Efficient and gentle delivery of molecules into cells with different elasticity via Progressive Mechanoporation

Abstract

Intracellular delivery of cargo molecules such as membrane-impermeable proteins or drugs is crucial for cell treatment in biological and medical applications. Recently, microfluidic mechanoporation techniques have enabled transfection of previously inaccessible cells. These techniques create transient pores in the cell membrane by shear-induced or constriction contact-based rapid cell deformation. However, cells deform and recover differently from a given extent of shear stress or compression and it is unclear how the underlying mechanical properties affect the delivery efficiency of molecules into cells. In this study, we identify cell elasticity as a key mechanical determinant of delivery efficiency leading to the development of "progressive mechanoporation" (PM), a novel mechanoporation method that improves delivery efficiency into cells of different elasticity. PM is based on a multistage cell deformation, through a combination of hydrodynamic forces that pre-deform cells followed by their contact-based compression inside a PDMS-based device controlled by a pressure-based microfluidic controller. PM allows processing of small sample volumes (about 20 μL) with high-throughput (>10 000 cells per s), while controlling both operating pressure and flow rate for a reliable and reproducible cell treatment. We find that uptake of molecules of different sizes is correlated with cell elasticity whereby delivery efficiency of small and big molecules is favoured in more compliant and stiffer cells, respectively. A possible explanation for this opposite trend is a different size, number and lifetime of opened pores. Our data demonstrates that PM reliably and reproducibly delivers impermeable cargo of the size of small molecule inhibitors such as 4 kDa FITC-dextran with >90% efficiency into cells of different mechanical properties without affecting their viability and proliferation rates. Importantly, also much larger cargos such as a >190 kDa Cas9 protein-sgRNA complex are efficiently delivered high-lighting the biological, biomedical and clinical applicability of our findings.

Fig. 1. Progressive mechanoporation: rapid and gradual cell deformation under controlled pressure and flow rate a) Left: schematic representation of the PDMS-based microfluidic platform for progressive mechanoporation (PM). The set-up consists of a pressure controller connected to a vial containing CO₂-independent cell culture media (CMV) which is further connected to a flow sensor via a tubing filled with the same media (CMT). Another tubing filled with cell suspension (CST) is attached between the flow sensor and microfluidic device inlet. To collect the mechanoporated cells, a tubing is connected at the outlet of the device. Right: Picture of the microfluidic chip made of a micro structured PDMS element bonded on a glass coverslip. Each chip includes three microfluidic devices. FEP tubing are inserted in correspondence of the inlet and outlet chambers. b) Schematic representation of the progressive cell membrane mechanoporation during cell flow inside a single channel and cell membrane recovery in the collection tube (reservoir). The channel comprises of three deformation regions: low (LDR), medium (MDR) and high deformation region (HDR). For each region, length (L) and width (W) of the corresponding channel are indicated. The arrows in the MDR, HDR and before the outlet, indicate the cell volume exchange resulting in convective molecular transport into cell cytoplasm at the outlet. The arrows in the reservoir show the diffusion of molecules from the surrounding medium to the cell cytosol before cell membrane repair. c) Pressure stabilization over time for four different operating pressures (3, 4 and 5 bar). The gradient grey zone shows the device delamination for operating pressure higher than 5.5 bar. d) Flow rate at the device inlet, Q_i, as function of the applied pressure for four different device geometries (L_c–W_c = 40–6μm; 60–6μm; 40–4μm; 60–4μm). The mean values and SD of three independent experiments are plotted.

Fig. 2. Analysis of cell size and elasticity. a) Cell diameter distribution for four different cell types: HeLa K, RPE-1, U2OS and BJ. b) Young's modulus distribution of HeLa K, RPE-1, U2OS and BJ cells measured by real-time deformability cytometry (RT-DC). The mean and SD are plotted.

Fig. 3. Intracellular delivery of small molecules into cells with comparable size and different elasticity. a) Delivery efficiency of 4 kDa FITC-dextran in HeLa K and RPE-1 cells, obtained by using 40–6μm (L_c–W_c) (top) and 60–6μm (L_c–W_c) (bottom) devices. The delivery efficiency was measured by FACS for the following conditions: not treated cells (Ctrl); cells treated with operating pressure of 3 bar and 5 bar. The symbol () represents the addition of 4 kDa FITC-dextran to the cell suspension. Individual measurements (circles) and mean values (line) are reported. Significance between 3 bar and 5 bar samples according to an unpaired T-test (ns = not significant). b) Cell viability of HeLa K and RPE-1 cells represented as propidium iodide (PI) negative cells (top), measured by FACS analysis of cells stained with PI directly after PM in 40–6μm and 60–6μm (L_c–W_c) devices (pressure 5 bar) and without PM (Ctrl). Fold increase in cell number (bottom) comparing HeLa K and RPE-1 cells 24 hours after PM in 40–6μm and 60–6μm (L_c–W_c) devices and without PM (Ctrl) measured by bright-field microscopy. The mean values and SD of three independent experiments are plotted. Significance according to Kruskal–Wallis one-way analysis of variance (ns = not significant).

Fig. 4. Progressive mechanoporation in narrower constriction enhances delivery efficiency of small molecules into stiffer cells. a) Delivery efficiency of 4 kDa FITC-dextran in HeLa K and RPE-1 cells, obtained by using 40–4μm (L_c–W_c) (top) and 60–4μm (L_c–W_c) (bottom) devices. The delivery efficiency was measured by FACS for the following conditions: not treated cells (Ctrl); cells treated with operating pressure of 3 bar and 5 bar. The symbol () represents the addition of 4 kDa FITC-dextran to the cell suspension. Individual measurements (circles) and mean values (line) are reported. Significance between 3 bar and 5 bar samples according to an unpaired T-test (ns = not significant). b) Cell viability of HeLa K and RPE-1 cells represented as propidium iodide (PI) negative cells (top), measured by FACS analysis of cells stained with PI directly after PM in 40–4μm and 60–4μm (L_c–W_c) devices (pressure 3 bar) and without PM (Ctrl). Fold increase in cell number (bottom) comparing HeLa K and RPE-1 cells 24 hours after PM in 40–4μm and 60–4μm (L_c–W_c) devices and without PM (Ctrl) measured by bright-field microscopy. The mean values and SD of three independent experiments are plotted and p values are indicated. Significance according to Kruskal–Wallis one-way analysis of variance (ns = not significant).

Fig. 5. Intracellular delivery of bigger molecules in a size range of biologically relevant cargo. Delivery efficiency of 70 kDa FITC-dextran in HeLa K (top) and RPE-1 (bottom) cells, obtained by using 40–4μm and 60–4μm (L_c–W_c) devices. The delivery efficiency was measured by FACS for the following conditions: not treated cells (Ctrl); cells treated with operating pressure of 3 bar. The symbol () represents the addition of 70 kDa FITC-dextran to the cell suspension. Individual measurements (circles) and mean values (line) are reported.

Fig. 6. Successful delivery of Cas9–sgRNA RNPs. a) Scheme of the Cas9–NLS protein-sgRNA RNPs delivery into U2OS cells (D0 = day 0), cell splitting (D1 = day 1), the microscopy (D3 = day 3) and FACS analysis (D4 = day 4). b) Delivery efficiency of Cas9–NLS protein–sgRNA RNPs targeting GFP (GFP targeting) or non-targeting in U2OS, obtained by using 40–4μm and 60–4μm (L_c–W_c) devices with operating pressure of 3 bar and analysed by FACS as percentage of GFP negative cells. Individual measurements (circles) and mean values (line) are reported. The data were obtained from two independent experiments (with three technical repeats for Cas9–sgRNA RNPs targeting GFP and two technical repeats for Cas9–sgRNA RNPs non-targeting within one independent experiment). c) Fluorescent images of cells after delivery of non-targeting or GFP-targeting Cas9–sgRNA RNPs as indicated in (a), by using 40–4μm (L_c–W_c) (top) and 60–4μm (L_c–W_c) (bottom) devices. DAPI was used for DNA staining. The scale bar is 20 μm.