PPNPs were generated using a modified procedure as described previously (20 (link)). First, an aqueous solution of 1% PVA (Sigma, P8136) was made. Then, a solution of PLGA (Lactel Polymers, B6010–4) in acetone (90 mg/5ml) was made in duplicate (PLGA control and PPNPs). Once both solutions were dissolved, 10 mg of PTX (Advanced ChemBlocks, F-4194) was added to one PLGA-acetone mixture to generate PPNPs. The PLGA-acetone mixture was added dropwise to the PVA solution then mixed overnight at 800 rpm while loosely covered in foil to allow for acetone evaporation. The next day, 5 mg/ml of PLL (Sigma, P2636) and F127 (Sigma, P2443) was made in Milli-Q water and was added dropwise to each beaker (2 ml each). This solution was mixed for seven hrs, then the volume was brought to 20 ml using Milli-Q. Samples were aliquoted into 1.5 ml tubes and stored at −20°C. One tube of control and loaded particles were taken and briefly sonicated using a probe sonicator (MISONIX Inc.) to ensure proper homogeneity for DLS analysis.
Probe sonicator
Manufactured by Bioventus
Sourced in United States
The Probe Sonicator is a laboratory instrument used to generate high-intensity ultrasonic waves. Its core function is to disrupt or homogenize samples through cavitation and acoustic streaming, which can be useful in various scientific and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
8 protocols using probe sonicator
1
Fabrication of PTX-loaded PLGA Nanoparticles
2
Paclitaxel-loaded eNP Nanoparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
eNP monomer (25 mg/mL) and paclitaxel (1.25 mg/mL, 0.05 wt/wt equiv.) were dissolved in 2.5 mL of dichloromethane and sodium dodecyl sulfate (12 mg/mL, 0.24 wt/wt equiv.) dissolved in 10 mM pH 7.4 phosphate buffer (10 mL). The aqueous and organic phases were sonicated under argon atmosphere for 30 min (1 s pulse with 2 s delay at 20% amplitude) in an ice bath using a Misonix probe-sonicator while stirring the solution with a stir-bar. Following emulsification, tetramethylethylenediamine (10 uL) and 200 mM ammonium persulfate (100 uL) were added to the reaction and this was allowed to stir for 4 h under argon and overnight under air. Particles were then dialyzed against 5 mM pH 7.4 phosphate buffer (1 L) and the buffer exchanged one time over 24 h.
3
Paclitaxel-loaded eNP Nanoparticles
4
Membrane Preparation of rNPC1L1-Expressing HEK293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 cells stably expressing rNPC1L1 were treated with 4 mM sodium butyrate (Sigma-Aldrich) for 24 h, and then used for membrane preparation essentially according the methods of Garcia-Calvo et al.[10] (link). Briefly, cells suspended in buffer containing 20 mM HEPES/Tris buffer (pH 7.4), 8% sucrose, and proteinase inhibitors (complete EDTA-free; Roche Applied Science) were disrupted with a probe sonicator (Misonix) on ice, and then centrifuged at 1,500×g for 10 min at 4°C to remove unbroken cells and nuclei. The supernatants were centrifuged at 125,000×g for 3 h at 4°C, and the resultant pellets were resuspended in ice-cold buffer containing 20 mM HEPES/Tris buffer (pH 7.4), 160 mM NaCl, and 5% glycerol. Samples were stored at −80°C prior to use.
5
Paclitaxel-loaded eNP Nanoparticles Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
eNP polymer (25 mg/mL) and paclitaxel (1.25 mg/mL, 0.05 wt/wt equiv.) were dissolved in 50 mL of dichloromethane and sodium dodecyl sulfate (12–48 mg/mL, 0.24–0.96 wt/wt equiv.) dissolved in 10 mM pH 7.4 phosphate buffer (200 mL). The aqueous and organic phases were sonicated under argon atmosphere for 30 min (1 s pulse with 2 s delay at 20% amplitude) in an ice bath using a Misonix probe-sonicator while stirring the solution with a stir-bar. The emulsion was then processed on the LV1 Microfluidizer at 15,000 PSI (100 mPa) using an F12Y reaction chamber cooled in an ice bath. After processing 250 mL of nanoparticles through three passes, these were set aside and stirred under air for 4 h to allow the dichloromethane to evaporate (quantified by headspace analysis). Additional 250 mL batches of particles were synthesized to generate 1 L. Particles were then dialyzed against 5 mM pH 7.4 phosphate buffer (4 L per batch) and the buffer exchanged once over 24 h.
Smaller scale (20 mL) production runs were used to evaluate the impact of surfactant concentration and number of LV1 processing passes on eNP diameter and PDI. Particle diameter and PDI were measured using a Brookhaven DLS. Samples were diluted 100× in deionized water prior to measurement.
Smaller scale (20 mL) production runs were used to evaluate the impact of surfactant concentration and number of LV1 processing passes on eNP diameter and PDI. Particle diameter and PDI were measured using a Brookhaven DLS. Samples were diluted 100× in deionized water prior to measurement.
6
Paclitaxel-loaded eNP Nanoparticles Synthesis
7
Quantifying Methionine Encapsulation Efficiency
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine %EE of the Met, MetNp were first digested for 30 min in a solution of acetic acid (2% v/v) by employing a probe sonicator (Misonix, Farmingdale, NY, USA). Afterward, the samples were subjected to centrifugation to develop pellets of NPs at 12,000 rpm for 0.5 h at 25 ± 0.5 °C [29 (link),30 (link)]. The supernatant after centrifugation was analyzed for Met concentration in a UV-visible spectrophotometer (Shimadzu, Kyoto, Japan) at λmax = 277 nm to determine the %EE of Met by using Equation (2),
where
where
X1 = Amount of Met added (mg);
X2 = Amount of free Met after centrifugation (mg).
8
Nanoceria Powder Preparation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nanoceria powder was procured commercially from M/S Sigma (#544841) for the present study as it exhibits suitable redox properties.15 (link) The nano powder was mixed with type I Milli Q-grade water at 5 mg/mL concentration. Mixture was sonicated with a probe sonicator (Misonix, USA) for 3 cycles of 5 min each (with 30 sec intervals in between), which resulted in milky white suspension. Larger particles and aggregates in the suspension were settled down by centrifugation at 200g for 5 min. Supernatant was collected in fresh vial and stored at 2–8 °C until further use.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!