To evaluate drug loading, ~10 mg of lyophilized MPs were weighed, and the drug was extracted using 4 mL of a DCM:DMSO solvent mixture (50:50 for OLA and 75:25 for SA, v/v) in a 20 mL scintillation vial. This solution was then vortexed until the MPs were fully dissolved in the solvent. Following this, the resulting solution was filtered (0.45 µm, Millex-HV PVDF Syringe Filters, Sigma Aldrich, CA) and appropriately diluted in methanol prior to drug quantification via high-performance liquid chromatography (HPLC). HPLC analysis of OLA and SA was performed using an Agilent Technologies 1260 Infinity II (Agilent Technologies, Santa Clara, CA, USA) with a photodiode array (PDA) detector and an XDB-C18 column (4.6 × 150 mm, ZORBAX Eclipse, Agilent). The mobile phase was composed of MilliQ water:ACN (70:30 wt%, with 0.1 wt% FA), and the flow rate was 1.2 mL/min. OLA and SA were analyzed at wavelengths of 254 nm and 303 nm, respectively. The experimental drug loading capacity (i.e., DLC) was calculated as follows:
Millex hv pvdf syringe filter
Manufactured by Merck Group
Sourced in United States
The Millex-HV PVDF Syringe Filters are a type of laboratory filtration device. They are designed to filter liquids using a hydrophilic polyvinylidene fluoride (PVDF) membrane. The filters have a disc-shaped configuration and can be attached directly to a syringe for convenient sample preparation.
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4 protocols using millex hv pvdf syringe filter
1
Quantifying Drug Loading in Microparticles
2
Sink Conditions for In Vitro Drug Release
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To ensure the in vitro drug release studies were conducted under sink conditions, the solubility of the drug in the release media (PBS with 0.5 wt% SDS) was measured. Specifically, an excess amount of drug (~10 mg of OLA and ~30 mg of SA) was added to 5 mL of release media (i.e., PBS with 0.5% Tween) in scintillation vials. The scintillation vials were incubated at 37 °C with gentle stirring (50–100 rpm) overnight, followed by filtration using a filter membrane (0.45 µm, Millex-HV PVDF Syringe Filters, Sigma Aldrich, CA). The drug concentrations were then quantified using HPLC as described in the previous section.
3
SPH Fractionation and Bioactivity Evaluation
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A solution of 10 mg/mL of SPH was prepared in ultra-filtered water and filtered through a Millex-HV PVDF syringe filter with a pore size of 0.45 mm (Merck Millipore, Billerica, MA, USA). The resulting crude SPH solution was subsequently fractionated using Amicon centrifugal filters, MWCO 3 kDa. The filtration procedure was carried out in accordance with the instructions provided by the manufacturer. Briefly, 15 mL (per single filtration) was loaded to the filters and centrifugation was performed at 4400 rpm for 20 min at 25 °C. Permeates and retentates from the filtration were collected and were freeze dried before further analysis.
Molecular-weight-based fractionation of the ProGo-permeate was performed using the same column and same method explained inSection 2.1 with higher loading. A total of three fractions were collected in the elution periods of 7–9 min (permeate I), 9–10 min (permeate II), and 10–13 min (permeate III). These three subfractions were freeze dried before further bioactivity evaluation.
Molecular-weight-based fractionation of the ProGo-permeate was performed using the same column and same method explained in
4
Retroviral Transduction of Bone Marrow Macrophages
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A total of 2 × 106 PlatE [58 (link)] cells were plated in 10‐cm dishes and left to adhere overnight. PlatE cells were transiently transfected with empty vector pMIGRMCS_GFP or specific TLR4_pMIGRMCS_GFP constructs (Supporting Information Table S2 ) using lipofectamine 2000 (Invitrogen). At 24 h post‐transfection, cells were washed and incubated at 32°C for 48 h to facilitate virus production. At the same time, mouse BM was collected from Tlr4−/− mice, plated in BMM complete media, and incubated at 37°C for 48 h. A total of 10 mL of viral supernatant was collected from transfected PlatE cells, filtered for retrovirus using 0.45 μm Millex‐HV PVDF syringe filter (Merck) into labeled 15 mL Falcon tubes. A total of 20 mM HEPES (Gibco), 60 ng polybrene, and 104 U CSF‐1 were added to each supernatant. BMM progenitors were collected from plates and added equally to retroviral supernatants, before being aliquoted into nontissue culture six‐well plates. Plates were centrifuged at 1000g at 35°C for 2 h to facilitate viral uptake. At 48 h post‐infection, media was replaced with BMM complete media. BMM were collected on day 6 and assessed for transduction efficiency by measuring GFP expression by flow cytometry, before being plated for further experiments.
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