Modulyod freeze dryer

The extraction of GEP was performed with a previously reported method [19 (link)]. Briefly, the dried Gastrodia elata (50 g) was powdered and defatted twice with 80% (v/v) ethanol for 2 h each time. Skimmed Gastrodia elata powder (10 mg) was added into deionized water (300 mL) (1:30 w/v) in a 500 mL beaker, followed by sonicating in an ultrasound bath (Kunshan Ultrasound Instrument Co., Ltd., Kunshan, China) at 70 °C at a power of 200 W and frequency of 40 kHz for 30 min. The ultrasound extraction process was repeated three times. The extract was centrifuged by an X-15R centrifuge (Beckman Coulter, Inc., Palo Alto, CA, USA). The supernatant was concentrated by a RE-2000A rotary vacuum evaporator (Yarong Biochemical Instrument Factory, Shanghai, China). Then, 95% (v/v) ethanol was added into the concentrated solution and placed at 4 °C overnight for polysaccharide precipitation. The precipitate of GEP was centrifuged and freeze-dried by a MODULYOD freeze-dryer (Thermo Fisher Scientific, Waltham, MA, USA). In this study, the phenol–sulfuric acid method [30 (link)] was employed to determine total polysaccharide content of GEP.

Fresh porcine hearts were obtained from Quinn's Meats Ltd. (Yarker, Canada). Decellularization was performed using methods adapted from Wainwright et al.²⁶ Briefly, the left ventricles from a total of 10 porcine hearts were excised and the fibrous outermost layers of the epicardium and endocardium were carefully removed to maximize tissue exposure to the decellularization reagents. The samples were then pooled and subjected to four cycles of freeze–thaw in hypotonic freezing buffer solution (pH 8.0) containing 10 mM Tris base and 5 mM ethylenediaminetetraacetic acid (EDTA). Next, the tissue was cut into 4–5-mm³ pieces and treated with two cycles of 6 h in 0.25% trypsin-EDTA (Life Technologies, Burlington, Canada), 20 h in 4% sodium deoxycholate, and 20 h in 3% Triton X-100 under orbital shaking at 37°C, with repeated rinsing in phosphate-buffered saline (PBS) between solution changes. All solutions were supplemented with 1% antibiotic–antimycotic solution (Life Technologies) and 0.01 mM phenylmethanesulfonyl fluoride (PMSF). The DLV was rinsed extensively in distilled water and lyophilized using a ModulyoD freeze dryer (Thermo Scientific, Waltham, MA).

Synthesis of PCL-AMB NPs was carried out using a modified emulsion solvent method [23 (link)]. Briefly, 20 mg of AMB was dissolved in 0.8 mL of DMSO. After complete dissolution, 3.2 mL of methanol was added to the solution to make up 4 mL. The mixture was vortexed for proper mixing and set aside. To obtain the polymeric solution, 300 mg of PCL pellets were dissolved in 6 mL DCM and the entire solution was added to the 4 mL of AMB solution and vortexed for 5 min. The mixture was completely added dropwise to 40 mL of 1% PVA prepared in MQ water, stirring at 500 rpm before sonication using a Branson 450 Sonicator (VWR, PA, USA) for 3 min on ice. The emulsion was left to stir overnight, to allow sufficient evaporation of the organic solvents. After overnight stirring, the nanoparticles were collected by ultra-centrifugation at 23,200× g for 30 min at 4 °C using a Thermo Scientific™ Sorvall™ RC 6 Plus Centrifuge (Thermo Fisher Scientific, MA, USA). Nanoparticles were washed three times with MQ water before lyophilization using a ModulyoD freeze dryer (Thermo Fisher Scientific, MA, USA) for 72 h and stored at 4 °C for further use. Similarly, unloaded, or blank nanoparticles (PCL NPs) were prepared in DMSO and methanol solutions, with PVA as the emulsifier. Blank nanoparticles were stored similarly to PCL-AMB NPs.

YS1646-derived vaccine candidates and control strains were first cultured in LB medium and then formulated in a lyophilization buffer consisting of 28% (w/v) sucrose (BioShop, Burlington, Canada) and 1% gelatin (Sigma-Aldrich, St. Louis, MO) in 25 mM potassium phosphate (KPO₄) at pH 7^{75 (link)}. Samples were aliquoted and frozen at −80 °C overnight. The following day, the strains were freeze-dried using a ModulyoD Freeze Dryer (Thermo Fisher Scientific) for 24 h. Samples were later stored at room temperature for various times ranging from 2 weeks to 4 months and cultured on LB plates to determine viability through the colony-forming unit (cfu) counts. A sample that did not undergo the freeze-drying process was cultured to determine the initial viability of strains (determined by cfu counts) prior to lyophilization.

A powder of cultivated strain TM02 of L. rhinocerus (LR) was obtained from LiGNO Biotech™ Sdn Bhd, Selangor, Malaysia. This powder was extracted into three fractions with ethanol, cold water and hot water using the maceration technique. Briefly, 100 g of LR powder was macerated with 1 L of ethanol and the extract was placed on the shaker at 4 °C for 24 h. After that, it was filtered by using Whatman® No.2 filter paper and ethanol was removed by rotary evaporation (Heidolph, Laborota 4011) to yield the crude ethanol extract (LRE). Cold water extraction: 100 g of LR powder was suspended in sterile water and placed on the shaker at 4 °C for 24 h. For hot water extraction, sclerotial powder was extracted with water at 95–100 °C for 2 h. After that, the mixture was filtered and freeze dried by lyophilizer (ModulyoD freeze dryer, Thermo Fisher Scientific, Waltham, MA, USA) to give a crude cold-water extract (LRC) and a crude hot-water extract (LRH). Yields of LRE, LRC and LRH were 0.73 g, 11.07 g and 10.13 g, respectively. Before starting the experiment, the crude extract of LRE was dissolved in DMSO, while the crude extracts of LRC and LRH were dissolved in sterile water to make the 100 mg/mL stock solution.

The bosutinib-HDL NPs were prepared using a film hydration technique. DPPC and bosutinib (5 DPPC: 2 bosutinib w/w) were dissolved in methanol in a round bottom flask. The methanol was evaporated using a rotary evaporator. The resulting thin film was hydrated with a ApoA-1 mimicking peptide solution (2.5 peptide: 5 DPPC w/w) in phosphate buffered saline (PBS). The solution was sonicated for 15–30 min followed by 4 rounds of thermal cycles at 25 °C (5 min) and 50 °C (5 min). The resulting nanoparticle solutions were concentrated and washed twice using Amicon centrifuge inserts (molecular weight cut-off (MWCO), 10 kDa). The final nanoparticle solutions were filtered through a 0.22 µm polyethersulfone (PES) filter. The bosutinib-HDL NPs were lyophilized (ModulyoD Freeze Dryer, Thermo-Fisher, USA) and stored at − 20 °C. Reconstituted bosutinib-HDL NP samples were filtered with 0.22 µm filter before use.

A powder of the cultivated strain TM02 of LR or tiger milk mushroom was obtained from LiGNO Biotech^TM Sdn Bhd, Selangor, Malaysia. The three extractions of LR were ethanol extract (LRE), cold-water extract (LRC), and hot water extract (LRH). One hundred grams of LR powder were macerated with 1 L of ethanol and the extract was shaken at 4 °C for 24 h. After incubation, the extract was filtered using Whatman^® No.2 filter paper and ethanol was removed by rotary evaporation (Heidolph Laborota 4011, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). The procedure of cold-water extraction is similar to LRE, but sterile water is used instead of ethanol. Although the powder was boiled in hot water at 95–100 °C for 2 h, both LRC and LRH were filtered and freeze dried in a lyophilizer (ModulyoD freeze dryer, Thermo Fisher Scientific, Waltham, MA, USA). Finally, about 0.73 g, 11.07 g, and 10.13 g were obtained from LRE, LRC, and LRH, respectively.