5.1 Materials
Standard rhodojaponin III (>98% purity) was acquired from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); Rhodojaponin III was obtained from the Catch Bio-Science & Technology Co., Ltd. (Jiangsu, China) with purity more than 92%. PC-98T egg yolk lecithin (AL15018, Purity = 98%) was procured from A.V.T. Pharmaceutical Co., Ltd (Shanghai, China). GM, GT, SA, and Tween® 80 were bought from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). ATO 5 and 888 ATO were gifts from GATTEFOSSé (Saint-Priest, France). HACC (Lushen bioengineering Co, Ltd. (Nantong, China)), Dulbecco's Modified Eagle's Medium (DMEM), Penicillin/Streptomycin, Fetal Bovine Serum (FBS), Hank's Balanced Salt Solution (HBSS), and 3-(4,5-Dimethylthiazolyl-2)-2,5-Diphenyltetrazolium Bromide (MTT) were the products of Thermo Fisher (Massachusetts, USA).
5.2 Cell culture
Caco-2 Cell lines were purchased from American Type Culture Collection (Virginia, USA) and cultured in 10% (v / v) FBS-supplemented DMEM with 1% penicillin-streptomycin as well as 1% (v / v) non-essential amino-acids in a 5% CO2, 90% relative humidity atmosphere at 37°C. Medium change was done every other day while cell passaging was done every 4 ~ 6 days through dissociation utilizing trypsin (0.25%) – EDTA (0.02%) solution.
5.3 Animals
The ICR female and male mice (Grade II, 18 ~ 22 g) were from Zhejiang Wei-Tong-Li-Hua laboratory animal technology Co. LTD (Zhejiang, China), which has a production license number of SCXK (Zhejiang) 2019-0001. Mice were kept in air-conditioned rooms at 22 ~ 24 ºC, 12 h light/dark cycle and provided with food and water ad libitum. Prior to experiments, mice were fasted overnight. The Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine approved this study (Ethical Accreditation No. PZSHUTCM200612005).
5.4 Preparation of RJ-III@HACC-SLNs
Rhodojaponin III-loaded solid lipid nanoparticles (RJ-III@SLNs) were prepared via an emulsification-diffusion approach with minor modification [34]. Before the optimization, the main preparation process is as follows: 20 mg egg yolk lecithin and 3 mg RJ-III were completely dissolved in absolute alcohol and thereafter mixed with glycerol monostearate (30 mg) to form an oil phase. Vacuum rotary evaporation was used to remove the organic solvent to obtain a lipid film layer. Under sustained ultrasound, 10 mL water phase supplemented with 0.2% (w/v) Tween-80 was added to the lipid film in 30 min using a needle. The RJ-III@SLNs were obtained after intermittent sonication by a probe sonicator (Xinzhi, Ningbo, China) at 400 w for 4 min (2s/3s). RJ-III@HACC-SLNs was obtained by binding HACC to the surface of RJ-III@SLNs via electrostatic adsorption [31]. In brief, 4 mL RJ-III@SLNs was added to 8 mL HACC solution (0.1%, w / v), followed by further stirring for 1 h.
5.5 Optimization of RJ-III@HACC-SLNs
To develop efficient RJ-III@HACC-SLNs, a single factor experiment was conducted. The design of RJ-III@HACC-SLNs in basic terms, is dependent on the HACC coating and type of lipid used. Previous works showed that the particle sizes of SLNs were markedly affected by volume ratios of VH to VS and stirring time in the modification process [31]. However, the zeta potential and EE, important factors for stability and effectiveness, were not studied in the modification process. Therefore, we optimized the process of preparing modified RJ-III@SLNs, several parameters, including volume ratio of VS to VH and stirring time on particles size, zeta potential, and EE, were investigated. The solid lipid had a marked effect on the EE and particle sizes of SLNs due to their different molecular weight and solubility [22]. So, we selected core lipids by comparing the EE, zeta potential, and particle size of RJ-III@HACC-SLNs based on five commonly and different molecular weight used solid lipids (GM, GT, SA, ATO 5, and 888 ATO). Table 3 shows formulations of the SLNs.
Table 3
Formulations of the various solid lipid nanoparticles
Number
|
Lipid
|
VH: VS
|
Stirring time (h)
|
1
|
Glycerol monostearate
|
4:1
|
1
|
2
|
Glycerol monostearate
|
2:1
|
1
|
3
|
Glycerol monostearate
|
1:1
|
1
|
4
|
Glycerol monostearate
|
2:1
|
1
|
5
|
Glycerol monostearate
|
2:1
|
2
|
6
|
Glycerol monostearate
|
2:1
|
4
|
7
|
Glycerol monostearate
|
2:1
|
1
|
8
|
Glyceryl trioleate
|
2:1
|
1
|
9
|
Stearic acid
|
2:1
|
1
|
10
|
Precirol ATO 5
|
2:1
|
1
|
11
|
Compritol 888 ATO
|
2:1
|
1
|
5.6 Characterization of RJ-III@HACC-SLNs
The morphology of RJ-III@HACC-SLNs was performed by TEM (FEI Talos, Thermo Fisher Scientific, USA), while zeta potentials and particle sizes were measured using a Zeta Potential / Particle Sizer (Nicomp 380 ZLS, PSS⋅NICOMP, USA). The changes in solid-state forms of RJ-III in SLNs were evaluated by XRD (Rikagu, D / Max-3C, Japan). FT-IR spectrophotometer (IRAffinity-1S, SHIMADZU, Japan) was used to evaluate the surface chemistry structure of RJ-III, SLNs, RJ-III@SLNs, RJ-III@HACC-SLNs, to assess the surface characterization of RJ-III@HACC-SLNs. The EE of RJ-III@HACC-SLNs were assessed by high performance liquid chromatography (HPLC) and ultrafiltration centrifugation. Briefly, the unencapsulated RJ-III was isolated from RJ-III@HACC-SLNs through 20 KD ultrafiltration centrifuge tube (Millipore, USA) at 10000 rpm for 30 min. Total drug amounts in drug-loaded SLNs were evaluated by dissolving SLNs in methanol to release encapsulated RJ-III, and assayed by HPLC. The conditions for the HPLC system (Agilent-1260B, Agilent, USA) equipped with an evaporative light scattering detector (Agilent-G460B, Agilent, USA) were: CAPCELL PAK C18 column (4.6 × 150 mm, 5 µm); mobile phase, acetonitrile-water (30:70, v/v, 1.0 mL/min); injection temperature, 25°C; evaporation temperature, 60°C; atomization temperature, 30 ℃; sample volume, 20 µL. The equation for calculating EE was:
5.7 In vitro stability and release of RJ-III@ HACC-SLNs
The stability in vitro of RJ-III@SLNs and RJ-III@HACC-SLNs were studied by previous reports [31]. Incubation of nanoparticles was done at 37°C in SGF. Samples were obtained at 0, 1, 2, and 3 h, and assessed for changes in EE. SGF was constituted using 2 g sodium chloride, 36.5% 7 mL hydrochloric acid, and pepsin (3.2 g) in 1000 mL of water.
The In vitro release investigation of RJ-III@HACC-SLNs, RJ-III@SLNs, and RJ-III were performed in PBS of pH 6.8 by the dialysis bag (molecular weight cut off 8~14kDa) diffusion technique. The test samples were stored inside the bag (equivalent to 3 mg RJ-III), dipped into 200 mL medium at (37 ± 0.5) ℃ in a conical flask with a stirring speed of 100rpm. 2 mL samples were taken and instantly replaced with an equal volume of fresh release medium at pre-set time 0.25, 0.5, 1, 2, 4, 8, 12, 24 h. Amounts of RJ-III in the medium were also determined using the HPLC method mentioned above in 5.6.
5.8 Pharmacokinetic studies
The RJ-III@HACC-SLNs oral absorption was evaluated by pharmacokinetic parameters, the pharmacokinetic study was performed in mice. The mice were randomized into several groups (6 mice per group) and intragastrically administered with RJ-III or RJ-III@HACC-SLNs containing RJ-III 0.2 mg/kg [19]. With regards to pharmacokinetic assays, mice have anesthetized using diethyl ether at 0.033, 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, and 8h time points, after administration, venous blood samples were obtained from saphenous veins of the thighs into tubes containing EDTA-K2. Plasma samples were obtained by centrifugation (6,000 rpm, 8 min, 4 ℃), and were then quantified according to the LC-MS/MS method previously studied by the research group [19]. Chromatographic separation was performed using an ACQUITY UPLC HSS T3 (1.8 µm, 2.1 × 50 mm) reverse-phase column (Waters technology (Shanghai) Co., LTD (Shanghai, China)). The column was equipped with an AF0-8497 guard column (Phenomenex, CA, USA) and maintained at room temperature. The flow rate and sample injection volume were 0.5 mL/min and 10 µL, respectively [19]. Non-compartmental analysis was conducted using the WinNonlin®8.2.0 software (Pharsight, CA, USA) to obtain pharmacokinetic parameters.
5.9 Multimodal analgesia studies of RJ-III@HACC-SLNs
The multimodal analgesic effects of RJ-III@HACC-SLNs were determined using the acetic acid writhing and hot plate, and formalin tests. The acetic acid writhing test was separated into two parts. In the first part, 40 mice were assigned into 4 groups (n = 10) and pretreated with RJ-III@HACC-SLNs (0.10 mg/kg of RJ-III, i.g), RJ-III (0.10 mg/kg, i.g), aspirin (200 mg/kg, i.g) or normal saline (0.9% NaCl, i.g). After 15 min, mice were administered with 0.8% acetic acid (10 mL/kg, ip) and the nociception intensity evaluated by counting the number of abdominal contortions, such as abdominal muscle contractions and extensions of hind paws for 30 min. In the second part of the experiment as before, but the time of acetic acid intervention was 45 min after administration. The pharmacodynamics of every preparation was evaluated by comparing the number of writhing.
In the hot plate test, firstly, each mouse was placed thrice on the heated plate (53 ± 0.5) ℃ at a 15 min interval to obtain a basal pain threshold which is the reaction time about paw lick time or jump. Mice with reaction < 5 s or > 30 s longer were omitted. Then, the animals (n = 10) received RJ-III@HACC-SLNs (contain 0.20 mg/kg RJ-III, i.g), RJ-III (0.20 mg/mg, i.g), aspirin (200 mg/kg, i.g) or normal saline (0.9% NaCl, i.g). Reaction times were evaluated at 15 and 45 min after administration, with 30 s as the cutoff time to avoid injury to the paw.
The formalin test was divided into two parts as similar to acetic acid writhing test. In the first part, the mice (n = 10) were treated with RJ-III@HACC-SLNs (contain 0.10 mg/kg RJ-III, i.g), RJ-III (0.10 mg/kg, i.g), aspirin (200 mg/kg, i.g) or normal saline (0.9% NaCl, i.g) 15 min before 25 µL of 2.5% formalin subcutaneous injection. The licking as well as biting time about the injected left hind paw, indicating pain, was documented from 0 to 5 min (phase I, neurogenic phase) and from 15 to 30 min (phase II, inflammatory phases). Findings were presented as licking time, in seconds (s). In the second part of the experiment as before, but the time of formalin intervention was 45 min after administration. Finally, the antinociceptive effect of each preparation was determined by comparing the time of licking and biting.
5.10 In vitro cytotoxicity
The biocompatibility of RJ-III@HACC-SLNs was assessed by cytotoxicity of Caco-2 cells [55]. In the cytotoxicity study, the Caco-2 cells were cultured at 1.5 × 105 cells/well in 96-well plates, and incubated for form cell layers. Test solutions were assigned into five groups of Blank SLNs (SLNs), Blank SLNs modified by HACC (HACC-SLNs), RJ-III, RJ-III@SLNs, and RJ-III@HACC-SLNs. The cell layers were obtained and rinsed 3 times using HBSS. Then, cells were incubated with 200 µL of various concentrations of blank nanocarriers (1, 2, 5, 10, 50, 100, and 500 µg/mL), and nanoformulations with RJ-III at different doses (total amount of RJ-III in SLNs of 0.1, 0.2, 0.5, 1, 2, 5, and 10 µg/mL) for 24 h. Cell layers treated with blank culture medium (200 µL) were the controls of 100% viability. After incubation, the addition of MTT solution (20 µL; 5 mg/mL) was followed by further incubation for 4 h. Subsequently, the MTT dye was removed from wells and 200 µL of dimethyl sulfoxide was added to each well to solubilize the formazan crystals. The results were quantified using a microplate reader at 570 nm.
OD1 is the absorbance intensity of the untreated cells, while OD2 is the absorbance intensity of the treated cells.
5.11 In vivo acute toxicity test
To evaluate the safety of RJ-III-HACC@SLNs in vivo, acute lethal characteristics were assayed as previously reported, with minor changes [14]. Briefly, after 3 days of adaptation, 80 mice were randomized into 10 groups (n = 8). Ever group was administered RJ-III (1.711, 2.312, 3.125, 4.223, 5.707 mg/kg, obtained from our previous studies) or RJ-III@HACC-SLNs (total amount of RJ-III in SLNs of 3.423, 4.625, 6.250, 8.446, 11.413 mg/kg, based on a preliminary experiment) by single intragastric administration, and cumulative mortality within 7 days was recorded to calculate median lethal dose (LD50) by Bliss method [56].
5.12 Statistical analyses
Results are shown as mean ± standard error (n = 3). The one-way or two-way ANOVA were performed using Origin 2021b software. P < 0.05, P < 0.01 and P < 0.001 were significance thresholds.