Andrographolide/ Phospholipid/ Cyclodextrin complex-loaded Nanoemulsion: Preparation, Optimization, in vitro and in vivo Evaluation

Andrographolide (AG), a natural product with various pharmacological effects, exhibited low oral bioavailability owing to its poor solubility, stability and low absorption. Previous studies have suggested that phospholipid (PC) and hydroxypropyl-β-cyclodextrin (HPCD) could improve the drug solubility and absorption. Moreover, nanoemulsion (NE) has been confirmed as an appropriate enhancer for oral bioavailability. Therefore, AG/HPCD/PC complex (AHPC) was synthesized, and AHPC-loaded nanoemulsion (AHPC-NE) was optimized and prepared using central composite design combined response surface methodology. The average droplet size and PDI were 116.50±5.99 nm and 0.29±0.03, respectively. AHPC-NE with a loading capacity of 0.32 ± 0.01% and an encapsulation efficiency of 96.43 ± 2.27% appeared round and uniformly dispersed based on transmission electron microscopy. I n vivo release studies demonstrated that AHPC-NE had good sustained-release effects. Further, AHPC-NE significantly enhanced the absorption of AG with a relative bioavailability of 550.71% compared to AG suspension. Such findings reveal AHPC-NE as a potential strategy for sustained-release and oral bioavailability enhancement. 349.109→287.04 (AG) and m/z 325→162.968 (Bilobalide).


INTRODUCTION
Andrographolide (AG), a diterpene lactone compound, is one of the main active constituents in the aerial parts and leaves of the plant Andrographis paniculata. 1) Modern pharmacological studies revealed that AG has good performance in antibacterial, 2) antivirus, 3) antitumor, 4) anti-inflammatory, 5) and immune regulation. 6) However, AG is considered as a class Ⅳ drug according to the biopharmaceutics classification system (BCS) with low solubility and low permeability. 7) Despite the traditional preparations of AG used in clinical treatment such as dropping pills, tablets, and capsules have a certain therapeutic effect, the oral bioavailability remains at a low level, which is owing to their poor water solubility, rapid drug release and metabolism, and low intestinal absorption. Therefore, development of a new oral preparation of AG with high bioavailability has great potential for clinical application.
According to the previous reported literatures, [18][19][20] the alternative phase solvents were selected as follows: Oil phase: GMC, IPM, Soybean oil, IPP; Surfactant: Span-20, Span-80, RH-40; Co-surfactant: Transcutol HP, PEG-400, Glycerin, 1,2-Propanediol. Excessive amounts of AHPC were respectively added to 5 mL of the above phase solvents. The samples were vortexed for 5 minutes, sonicated for 20 minutes, and equilibrated for 24 hours at 37 ℃ using a constant temperature shaker. The saturated solution was taken for centrifugation at 8000 rpm for 10 min. Thereafter, 1 mL of supernatant was placed in a 5 mL volumetric flask, fixed with methanol, and filtered by the 0.45μm microporous membrane, the solubility of AG in different phase solvents was measured using the HPLC method described above.

Construction of Pseudo-ternary Phase Diagram
The mixed surfactant (S-mix) 21) was composed of surfactant and co-surfactant, which were selected based on the results from the phase solubility experiment. Pseudo-ternary phase diagram was performed by water titration method combined emulsification method 22,23] for the selection of Km (the mass ratio of surfactant and co-surfactant). The oil phase and S-mix with different Km (3:1, 2:1, 1:1, 1:2, 1:3) were mixed at a weight ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 respectively. The samples were homogenized by magnetic stirring for 5 min, the distilled water was then slowly added until the system became turbid. The amount of added distilled water was recorded, and the quality score of the three phase was calculated to construct the pseudo-ternary phase diagram.

Experimental Design
According to the results of the univariate experiment, the magnetic stirring speed (A), the amount of distilled water (B), and the amount of AHPC (C) were selected as the factors with value ranges of 1000-1800 rpm, 0.6-1.2 g, 50-450 mg, respectively. The average droplet size (Y1) and PDI (Y2) were selected as evaluation indicators in this experiment. In addition, overall desirability (OD) was set to reasonably reflect the influence of each factor 24) according to the optimization requirements. The normalization method was used to fit each indicator, we

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firstly standardized all indicators to a normalized value (desirability) between 0 and 1.0. The formula for calculation of desirability and OD are as follows: OD = (d 1 ×d 2 ×d 3 ×…×d k ) 1/k (2) where d min is the desirability of the indicator more favorable with a smaller value such as the droplet size and PDI, Yi is the value of them in different experimental design, k is the number of evaluation indicators.
Thereafter, the horizontal code value and corresponding value of the three factors were listed in Table 1 and a total number of twenty runs was performed and their OD values were calculated in Table 2 based on the principle of CCD-RSM. 25) The runs were operated in a randomized arrangement to avoid systematic bias.

Optimization and Validation
The experimental design, analysis of variance (ANOVA), response surface analysis, and prediction of the optimal process were performed using the Design Expert (version 8.0.6.1).
The quality of the fitting model was expressed by the P-value and R 2 (P-value＜0.1×10 -3 and a high R 2 value close to 1), which confirms the significance and accuracy of the applied model. Finally, AHPC-NE was prepared using the suggested optimal process to validate the prediction of model.

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3mL of AHPC-NE prepared by the optimal process was used to determine the droplet size, polydispersity index (PDI) with a Malvern Instrument (ZEN3700, Malvern Instruments Co., Ltd., Germany).

Transmission Electron Microscopy
To confirm the droplets size and investigate the morphological characteristics of AHPC-NE, transmission electron microscopy (TEM; HT-7700, Hitachi, Japan) was performed. 1mL of the NE was diluted by 3 times with GMC and then added dropwise on the copper mesh covered with carbon film. After natural drying, the microscopic appearance of the NE was observed using TEM.

Determination of LC and EE
The loading capacity (LC) and encapsulation efficiency (EE) were determined by following slight modifications of a previously reported indirect method 26) and were calculated using the formulas given below. Briefly, 3mL of the NE and the NE without addition of AG were measured and weighed. The difference between the above two samples was the mass of AG, and the LC was calculated according to formula (3). On the other hand, 1 mL of AHPC-NE was placed in a 10 mL volumetric flask, 5 mL of methanol was added and sonicated for 30 minutes to demulsify the NE. The samples were fixed with an appropriate amount of methanol and then centrifuged in a low-temperature high-speed centrifuge (4 ℃, 10000 r/min, 5 min). 1 mL of the supernatant was placed in a 10 mL volumetric flask, diluted with methanol to meet the volume and then measured by using the HPLC method mentioned above. The measured value indicates the amount of AG encapsulated in the NE, and EE was calculated according to formula (4).

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Drug release from AHPC-NE was assessed using the dialysis bag method. 27) In brief, the dialysis bag (molecular retention capacity 8000-14000 Da) was boiled in water for 15 minutes, rinsed three times with pure water, shaken dry, and set aside. The dialysis bag (5 cm) was placed in the simulated gastric fluid (pH 1.2) and intestinal fluid (pH 6.8) for 12 h to reach the equilibrium of osmotic diffusion. 28) After one end was clamped, 1 mL of AHPC-NE, AHPC solution, and AG suspension measured accurately were placed in a dialysis bag preliminarily filled with 1 mL release medium. The other end was quickly clamped and placed in a 250 mL beaker containing 100 mL release medium, The samples were shaken at a speed of 70 rpm and the temperature was controlled at 37±0.5°C. 1 mL of the release medium containing the diffused drug was collected after shaking for 0.5, 1, 2, 3, 5, 7, 9, 12, 24, 30, and 48 h. The same volume of release medium was replenished, and the sample solution was diluted 5-fold with the mobile phase, mixed well, and filtered using a microporous membrane (0.22um). The drug concentration was determined using the HPLC method and the cumulative drug release percentage (Q) was calculated using the following formula: (n=3) where C i is the concentration of the samples collected at different time points, V r is the volume of the release medium (100 mL), V s is the volume of the sample (1mL), and m is the mass of AG initially added.

Pharmacokinetic Studies
The plasma concentration of AG was determined using an HPLC Twenty-four SD rats, which were fasted overnight with free access to water, were randomly divided into the following three groups: the first group was orally administered with

Data analysis
SPSS 25.0 was used for statistical analysis. Data obtained in triplicate at least three times repeated were expressed as mean ± SD. Differences between formulations were compared by one-or two-way ANOVA, p＜0.05 was considered significant.

Solubility of AHPC
As shown in Table 3, AHPC greatly increased the solubility of AG by 200-300 folds in solutions with different pH values, especially in the distilled water, the solubility of AG

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reached the highest of 13.45±0.47 mg/mL when loaded in the PC/HPCD complex. There was a trend that the solubility of AHPC increased with increasing pH. However, the pure AG did not exhibit a significant change with the changing pH, and the highest solubility of 0.053±0.0009 mg/mL was still at a low level.

Phase Solubility of AHPC
The solubility results of AHPC in different oil phases can be seen from Fig 1A,

Pseudo-Ternary Phase Diagram
It can be clearly seen that the NE area (area under the curve) was affected by the Km (Fig.2), and the largest NE area was founded when the Km was 1:3. As depicted in Fig. 2E, the formed NE performed a clarified, transparent appearance with the maximum addition of distilled water when the mass ratio of GMC and S-mix was 2:3. Therefore, the final ratio of the ingredients was GMC (30.77%, w/w), S-mix (46.15%, w/w), and Distilled water up to 23.08% (w/w). In such conditions, the value range of the distilled water addition would increase in the univariate experiment, which is potentially helpful in optimizing the prescription with higher LC due to the greatly enhanced water solubility when AG prepared into AHPC. For the present, S-mix with a Km of 1:3 and the mass ratio of GMC and S-mix (2:3) were selected for further studies. Km, the mass ratio of surfactant and co-surfactant.

Fitting of Model and Response Analysis
The Moreover, R 2 and adjusted R 2 values of 0.9702 and 0.9435 are closed to 1, which indicated that the quadratic model was valid for the present study. 29) The 3D surface plot in Fig. 3A showed the combined effect of distilled water addition and magnetic stirring speed on the OD value while the third factor was set to its middle value. It can be seen that the OD value decreased when both distilled water addition and magnetic stirring speed decreased. However,

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if the amount of distilled water added got lower than 0.99 g, the OD value started to decrease rapidly. The interaction between magnetic stirring speed and AHPC addition when the other factor at its middle value was shown in Fig. 3B, the OD value increased inversely proportional to the amount of AHPC added. In addition, the interaction of distilled water addition and the AHPC addition was depicted in Fig. 3C, which indicated that the increase of distilled water addition and decrease of AHPC addition led to an increase of the OD value until the distilled water addition exceeded 0.99g; thereafter, the OD value started to decrease slightly. The results from Fig 3A and Fig 3C showed that too high or too low distilled water addition might cause a decrease in the OD value.

Method Optimization and Validation
In order to maximize the drug loading of the NE, we selected the prescription with the largest amount of AHPC added when the expected OD value was set as 1, the predicted optimization result associated with the maximum OD value of 1.0221 was reached when the experimental factors to their optimum values was set as follows: speed of magnetic stirring was 1453.33 rpm; amount of distilled water was 1.08 g, amount of AHPC was 223.33 mg.
The OD value of AHPC-NE prepared via the optimized process is 1.013, which is in good concordance with the predicted OD value (deviation was 0.89%), supporting the accuracy of the optimization in the studied range using this model.

Characterization of AHPC-NE
As shown in Fig. 4 Figure 6A shows the characteristics of AG release from AHPC-NE in simulated gastric fluid (pH=1.2). It can be seen that the drug in AG suspension group released almost completely at 12h, which was higher than that of AHPC solution and AHPC-NE. AHPC Similar to drug release in the simulated gastric fluid (pH=1.2), the AG release from AHPC-NE also showed a characteristic of sustained-release.

DISCUSSION
The clinical applications of AG have been markedly limited owing to its poor solubility, stability, and low oral absorption. Therefore, it has a promising perspective to prepare a novel AG-loaded formulation with high oral bioavailability. To this aim, AHPC-NE drug delivery system was constructed to achieve efficient delivery for AG.
The synthesized AHPC significantly enhanced the solubility of AG in the solutions with different pH. Ren et al. 30) and Singh et al. 31) have prepared AG-CD complex and noted that the solubility of AG has a linear relationship with the concentration of CD, the lineal host-guest correlativity with slope (<1) advised synthesis of a 1:1 ratio complex of AG with CD. Zhang et al. 32) have prepared AG-CD complex with an increased solubility of AG by approximately 31 folds. Unlike the binary complex, the AHPC consists of AG, PC, and HPCD with a molar ratio of 1:1.5:1.5 showed a 200-300 folds improvement at different pH ranges in the solubility test, which may be associated with the solubilization effect of HPCD and PC. Studies have reported that PC, an amphiphilic molecule with a hydrophilic nitrogenor phosphorus-containing head and a long hydrophobic (lipophilic) hydrocarbon chain, could enhance the solubility of poorly soluble drugs. 11,12) For another, the hydrophilic exterior surface and a non-polar interior cavity of HPCD would provide the suitably sized drug with a lipophilic microenvironment for hydrophobic drugs inclusion and finally improve their solubility. 33) In addition, the solubility of AG in the AHPC group showed a trend of first increasing and then decreasing with the pH increasing, the possible reason was that HPCD at a strong acidic condition was unstable, causing the drug release from the interior cavity so that the inclusion solubilization effect was reduced at the acidic solution. 34) NE with a high LC is beneficial for clinical treatment due to the reduction of drug administration frequency. In order to enable more AHPC to be loaded into NE, we investigated the saturated solubility, an important criterion to choose each phase solvents, of AHPC in the alternative phase solvents mentioned previously. According to Fig. 1, GMC exhibited the highest solubilization of AHPC, the similar result was observed in a recent study by Elbardisy et al. 35) Interestingly, the solubility of AHPC gradually decreases with the hydrophilic-lipophilic balance (HLB) value of the surfactant decreased, one possible explanation for this phenomenon was that higher HLB value indicated more hydrophilicity of the surfactant, 36) and thus more AHPC can be dissolved due to their enhanced water solubility.
However, Span-20 was too viscous as reported 37) to achieve the expected emulsification effect in the actual production process while Span-80 displayed better. Therefore, GMC, Span-80, and Transcutol HP were selected as the oil phase, surfactant and co-surfactant, respectively.
Thereafter, Pseudo-Ternary Phase Diagram was plotted to aid in selection of the Km and to determine the optimum mass ratio of GMC and S-mix.
In the analysis of response surface, we can find that the OD value was influenced greatly by the addition of distilled water. Such findings are in good agreement with the observations of Mayer et al. 38) that the increase of the amount of distilled water added may change the droplet size and the PDI of NE. Meanwhile, the increase of AHPC addition would be helpful for the drug loading at a certain point. However, the OD value showed an obvious downward trend when the amount of AHPC added more than 250 mg. Therefore, the amount of AHPC addition should be carefully considered because its integrative effects on droplet size, PDI, and the LC.
The process of AHPC-NE was optimized using CCD-RSM and AHPC-NE with excellent physicochemical properties were successfully prepared and characterized.
Compared with the AG-loaded NE prepared by Sooksai et al, 39) AHPC-NE exhibited smaller droplet size and higher LC. Notably, the droplet size is critical to the efficacy of the NE. Win et al. 40) found that the smaller the particle size, the faster the release of the standard polystyrene nanoparticles, which was related to the larger surface area per unit mass or volume. And the small droplet size could yield a favorable absorption for the application in vivo. 41) Moreover, the higher LC may be helpful for reducing the dosing volume in the clinical applications.
In vitro drug release experiments were performed to preliminarily explore AG release properties from AHPC-NE, AHPC solution, and AG suspension in simulated gastric fluid and intestinal fluid. The absorption and metabolism of the drug in the gastrointestinal tract can be

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estimated via the analysis of the drug release profiles. According to the drug release curve, we found that the AG loaded in AHPC released faster than that of AHPC-NE, which may be related to the encapsulation effect of oil phase of AHPC-NE slowed down the drug release rate. Another possible explanation for this phenomenon was that the encapsulation effect of the oil phase could also increase the time for fully mixed with the release medium. In addition, the cumulative drug release rate of each group increased with the pH increases to 6.8, this result was consistent with a study reported by Lee et al. 42) This may be potentially relevant to the low stability of PC and HPCD in a strong acidic environment, the destruction of their structures might hinder the release of AG. 43,44) Moreover, the result also indicated that the amount of AG released in the gastric fluid was much lower than that in the intestinal fluid, which is of great help to avoid AG being metabolized prematurely. Meanwhile, the cumulative release rate curves of AHPC solution as well as AHPC-NE showed a downward trend at the later time points, and this downward trend in the AHPC solution group was more pronounced, we speculated that this may be caused by the instability of AG in the release medium, since the continuous phase of AHPC-NE has a protective effect on AG 45) and thus slowed down its degradation.
Pharmacokinetic studies revealed that AHPC-NE greatly improved the oral bioavailability of AG, which is consistent with those of Gao et al. and Yen et al. 46,47) The possible reason was that the hydrophilic hydroxypropyl groups of HPCD increased the water solubility of AG, which was aided significantly in drug absorption, further, the amphiphilic PC molecule might stimulate the outer membrane of intestinal epithelial cells to guide the absorption of AG. 48) In addition, the oil phase molecules in the prescription of AHPC-NE may penetrate into biological membranes and interact with phospholipid polar groups to change the fluidity of the membranes, 49) and the formation of nano-sized droplets loaded with AHPC could further increase the transport of AG and intestinal absorption. Moreover, the nano-sized droplets were evenly dispersed in the continuous phase and this wrapping effect of the oil phase forms a natural barrier to reduce the metabolism of AG by gastric acid or other enzymes, thereby increasing the absorption of AG in the intestine, 50) which was corresponds to the speculation in the drug release experiment. It is also worth noting that the C max of

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AHPC was higher than that of AHPC-NE while the AUC 0→t was lower, which may be due to the sustained-release effect of AHPC-NE affecting the plasma concentration. 51) Although the AHPC absorbed quickly to reach a higher C max , the elimination rate was also fast, thereby causing a lower bioavailability.
In conclusion, we prepared the AHPC first and investigated its solubilization. The process of AHPC-NE was optimized using CCD-RSM to further increase the oral bioavailability. We assessed the characteristics and efficiency of the AHPC-NE in vitro and in vivo. Our results indicated that AHPC markedly enhanced AG solubility. In vitro release experiments exhibited a sustained-release of AG when loaded in the AHPC-NE, which is in agreement with our in vivo observations. In addition, pharmacokinetic studies showed that the optimized AHPC-NE significantly increased the oral bioavailability of AG with a relative bioavailability of 550.71% compared with the AG suspension. Such findings demonstrate that the AHPC-NE is effective for improving the oral bioavailability and sustained-release of AG, exhibiting great potential for clinical applications.