Immunoisolation of Nanoparticles Containing Endocytic Vesicles for Drug Quantitation

Abstract

Cell-mediated nanoparticle delivery has recently emerged as an efficacious method of delivering therapeutic agents across physiological barriers. Use of cells as nanodelivery vehicles requires accurate assessment of their loading capacity and identification of intracellular compartments where nanoparticles are sequestered. This is of great interest since specific endocytic trafficking routes can ultimately influence the mode of nanoparticle release and their efficacy and function. Here, we describe a technique that allows for the isolation of individual populations of nanoparticle-containing endosomes for subsequent quantitative analysis and more accurate description of where nanoparticles are stored on a subcellular level.

1 Introduction

For over 20 years, nanoparticles (NP) have been researched for their use in drug delivery (1, 2). Drug-loaded NP have the potential to increase efficacy, reduce toxicity, and improve clinical outcomes of diseases. These nanoparticles tend to be designed to deliver drugs or other therapeutic compounds, such as protein or DNA, to specific cell populations. One of the important questions when researching drug-carrying nanoparticles is determining precisely how much drug the target cells are able to take up. Generally, this question is not difficult to answer. However, of even greater importance than how much drug the target cells are able to pick up is where within the cells are the nanoparticles being trafficked and stored.

2 Materials

Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ cm at 25°C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials.

2.1 Conjugation of Magnetic Beads to Antibodies

1. PureProteome Protein A and Protein G Paramagnetic Beads (Millipore).

2. Antibodies to endosome surface markers of interest (see Note 1).

3. Bovine serum albumin fraction V (10%).

4. Sterile 1× phosphate-buffered saline (PBS).

5. Microcentrifuge tubes (1.7 mL).

6. Microcentrifuge tube tumbler rotator.

7. Magnetic separator rack.

8. Refrigerated tabletop centrifuge.

2.2 Cellular Treatment Components

1. Cells in culture (see Note 2).

2. Cell incubator.

3. Serum-free DMEM (or other appropriate serum-free culture medium).

4. Nanoparticles (see Note 3).

5. Sterile PBS.

2.3 Homogenization of Nanoparticle-Loaded Cells

1. Homogenization buffer: 10 mM HEPES–KOH, pH 7.2, 250 mM sucrose, 1 mM EDTA, and 1 mM Mg(OAc)₂.

2. Cell scrapers.

3. Dounce homogenizer (7 mL).

4. 15 mL centrifuge tubes.

5. Refrigerated centrifuge.

2.4 Isolation of Nanoparticle-Containing Endosomes

1. Homogenate from nanoparticle-treated cells (from Subheading 2.3).

2. Magnetic beads with attached antibodies (from Subheading 2.1).

3. Magnetic separator rack.

4. Sterile PBS.

5. Refrigerated tabletop centrifuge.

2.5 Quantification of Drug Content by HPLC

1. HPLC-grade methanol.

2. Sonicator disruptor with probe tip.

3. Refrigerated tabletop centrifuge.

4. 0.5 mL microcentrifuge tubes.

5. HPLC autoinjector vials with low-volume inserts.

3 Methods

3.1 Conjugate Antibodies to Magnetic Beads

1. In a 1.7-mL microcentrifuge tube, combine 1 mL of 10% bovine serum albumin in PBS with 20 μL of magnetic bead slurry and 20 μg of antibody of interest (see Note 1).

2. Place tubes on a microcentrifuge tube tumbler rotator and rotate at 15 rpm for 12 h at 4°C.

3. Place tubes in the magnetic separator rack for up to 1 h at 4°C. Remove supernatant and resuspend antibody-bead conjugates in sterile PBS. Repeat wash cycle two more times. Finally, resuspend antibody-bead conjugates in 500 μL of sterile PBS and use within 24 h.

3.2 Treat Cells with Nanoparticles

1. Wash cells three times for 10 min with 37°C serum-free medium to remove residual serum protein.

2. Add nanoparticles in sterile serum-free DMEM (or other appropriate cell medium) to cells at desired concentration (Fig. 1a).

3. Incubate at 37°C to allow cells to take up nanoparticles (see Note 4).

4. Once maximum nanoparticle uptake has been reached, wash the cells three times with 37°C sterile PBS to remove any nano-particles that have not been taken up. Keep cells in PBS and immediately begin homogenization.

Fig. 1.

Schematic diagram of immunoisolation of nanoparticle-containing endosomes. Cells are first treated with nanoparticles (a). After maximum nanoparticle endocytosis, cells are ruptured in homogenization buffer using a Dounce tissue homogenizer (b). Cell homogenate is then slowly centrifuged in order to remove nuclei, organelles, and cellular debris (c). Following enrichment, the endosome fraction is exposed to protein A/G paramagnetic beads conjugated to antibodies and allowed to bind with the endosomes carrying the surface markers of interest. (d) The endosome population bound to the beads is isolated by magnetic separation (e). The isolated fraction undergoes several washes with sterile cold PBS prior to drug quantitation (f)

3.3 Homogenization of Nanoparticle-Loaded Cells

1. Remove PBS and add enough homogenization buffer to cover the bottom of each well or flask being used.

2. Detach cells from bottom of well or flask using cell lifter.

3. Add scraped cells to Dounce homogenizer and grind cells with 15 strokes (Fig. 1b) (see Note 5).

4. Add entire volume to a 15 mL centrifuge tube and remove nuclei and unbroken cells by centrifuging at 400 × g for 10 min at 4°C (Fig. 1c).

5. Remove supernatant, place in 1.7 mL microcentrifuge tubes, and use for immune isolation of endocytic compartments.

3.4 Isolate Nanoparticle-Containing Endosomes

1. In a 1.7-mL microcentrifuge tube, combine 1 mL of the cellular homogenate from Subheading 3.3, step 5, and the entire volume (500 μL) of one antibody-bead combination from Subheading 3.1, step 3 (Fig. 1d). Be sure to include one tube with blank beads (i.e., fresh beads with no antibody conjugate) as a control.

2. Place tubes on a microcentrifuge tube tumbler rotator and rotate at 15 rpm for 18–24 h at 4°C.

3. Place tubes on microcentrifuge tube magnetic separator and allow for magnetic separation for 1 h at 4°C (Fig. 1e). The solution should become clear as the beads form a layer in the tube wall facing the magnet.

4. Carefully remove the solution without disturbing the beads. Add 1 mL of cold sterile PBS and resuspend the beads (see Note 6). Repeat this wash cycle two more times. Endosomes are now ready for quantitative analysis (store at 4°C until ready for analysis).

3.5 Quantification of Drug Content of Nanoparticle-Containing Endosomes by HPLC

1. Take samples from Subheading 3.4, step 4, and centrifuge at 10,000×g for 10 min at 4°C. Remove the supernatant and add 400 μL of 100% methanol (see Note 7).

2. Sonicate solution with sonicator probe for 3 s at 20% amplitude.

3. Centrifuge solutions at 20,000 × g for 10 min at 4°C. Remove supernatant and add to a clean 0.5 mL microcentrifuge tube.

4. Transfer 70 μL to an HPLC autosampler vial and inject three 20 μL aliquots onto the HPLC for drug quantitation (see Notes 8 and 9).

5. Determine drug content by comparing peak area of drug in sample to peak areas of known concentrations of drug standards.