The synthesis of conjugated Cy7.5 silica NPs (conCy7.5-NPs) was carried out using a slightly modified oil-in-water microemulsion method previously reported by Kumar et al.13 (link),14 (link) To conjugate the Cy7.5-N-hydroxysuccinimide (NHS) ester fluorophore into the NPs, Cy7.5-silane was prepared by conjugating Cy7.5-NHS ester (Lumiprobe Corporation, Hallandale, FL, USA) to a silane precursor using (3-aminopropyl) triethoxysilane (Sigma-Aldrich Co., St Louis, MO, USA) under argon atmosphere. The NPs were prepared at room temperature with 2.2% dioctyl sulfosuccinate sodium salt 96% (AOT) (Sigma-Aldrich Co.) (wt/v) in 10 mL high performance liquid chromatography water, followed by the addition of 1-butanol and Cy7.5-silane at 1,200 rpm stirring. After 15 minutes, vinyltrimethoxysilane (Sigma-Aldrich Co.) was added and the mixture was continuously stirred for 45 minutes, at which point, ammonium hydroxide (Sigma-Aldrich Co.) was added and the reaction was covered and left to stir overnight. The NPs’ suspension was then dialyzed against distilled water for 48 hours at room temperature, using a cellulose membrane (Thermo Fisher Scientific, Waltham, MA, USA) with a cutoff size of 12–14 kDa. Following dialysis, the NPs were sterile filtered (0.45 μm) and stored at 4°C for future use.
Cellulose membrane
Manufactured by Thermo Fisher Scientific
Sourced in United States
Cellulose membranes are a type of filtration material used in various laboratory applications. They are composed of cellulose, a naturally occurring polymer. Cellulose membranes are designed to separate and retain specific components from liquid or gas mixtures based on their physical and chemical properties.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Anhydrous ethylene dichloride, by Merck Group (2 mentions)
Milli q instrument, by Merck Group (2 mentions)
Vinyltrimethoxysilane, by Merck Group (1 mentions)
3 aminopropyltriethoxysilane, by Merck Group (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
Cy7.5 nhs ester, by Lumiprobe (1 mentions)
Na2co3, by Merck Group (1 mentions)
Branched pei, by Merck Group (1 mentions)
Endofree plasmid maxi, by Qiagen (1 mentions)
Pei 1800, by Merck Group (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
7 protocols using cellulose membrane
1
Synthesis of Conjugated Cy7.5 Silica Nanoparticles
2
Silk Fibroin Extraction from Bombyx mori
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Silk fibroin was prepared from Bombyx mori cocoons according to previously established protocols with minor modifications (Rockwood et al., 2011 (link)). B. mori silk cocoons were cut into small pieces and boiled in 0.02 M Na2CO3 (Sigma-Aldrich) for 30 min to remove the glue-like sericin coating layer from the structural fibroin protein which was then rinsed 3x with distilled water (diH2O). The obtained silk fibroin fibers were dried overnight, dissolved in a LiBr (Sigma-Aldrich) solution (9.3 M) at 60°C for 4 h, and dialyzed through a cellulose membrane (ThermoFisher, Waltham, MA, 3500, MWCO) across distilled water for 4 days. The obtained silk solutions were centrifuged thrice at 4200 g and lyophilized for 4 days before resuspending in formic acid for a final concentration of 8% silk/formic acid.
3
Extraction and Purification of Keratin from Human Hair
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The keratin extraction process was adapted from a previous method [6 (link)]. Human hair was washed with warm water and soap and rinsed off thoroughly with deionized (DI) water. Then, the hair was treated with peracetic acid solution (PAS) 2% (w/v) for 12 h at room temperature. Afterwards, the hair was separated from the PAS using a 500-µm sieve and then rinsed thoroughly with DI water to remove residual acids. PAS was used to break the disulfide bonds that are prevalent in keratin. Free proteins were extracted in 100 mM Tris base solution (TBS) for one hour, followed by a second extraction in DI water for another hour. These extractions were carried out in a Dubnoff Metabolic Shaking Incubator (Model 2876, Marietta, OH, USA) that was set to 37 °C and 65 rpm, and retained by passage through a 500-µm sieve. Extracts were neutralized to a pH of 7 using diluted HCL (1 mM), concentrated using a rotary evaporator (Heizbad, Germany), and centrifuged (VWR Clinical 200, Germany) at 3000 rpm for 10 min in order to remove particles. Extracts were dialyzed against DI water using a cellulose membrane with a 12-kDa–14-kDa molecular weight cutoff (Fisher Scientific, Pittsburgh, PA, USA). After extracts were purified for 24 h by dialysis, they were lyophilized to form a keratin powder.
4
Non-invasive pH measurement setup
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To prepare the setup for noninvasive measurement of pH, a hole was created in the bottom wall of a T-flask. Subsequently, a cellulose membrane (Fisher Scientific, Hampton, NH, USA) was attached externally over the hole. A sampler, consisting of a pH sensing patch attached to a transparent layer, was attached to the cellulose membrane from outside. The sensing patch was aligned with the center of the hole. The semi-permeable membrane has a pore size of 4.8 nm and molecular weight cut off (MWCO) of 12,000 Daltons allowing the small-molecule components of the cell culture medium to move towards equilibrium concentration on both sides of the membrane. Different parts of the setup are shown in Fig. 1. To conduct the online measurement of pH, a reader was placed below the vessel. The LED light must be aligned with the pH patch in the sampler. After preparing the setup, a pH patch was attached to the bottom wall of the modified T-flask, as a control method for pH measurement. Different buffers with varying pH values were added to the T-flask. After adding each solution, the measurements through control and noninvasive methods were recorded. The response times, representing the time taken for the sensor to reach 90% of the output, were calculated for both methods.
5
Branched PEI-Mediated Gene Delivery
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Branched PEI (molecular weight 1.8k and 25k Da) and anhydrous ethylene dichloride (EDC) were purchased from Sigma-Aldrich. 2,6-pyridinedicarboxaldehyde (PDA) was obtained from TCI (Shanghai) Development Co., Ltd. Cellulose membranes (MWCO = 10,000 Da), Roswell Park Memorial Institute-1640 (RPMI-1640) medium, Fetal Bovine Serum (FBS), and Phosphate Buffered Saline (PBS, pH 7.4 basic) were purchased from Thermo Fisher Scientific (Shanghai). Escherichia coli bacterial strain DH5a was obtained from Tiangen Biotech (Beijing) CO., Ltd. The plasmids pVEGF165 and pGFP were constructed previously in our laboratory. Water was purified using a milli-Q instrument (Millipore).
6
Plasma and Liver Microsomal Protein Binding of SPN
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The plasma or liver microsomal protein bindings were performed using a rapid equilibrium dialysis device and cellulose membranes with a molecular weight cutoff of 8000 (Thermo Scientific, Rockford, IL, USA) [17 (link)]. The rat and human plasma samples (200 μL) containing SPN at 10 and 50 μM, respectively, were dialyzed against a dialysis buffer, phosphate-buffered saline (PBS, 400 μL). The loaded dialysis plate was covered with sealing tape, placed on an orbital shaker at approximately 200 rpm, and incubated at 37 °C for 4 h. Thereafter, samples (100 μL) from both PBS and plasma chambers were collected and mixed with an equal volume of blank plasma and PBS, respectively. All samples were stored at −80 °C until LC-MS/MS analysis. The unbound fraction of SPN in human (or rat) plasma was calculated by dividing the SPN concentration in PBS by that in plasma.
The human liver microsomal incubation mixtures (final concentration 0.1 mg/mL) without NADPH generating system were used to determine the unbound fraction of SPN. Other procedures were similar to those of plasma protein binding assay.
The human liver microsomal incubation mixtures (final concentration 0.1 mg/mL) without NADPH generating system were used to determine the unbound fraction of SPN. Other procedures were similar to those of plasma protein binding assay.
7
Synthesis and Characterization of PEI-PDA Nanoparticles
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PEI 1800 and 25 kDa were purchased from Sigma-Aldrich. Anhydrous ethylene dichloride was obtained from Sigma-Aldrich. 2,6-pyridinedicarboxaldehyde (PDA) was purchased from TCI (Shanghai) Development Co., Ltd. Cellulose membranes (MWCO = 10,000 Da) were purchased from Thermo Scientific. Water was purified using a milli-Q instrument (Millipore). Poly (ethylene glycol) (PEG) standards kit (ranging from 106 to 20100 Da in molecular weight) was purchased from Polymer Standards Service GmbH. All the reagents were used without further purification.
Plasmid DNA encoding GFP and mouse-VEGF-A siRNA expression vector was constructed from pGPU6/GFP/Neo vector (Bioroot Biology, Shanghai, China) according to a previous report. The targeted VEGF-A sequences were 5′- CGATGAAGCCCTGGAGTGC -3′. And the plasmid was amplified using EndoFreeTM Plasmid Maxi (Qiagen). High-capacity cDNA reverse transcription kits were purchased from Applied Biosystems. RNeasy Mini Kit was purchased from Qiagen. The primers were supplied from Biotnt (Shanghai).
Plasmid DNA encoding GFP and mouse-VEGF-A siRNA expression vector was constructed from pGPU6/GFP/Neo vector (Bioroot Biology, Shanghai, China) according to a previous report. The targeted VEGF-A sequences were 5′- CGATGAAGCCCTGGAGTGC -3′. And the plasmid was amplified using EndoFreeTM Plasmid Maxi (Qiagen). High-capacity cDNA reverse transcription kits were purchased from Applied Biosystems. RNeasy Mini Kit was purchased from Qiagen. The primers were supplied from Biotnt (Shanghai).
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