Ex vivo cytosolic delivery of functional macromolecules to immune cells

Abstract

Intracellular delivery of biomolecules, such as proteins and siRNAs, into primary immune cells, especially resting lymphocytes, is a challenge. Here we describe the design and testing of microfluidic intracellular delivery systems that cause temporary membrane disruption by rapid mechanical deformation of human and mouse immune cells. Dextran, antibody and siRNA delivery performance is measured in multiple immune cell types and the approach's potential to engineer cell function is demonstrated in HIV infection studies.

Fig 1. Delivery methodology and performance in mouse cells.

A) Illustration of device design and delivery mechanism. B) Illustration of the system setup and delivery procedure. C) Representative histograms of T cells, B cells and myeloid cells (CD11b⁺) treated by the CellSqueeze device to deliver APC-labeled IgG1. D) Delivery efficiency of Cascade blue-labeled 3 kDa dextran, fluorescein-labeled 70 kDa dextran, and APC-labeled IgG1. All results were measured by flow cytometry within an hour of treatment. Dead cells were excluded by propidium iodide staining. Viability is shown in S2 Fig. Data in D) (mean ± SD) are from 3 independent experiments. Untreated cells were not put through the device or exposed to the biomolecules. The ‘no device’ samples were incubated with the biomolecules, but were not treated by the device. This control is meant to account for surface binding, endocytosis and other background effects.

Fig 2. Delivery to human immune cells.

A) Human T cells and MDDCs were tested for delivery of cascade blue labeled 3kDa dextran, fluorescein labeled 70kDa dextran, and APC labeled IgG1. The representative histograms for a 30–4 (T cells) and 10–7 (MDDCs) device (left) and replicates across device designs (right) are displayed. B) SiRNA mediated knockdown of CD4 and DC-SIGN protein levels in CD4⁺ T cells and MDDCs respectively. C) Knockdown of CD4 expression in human regulatory T cells in response to treatment by a 30–4 device. Dead cells were excluded for delivery or knockdown analysis. D) Comparison of device performance in T cells to nucleofection by Amaxa. Protein expression 72hrs after siRNA delivery and cell viability after treatment are shown. E) Intracellular staining for the p24 antigen was used as an indicator of HIV infection level in treated human CD4⁺ T cells 24hrs after infection. In these studies, vif and/or gag, siRNA was delivered 24hrs prior to infection while CD4 siRNA was delivered 48hrs prior to infection. F) Median fluorescence intensity of the p24 antigen stain across repeats (min. N = 4) of the experimental conditions. Data are represented as mean + 1 standard error.