions were lyophilized using sucrose as a lyoprotector.
The size, dispersity and zeta potential of drug-loaded micelles were determined by dynamic and electrophoretic light scattering using a Zetasizer NanoBrook 90Plus PALS, equipped with a 35 mW red diode laser, (λ = 640 nm) at a scattering angle of 90°. The zeta potential was calculated from the obtained electrophoretic mobility. All samples were measured at 25 °C.
Atomic force microscopy (AFM) images were obtained using a Bruker NanoScope V9 Instrument operating at 1.00 Hz scan rate under ambient conditions. The micelle solution (0.5 mg/ml) was spin-casted (2000 rpm) on a freshly cleaned glass substrate. AFM measurements were performed in Peak Force Tapping mode.
Kaempferol encapsulation was calculated as a difference between the initial concentration of the drug and the concentration found in the ethanol fractions collected after the filtration of the fresh micellar dispersion. Kaempferol was determined by UV-Vis spectrophotometry at a wavelength of 266 nm (ThermoScientific) according to a standard curve (5–25 µg/ml, r>0.9992). The encapsulation efficiency (EE) was calculated using the following equation:
The antioxidant activity of free kaempferol and kaempferol-loaded micelles was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and potassium superoxide scavenging assay (KO2). DPPH assay was performed according to a previously reported procedure (
Potassium superoxide scavenging assay (KO2) was performed by detection of the luminol-dependent chemiluminescence in a system of KO2 produced O2‒●. The apparatus (LKB 1251 luminometer, BioOrbit, Finland) was connected with AT-type computer via serial interface and MultiUse program ver. 1.08 for the collection of the obtained experimental data. The tested solutions of pure and micellar drug were mixed with 1 ml phosphate saline buffer (pH=7.4) containing 0.1 mM luminol. In parallel, control solutions without the tested pure or micellar kaempferol were prepared. The chemiluminescence response was measured immediately after the addition of 20 µl KO2 solution in DMSO. The chemiluminescence was registered for 1 min. every 50 milliseconds after the addition of KO2. The chemiluminescent response was calculated by determination of the area under the obtained chemiluminescent curve. The chemiluminescent ratio in the presence/absence of the tested compounds in percentage was used for calculation of the scavenging properties of the samples (
In the present study triblock copolymers containing blocks of PDMAEMA, in particular PDMAEMA9-b-PCL70-b-PDMAEMA9 or PDMAEMA13-b-PPO69-b-PDMAEMA13, were examined as micellar carriers of kaempferol taking in account the safety profile of copolymers containing short chains of PDMAEMA (
Size distribution of kaempferol loaded polymeric micelles prepared from PDMAEMA9-b-PCL70-b-PDMAEMA9 and PDMAEMA13-b-PPO69-b-PDMAEMA13 triblock copolymers.
Physicochemical properties of empty and kaempferol loaded micelles. Mean ± SD.
Micelles | Mean diameter (nm) | Dispersity | Zeta-potential (mV) |
---|---|---|---|
PDMAEMA9-b-PCL70-b-PDMAEMA9 | 134 ± 7 | 0.13 | 36.5 ± 5 |
KF-PDMAEMA9-b-PCL70-b-PDMAEMA9 | 161 ± 3 | 0.14 | 45.6 ± 2 |
PDMAEMA13-b-PPO69-b-PDMAEMA13 | 170 ± 4 | 0.15 | 34.9 ± 4 |
KF-PDMAEMA13-b-PPO69-b-PDMAEMA13 | 247 ± 4 | 0.17 | 40.3 ± 2 |
AFM analysis revealed that PDMAEMA13-b-PPO69-b-PDMAEMA13 copolymer formed a mixture of spherical and anisotropic (elongated structures) micelles, unlike the PDMAEMA9-b-PCL70-b-PDMAEMA9 copolymer which formed only spherical micelles (Fig.
The physicochemical properties of the micelles after lyophilization and redispersion are very important for their in vivo administration, efficiency and safety. The main characteristics that could be changed by lyophilization are the micellar size and the tendency for aggregation (
The two copolymers are similar regarding their macrochain architecture and the type and length of the hydrophilic segments; so their distinctive feature is the type of the hydrophobic block. Since kaempferol is a hydrophobic substance it is expected that drug molecules will be embedded into the hydrophobic micellar cores. Therefore, the affinity of the active molecule to the core-forming polymer is of a big importance for the efficient loading, release of the active substance and in vitro and in vivo stability of micelles (
The in vitro release of kaempferol from the micelles was performed in distilled water. The study showed the presence of initial burst effect and sustained release in the second phase (Fig.
AFM images of kaempferol loaded PDMAEMA9-b-PCL70-b-PDMAEMA9 (left) and PDMAEMA13-b-PPO69-b-PDMAEMA13 (right) micelles.
Calculated values for solubility parameters (δ), drug-polymer compatibility (χsp) for PCL- and PPO-containing copolymers and encapsulation efficiency (EE).
Kaempferol / Copolymer | δ (MPa1/2) (Fedors method) | χsp | EE (%) |
---|---|---|---|
Kaempferol | 34.2 | – | |
PDMAEMA-b-PCL-b-PDMAEMA | 19.7 | 14.5 | 66 |
PDMAEMA-b-PPO-b-PDMAEMA | 16.1 | 22.4 | 61 |
In vitro release of kaempferol from PDMAEMA9-b-PCL70-b-PDMAEMA9 and PDMAEMA13-b-PPO69-b-PDMAEMA13 micelles in distilled water.
The antioxidant activity of free and micellar kaempferol was evaluated in two model systems, in particular systems containing stable DPPH radicals or superoxide radicals (KO2). It is known that superoxide radicals participate in the formation of peroxynitrite, which is the most reactive form of the active forms of nitrogen. The radical scavenging activity of kaempferol loaded micelles and free kaempferol are presented in Fig.
DPPH and anion superoxide scavenge capacity of free kaempferol (KF) and micellar kaempferol;
The data in the present study suggest that micelles formed by PDMAEMA-PCL-PDMAEMA copolymer are appropriate system for delivery of kaempferol as the drug was well dissolved in aqueous media with the aid of micelles. The good compatibility between PCL block and kaempferol favoured a sustained drug release profile and contributed to preserve its antioxidant activity. In addition, the micelles maintained their structural integrity and nanosized dimensions after lyophilization and redispersion that encourage their further evaluation as drug delivery system of kaempferol.