Physicochemical, Pharmacokinetic, and Toxicity Evaluation of Methoxy Poly(ethylene glycol)-b-Poly(d,l-Lactide) Polymeric Micelles Encapsulating Alpinumisoflavone Extracted from Unripe Cudrania tricuspidata Fruit

Alpinumisoflavone, a major compound in unripe Cudrania tricuspidata fruit is reported to exhibit numerous beneficial pharmacological activities, such as osteoprotective, antibacterial, estrogenic, anti-metastatic, atheroprotective, antioxidant, and anticancer effects. Despite its medicinal value, alpinumisoflavone is poorly soluble in water, which makes it difficult to formulate and administer intravenously (i.v.). To overcome these limitations, we used methoxy poly(ethylene glycol)-b-poly(d,l-lactide) (mPEG-b-PLA) polymeric micelles to solubilize alpinumisoflavone and increase its bioavailability, and evaluated their toxicity in vivo. Alpinumisoflavone-loaded polymeric micelles were prepared using thin-film hydration method, and their physicochemical properties were characterized for drug release, particle size, drug-loading (DL, %), and encapsulation efficiency (EE, %). The in vitro drug release profile was determined and the release rate of alpinumisoflavone from mPEG-b-PLA micelles was slower than that from drug solution, and sustained. Pharmacokinetic studies showed decreased total clearance and volume of distribution of alpinumisoflavone, whereas area under the curve (AUC) and bioavailability were significantly increased by incorporation in mPEG-b-PLA micelles. In vivo toxicity assay revealed that alpinumisoflavone-loaded mPEG-b-PLA micelles had no severe toxicity. In conclusion, we prepared an intravenous (i.v.) injectable alpinumisoflavone formulation, which was solubilized using mPEG-b-PLA micelles, and determined their physicochemical properties, pharmacokinetics, and toxicity profiles.


Introduction
Cudrania tricuspidata, also known as the silkworm thorn and storehousebush, is a thorny and perennial tree of the family Moraceae, which is widespread in East Asia. It has multiple nutritional and medicinal applications, so this tree has been used as a source of traditional medicine for the cure of some conditions such as contusions, insomnia, eczema, mumps, tuberculosis, and acute arthritis [1]. The fruit of C. tricuspidata is a berry that is rich in various active ingredients, including major functional ingredients such as polyphenols and flavonoids that possess diverse biological activities [2]. Despite its numerous nutritional and medicinal applications, the use of this fruit has been limited to simple oral intake including as juice, wine, jam, and folk medicine.
The concentration of AIF in samples obtained from in vitro and in vivo assays in this study was determined using a Waters high-performance liquid chromatography (HPLC) system (Waters, Milford, MA, USA) equipped with Waters 2695 separations module and Waters 2996 photodiode array detector. The Fortis C18 chromatography column (5 µm, 4.6 × 250 mm) was used and maintained at 30 • C during the analysis. AIF and genistein (internal standard) were eluted using the isocratic mode with a mobile phase composed of ACN/water (70:30, v/v), which was freshly prepared for each run and degassed before use. The sample injection volume was 10 µL and flow rate was 1.0 mL/min for the mobile phase. The retention times of genistein and AIF were 3.4 and 12.7 min, respectively and their concentrations were calculated by comparing the peak areas with the standard curve ( Figure 1).

Determination of AIF Solubility
The solubility of AIF in DW or representative solubilizing agent was measured using a previously reported method [28]. To determine the solubility of AIF in water and a Cremophor EL ® /EtOH mixture (representative solubilizing agent commercially used in Taxol ® ), AIF was added to 1 mL water, 100% EtOH, 50% Cremophor EL ® /50% EtOH until saturation occurred. The solution was vortexed and centrifuged at 13,000 rpm for 5 min. The supernatant was filtered using a 0.2 µm regenerated cellulose filter (Corning Inc., Corning, NY, USA). The filtered clear solution was 100-fold diluted and 10 µL was injected into the HPLC system to determine the solubility.

Determination of AIF Solubility
The solubility of AIF in DW or representative solubilizing agent was measured using a previously reported method [28]. To determine the solubility of AIF in water and a Cremophor EL ® /EtOH mixture (representative solubilizing agent commercially used in Taxol ® ), AIF was added to 1 mL water, 100% EtOH, 50% Cremophor EL ® /50% EtOH until saturation occurred. The solution was vortexed and centrifuged at 13,000 rpm for 5 min. The supernatant was filtered using a 0.2 μm regenerated cellulose filter (Corning Inc., Corning, NY, USA). The filtered clear solution was 100-fold diluted and 10 μL was injected into the HPLC system to determine the solubility.

Preparation of AIF-Loaded Micelles
AIF-loaded polymeric micelles were prepared from various polymers using the thin-film hydration method [29]. Briefly, 6 mg AIF and various amounts of polymers (Pluronics ® F127 or mPEG(4k)-b-PLA[2.2k]) were dissolved in 1 mL ACN. For Soluplus ® , 6 mg of AIF and various amounts of Soluplus ® were dissolved in a mixture of 0.8 mL MeOH/0.4 mL ACN. The drug-polymer mixture was added into round-bottomed flask and the solvent was evaporated using a rotary evaporator (EYELA, Bohemia, NY, USA) at 60 °C for 10 min under reduced pressure to obtain a thin film. After complete evaporation, the film was hydrated with 1 mL DW for 30 min to obtain a clear micelle solution. The micelle solution was then centrifuged at 13,000 rpm (Hanil Science lnc., Gimpo, Korea) for 5 min and filtered using a sterile regenerated cellulose filter (0.2 μm pore size) to remove the unincorporated drug and polymer ( Figure 2).

Preparation of AIF-Loaded Micelles
AIF-loaded polymeric micelles were prepared from various polymers using the thin-film hydration method [29]. Briefly, 6 mg AIF and various amounts of polymers (Pluronics ® F127 or mPEG(4k)-b-PLA[2.2k]) were dissolved in 1 mL ACN. For Soluplus ® , 6 mg of AIF and various amounts of Soluplus ® were dissolved in a mixture of 0.8 mL MeOH/0.4 mL ACN. The drug-polymer mixture was added into round-bottomed flask and the solvent was evaporated using a rotary evaporator (EYELA, Bohemia, NY, USA) at 60 • C for 10 min under reduced pressure to obtain a thin film. After complete evaporation, the film was hydrated with 1 mL DW for 30 min to obtain a clear micelle solution. The micelle solution was then centrifuged at 13,000 rpm (Hanil Science lnc., Gimpo, Korea) for 5 min and filtered using a sterile regenerated cellulose filter (0.2 µm pore size) to remove the unincorporated drug and polymer ( Figure 2). Extraction of alpinumisoflavone (AIF) from unripe C. tricuspidata fruits and preparation of AIF-loaded polymeric micelles using thin-film hydration method.

Physicochemical Characterization of Micelles
The particle size of the AIF-loaded micelles was measured using a dynamic light-scattering (DLS) device (Otsuka Electronics, Osaka, Japan). The prepared micelle solution was 10-fold diluted before determination. Drug-loading (DL, %) and encapsulation efficiency (EE, %) of the AIF-loaded micelles were determined using HPLC analysis and were calculated using the following equations: DL% = Weight of drug in micelles/weight of feeding polymer and drug × 100 EE% = Weight of drug in micelles/weight of feeding drug × 100

Physicochemical Characterization of Micelles
The particle size of the AIF-loaded micelles was measured using a dynamic light-scattering (DLS) device (Otsuka Electronics, Osaka, Japan). The prepared micelle solution was 10-fold diluted before determination. Drug-loading (DL, %) and encapsulation efficiency (EE, %) of the AIF-loaded micelles were determined using HPLC analysis and were calculated using the following equations: DL% = Weight of drug in micelles/weight of feeding polymer and drug × 100 EE% = Weight of drug in micelles/weight of feeding drug × 100 The results of each sample analysis were expressed as the mean ± standard deviation (SD) of three separate experiments.

In Vitro Drug Release Assay
The in vitro release behavior of AIF in the micelles was assessed using the dialysis method with phosphate-buffered saline (PBS, pH 7.4) as the release medium, over 336 h. In brief, samples of AIF-loaded mPEG-b-PLA micelles (5 mg/mL) and AIF solution (5 mg dissolved in Cremophor EL ® :EtOH, 50:50, v/v) as the control were inserted into a pre-wetted dialysis membrane bag (MWCO 20 kD), which was tied and placed in 2.0 L release medium on a hot plate stirrer at 37 • C. The release medium was replaced with fresh medium after 8, 48, 120, 168, 216, and 288 h. At predetermined time intervals (0, 2, 4, 6, 8, 24, 48, 72, 168, 240, and 336 h), 20 µL of the sample was collected, 10-fold diluted with ACN, and the concentration of AIF was measured using HPLC analysis. Three experiments were conducted.

Pharmacokinetic Study
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University (CBNUR-1141-17, 24 October 2017). Male, Sprague-Dawley rats (7-week-old) were purchased from Orient Bio Inc. (Seongnam, Korea) and used for all animal experiments. The rats were housed in ventilated cages with free access to water and food. The rats in each group were i.v. injected with AIF (20 mg/kg) in 25% Cremophor EL ® /EtOH (AIF solution) and AIF-loaded mPEG-b-PLA micelle. Despite its toxicity, Cremophor EL ® /EtOH was used as a solubilizing agent for AIF solution. Blood samples were obtained from the retro-orbital plexus of the rats 5, 15, 30, 60, 120, and 240 min after administration and centrifuged at 3000 rpm for 5 min to obtain plasma samples, which were immediately frozen and stored at -70 • C until analysis. A non-compartmental model was used for the pharmacokinetic analysis to calculate relevant parameters: area under the concentration-time curve (AUC), total clearance (CL t ), apparent volume of distribution (V d ), and initial blood concentration (C 0 ) of AIF after injection of each formulation.

Biodistribution Study
The biodistribution study was conducted after i.v. injecting rats with the various formulations. At 8 h after administration, the rats were euthanized using carbon dioxide (CO 2 ) and the liver, kidneys, spleen, heart, lungs, and muscle tissues were dissected. The tissue samples were washed in saline, wiped with paper towels to remove excess fluid, weighed, and then stored at −70 • C until analysis.

Pre-Treatment of Biological Samples for HPLC Analysis
The frozen biological samples were thawed at room temperature. A 200 µL aliquot of the plasma sample was extracted with MeOH and 50 µL genistein (IS) was added, followed by centrifugation for 5 min at 13,000 rpm. The supernatant was filtered through a regenerated cellulose filter (0.2 µm pore size) and 10 µL of the sample was injected into the HPLC system for analysis [30]. The amount of AIF in the liver, kidney, spleen, heart, lung, and muscle samples was measured using homogenization method [31]. Briefly, the tissues were homogenized with a glass Potter-Elvehjem-type homogenizer with a Teflon pestle. After extraction of 200 µL of 20% homogenate with MeOH, the concentration of AIF in the supernatant was determined as described above.

In Vivo Toxicity Assay
Five groups of mice (n = 3 per group) were used to evaluate in vivo toxicity. The rats in each group were i.v. injected with Dulbecco's phosphate-buffered saline (DPBS, control), AIF (20 mg/kg) in 100% Cremophor EL ® /EtOH solution, AIF (20 mg/kg) in 50% Cremophor EL ® /EtOH solution, AIF (20 mg/kg) in 25% Cremophor EL ® /EtOH solution, and AIF-loaded mPEG-b-PLA micelles (20 mg/kg). The body weight changes of all groups were monitored once every two days for a total of 14 days. The i.v. injections of AIF solution and micelles were carried out on day 0, 4, and 8. The toxicity of formulations was defined as loss of body weight loss (over 10%), abnormal behavior, signs of discomfort, or death. The body weight changes were normalized and displayed as percentages. In the toxicity test, rats judged to have moribund conditions were euthanized [32].

Statistical Analysis
All data are expressed as the means ± SD. Distribution parameters are expressed as the means ± standard error (SE). Statistical analyses were performed using the Student's t-test and statistical significance was accepted at p < 0.05 or < 0.01 (95% and 99% confidence levels, respectively).

Isolation and Identification of AIF
The isolated AIF from extract of unripe C. tricuspidata fruits was identified as follows:   Table 1 shows the solubility in water, Cremophor EL ® /EtOH ratio (5:5, v/v), and Cremophor EL/EtOH ratio (0:10, v/v). The measured solubility of AIF in water was only 0.178 ± 0.121 μg/mL, confirming its poor water solubility. The solubility of AIF when dissolved in representative solubilizing agent (Cremophor EL ® and EtOH in half volume ratio) was 82.143 ± 9.989 mg/mL, indicating that the solubility could be elevated by using solubilizing agents, but the toxicity of these solubilizing excipients becomes a problem when designing an intravenously injectable formulation [16,[33][34][35].   Table 1 shows the solubility in water, Cremophor EL ® /EtOH ratio (5:5, v/v), and Cremophor EL/EtOH ratio (0:10, v/v). The measured solubility of AIF in water was only 0.178 ± 0.121 µg/mL, confirming its poor water solubility. The solubility of AIF when dissolved in representative solubilizing agent (Cremophor EL ® and EtOH in half volume ratio) was 82.143 ± 9.989 mg/mL, indicating that the solubility could be elevated by using solubilizing agents, but the toxicity of these solubilizing excipients becomes a problem when designing an intravenously injectable formulation [16,[33][34][35].

Physicochemical Characterization of AIF-Loaded Micelles
The EE (%), DL (%), and particle diameters of the AIF-loaded micelles prepared using various polymers (mPEG-b-PLA, Pluronics ® F127, and Soluplus ® ) are shown in Table 2. As demonstrated in the solubility section, measured solubility of AIF in water was very low (0.178 ± 0.121 µg/mL), indicating that a negligible portion (less than 0.01%) of the drug could exist in the aqueous phase of micellar solution. Furthermore, the unincorporated drug and/or polymer precipitates were removed by the centrifugation and filtration during the process. In this point of view, encapsulation of AIF in the polymeric micelles was well-characterized, as previous researches have done so far [16,17,36]. AIF-loaded mPEG-b-PLA and AIF-loaded Pluronics ® F127 micelles showed relatively high EE (39.5-81.0%) and DL (2.24-4.59%) values, whereas values of AIF-loaded Soluplus ® micelles were very low (AIF concentration, <0.1 mg/mL). In the DLS analysis, the average particle diameter of AIF-loaded mPEG-b-PLA micelles was 28.0-34.1 nm while those of other micelles prepared using Pluronics ® F127 or Soluplus ® were not detectable or showed multiple distribution with high (>0.5) polydispersity index (PDI) because of precipitation or abnormal conditions (Figure 4). The <100 nm particle size of the micelles indicates that phagocytosis and uptake by the reticuloendothelial system (RES) could be avoided, thereby prolonging its systemic circulation [37]. Considering the high EE and DL values and appropriate particle diameter, the mPEG-b-PLA polymer was selected for solubilization of AIF. At 135 and 150 mg, mPEG-b-PLA showed no significant differences in the EE, DL, and particle size. To minimize a potential unexpected effect of the excipient, the lower amount (135 mg) of the mPEG-b-PLA polymer and 6 mg AIF were the fixed amounts used in the preparation of AIF micelles, which will be used in further in vitro and in vivo studies.

In Vitro Drug Release Profile
In vitro drug release profiles of AIF-loaded mPEG-b-PLA micelle and AIF dissolved in Cremophor EL ® /EtOH solution are shown in Figure 5. The drug release of AIF-loaded mPEG-b-PLA micelles and AIF solution reached 32% and 63%, over 6 h, respectively. At 336 h, 84.6% of AIF was released from AIF solution, while 67.3% of AIF was released from AIF-loaded mPEG-b-PLA micelle, indicating that AIF in the mPEG-b-PLA micelles was released slower than that in the solution. This result indicated that the micelle carrier not only solubilizes poorly soluble drugs, but also restricts the rapid release of drugs and this is presumably due to intermolecular interaction of the hydrophobic drug and the hydrophobic segment of the polymer. These results showed that the micelle carrier not only solubilized the poorly soluble AIF, but it was also slowly released [38].

Pharmacokinetics of AIF in Rats
The mean plasma concentration time profiles of AIF in mPEG-b-PLA micelles or 25% Cremophor EL ®/ EtOH solution are shown in Figure 6. The plasma concentration of AIF decreased rapidly after intravenous injection of AIF-loaded mPEG-b-PLA micelles or AIF solution in rats. However, it was found that the plasma concentration of AIF at 4 h after injection of AIF solution was lower than limitation of detection (LOD), while concentration of AIF at 4 h after injection of AIF-loaded mPEGb-PLA was still detected in the plasma. The long circulation time of the micelles and sustained release of AIF contributed to the long retention of AIF in the blood. Pharmacokinetic parameters were calculated using a non-compartmental model. As shown in Table 3, when AIF was encapsulated in mPEG-b-PLA micelles, the CLt and Vd values of AIF were decreased. The AUC and bioavailability (BA, %) values were significantly higher with AIF-loaded mPEG-b-PLA micelles than with the AIF solution (* p < 0.05). These results indicated that mPEG-b-PLA micelles significantly increased blood

Pharmacokinetics of AIF in Rats
The mean plasma concentration time profiles of AIF in mPEG-b-PLA micelles or 25% Cremophor EL ® /EtOH solution are shown in Figure 6. The plasma concentration of AIF decreased rapidly after intravenous injection of AIF-loaded mPEG-b-PLA micelles or AIF solution in rats. However, it was found that the plasma concentration of AIF at 4 h after injection of AIF solution was lower than limitation of detection (LOD), while concentration of AIF at 4 h after injection of AIF-loaded mPEG-b-PLA was still detected in the plasma. The long circulation time of the micelles and sustained release of AIF contributed to the long retention of AIF in the blood. Pharmacokinetic parameters were Pharmaceutics 2019, 11, 366 9 of 13 calculated using a non-compartmental model. As shown in Table 3, when AIF was encapsulated in mPEG-b-PLA micelles, the CL t and V d values of AIF were decreased. The AUC and bioavailability (BA, %) values were significantly higher with AIF-loaded mPEG-b-PLA micelles than with the AIF solution (* p < 0.05). These results indicated that mPEG-b-PLA micelles significantly increased blood circulation time and decreased plasma clearance of AIF.
The mean plasma concentration time profiles of AIF in mPEG-b-PLA micelles or 25% Cremophor EL ®/ EtOH solution are shown in Figure 6. The plasma concentration of AIF decreased rapidly after intravenous injection of AIF-loaded mPEG-b-PLA micelles or AIF solution in rats. However, it was found that the plasma concentration of AIF at 4 h after injection of AIF solution was lower than limitation of detection (LOD), while concentration of AIF at 4 h after injection of AIF-loaded mPEGb-PLA was still detected in the plasma. The long circulation time of the micelles and sustained release of AIF contributed to the long retention of AIF in the blood. Pharmacokinetic parameters were calculated using a non-compartmental model. As shown in Table 3, when AIF was encapsulated in mPEG-b-PLA micelles, the CLt and Vd values of AIF were decreased. The AUC and bioavailability (BA, %) values were significantly higher with AIF-loaded mPEG-b-PLA micelles than with the AIF solution (* p < 0.05). These results indicated that mPEG-b-PLA micelles significantly increased blood circulation time and decreased plasma clearance of AIF.  Table 3. Pharmacokinetic parameters of alpinumisoflavone (AIF) after intravenous (i.v.) injection of AIF-loaded mPEG-b-PLA micelles and AIF dissolved in 25% Cremophor EL ® /ethanol (EtOH) solution.

Biodistribution of Micelles in Rats
The drug amounts of AIF in major organs at 8 h after i.v. injection of AIF-loaded mPEG-b-PLA micelles or AIF dissolved in 25% Cremophor EL ® /EtOH solution were determined and displayed in Figure 7. The amount of AIF (µg/g) in each tissue after i.v. injection of drug solution decreased in the following order of magnitude: the liver > kidney > lung > heart > spleen > muscle, while that after injecting the AIF-loaded mPEG-b-PLA micelles was: the liver > kidney > lung > spleen > heart > muscle. This finding confirmed that the AIF amount in both the solutions and mPEG-b-PLA micelles was high in the liver, which is a RES-related organ. However, a considerable amount of AIF also accumulated in non-RES organs, such as the kidneys, indicating that the tissue specificity of the micelles might also influence absorption by the RES system, as suggested by a previous study [39]. Overall, the mPEG-b-PLA micelles play a role as a vehicle of AIF, and the drug amount in major organs such as the liver, kidney, and lung were assumed to be relatively higher than that obtained with the drug solution, presumably because of the long circulation time in the blood [40,41].
muscle. This finding confirmed that the AIF amount in both the solutions and mPEG-b-PLA micelles was high in the liver, which is a RES-related organ. However, a considerable amount of AIF also accumulated in non-RES organs, such as the kidneys, indicating that the tissue specificity of the micelles might also influence absorption by the RES system, as suggested by a previous study [39]. Overall, the mPEG-b-PLA micelles play a role as a vehicle of AIF, and the drug amount in major organs such as the liver, kidney, and lung were assumed to be relatively higher than that obtained with the drug solution, presumably because of the long circulation time in the blood [40,41].  Figure 8 shows body weight changes of the rat groups after multiple injections of DPBS (control), AIF dissolved in 100% Cremophor EL ® /EtOH solution, 50% Cremophor EL ® /EtOH solution, 25% Cremophor EL ® /EtOH solution, and mPEG-b-PLA micelles. As demonstrated in Figure 8A, rats in the groups treated with AIF dissolved in 50% Cremophor EL ® /EtOH or 25% Cremophor EL ® /EtOH solution exhibited significantly lower mean body weight increase than that in the control group. This shows that even after dilution to minimize the solubilizing agent levels, the Cremophor EL ® /EtOH used as representative solubilizing agent was still toxic. In contrast, the mean body weight of rats in  Figure 8 shows body weight changes of the rat groups after multiple injections of DPBS (control), AIF dissolved in 100% Cremophor EL ® /EtOH solution, 50% Cremophor EL ® /EtOH solution, 25% Cremophor EL ® /EtOH solution, and mPEG-b-PLA micelles. As demonstrated in Figure 8A, rats in the groups treated with AIF dissolved in 50% Cremophor EL ® /EtOH or 25% Cremophor EL ® /EtOH solution exhibited significantly lower mean body weight increase than that in the control group. This shows that even after dilution to minimize the solubilizing agent levels, the Cremophor EL ® /EtOH used as representative solubilizing agent was still toxic. In contrast, the mean body weight of rats in the AIF-loaded mPEG-b-PLA micelle group increased at a similar level to that of the control group, showing the absence of toxicity. All rats in the group treated with AIF dissolved in 100% Cremophor EL ® /EtOH solution died immediately after i.v. administration, indicating the fatal toxicity of the solubilizing excipients ( Figure 8B). In the group treated with AIF dissolved in 50% Cremophor EL ® /EtOH solution, all the rats died 8 days after administration, presumably due to the cumulative toxicity of the solubilizing agents. In contrast, rats in the groups treated with DPBS (control), AIF-loaded mPEG-b-PLA micelles, and AIF in 25% Cremophor EL ® /EtOH solution showed a 100% survival rate for two weeks. All the toxicity data indicate that multiple i.v. injections of AIF exerted no severe toxicity to rats even when mPEG-b-PLA micelles were used for solubilization. the AIF-loaded mPEG-b-PLA micelle group increased at a similar level to that of the control group, showing the absence of toxicity. All rats in the group treated with AIF dissolved in 100% Cremophor EL ® /EtOH solution died immediately after i.v. administration, indicating the fatal toxicity of the solubilizing excipients ( Figure 8B). In the group treated with AIF dissolved in 50% Cremophor EL ® /EtOH solution, all the rats died 8 days after administration, presumably due to the cumulative toxicity of the solubilizing agents. In contrast, rats in the groups treated with DPBS (control), AIFloaded mPEG-b-PLA micelles, and AIF in 25% Cremophor EL ® /EtOH solution showed a 100% survival rate for two weeks. All the toxicity data indicate that multiple i.v. injections of AIF exerted no severe toxicity to rats even when mPEG-b-PLA micelles were used for solubilization.

Conclusions
In conclusion, we designed an i.v. formulation using C. tricuspidata. For this, we extracted AIF, a major compound of unripe C. tricuspidata fruits, and developed a preparation and solubilization method for a liquid dosage formulation using mPEG-b-PLA polymeric micelles. In addition, the physicochemical, pharmacokinetic, and toxicity properties of the AIF-loaded mPEG-b-PLA micelles were evaluated in vitro and in vivo. The nano-sized particles and slow release profile of the

Conclusions
In conclusion, we designed an i.v. formulation using C. tricuspidata. For this, we extracted AIF, a major compound of unripe C. tricuspidata fruits, and developed a preparation and solubilization method for a liquid dosage formulation using mPEG-b-PLA polymeric micelles. In addition, the physicochemical, pharmacokinetic, and toxicity properties of the AIF-loaded mPEG-b-PLA micelles were evaluated in vitro and in vivo. The nano-sized particles and slow release profile of the formulation prolonged the blood circulation of the drug after systemic administration, leading to increased bioavailability of AIF. Therefore, our designed micelle formulation warrants further pre-clinical investigation in preparation for subsequent evaluations in clinical trials for various pharmaceutical applications of AIF. We also expect the formulation to contribute to the added value of C. tricuspidata for medical use.