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Solid-Phase Reverse Transfection for Intracellular Delivery of Functionally Active Proteins

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Solid-Phase Reverse Transfection for Intracellular Delivery of Functionally Active Proteins Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Method Solid-phase reverse transfection for intracellular delivery of functionally active proteins Ruben Bulkescher,1 Vytaute Starkuviene,1,2 and Holger Erfle1 1BioQuant, Ruprecht-Karls-University Heidelberg, 69120 Heidelberg, Germany; 2Life Sciences Center, Vilnius University, Vilnius 10223, Lithuania Delivery of large and functionally active biomolecules across cell membranes presents a challenge in cell biological exper- imentation. For this purpose, we developed a novel solid-phase reverse transfection method that is suitable for the intracel- lular delivery of proteins into mammalian cells with preservation of their function. We show results for diverse application areas of the method, ranging from antibody-mediated inhibition of protein function to CRISPR/Cas9-based gene editing in living cells. Our method enables prefabrication of “ready to transfect” substrates carrying diverse proteins. This allows their easy distribution and standardization of biological assays across different laboratories. [Supplemental material is available for this article.] Numerous physical, chemical, and biological methods are avail- fore complexation with the cationic lipids (Zuris et al. 2015). able for the transfer of foreign material into cells (Kaestner et al. Furthermore, the net anionic nature of the Cas9–gRNA complex 2015). Diverse molecules pass the cellular membrane with varying allows its intracellular delivery via lipofection, resulting in targeted efficiency. Transfection of short nucleic acids (e.g., siRNAs or gene editing without additional fusion to polyanionic carriers miRNA mimics) is a well-established procedure, whereas cell entry (Liang et al. 2015; Zuris et al. 2015). So far, protein lipofection of large molecules (e.g., proteins) is inefficient due to its low mem- has been performed for only a few example molecules under the brane penetrating ability or low efficiency of the endosomal es- conditions of direct liquid transfection (liquid-phase). cape. The majority of available methods rely on diffusion of Here, we present a solid-phase reverse transfection method large cargo through transient openings in the cell membrane for proteins and demonstrate its utility for diverse application ar- that can be caused by electroporation, laser pulsing (Wu et al. eas. Our approach can be applied for gene engineering and high- 2015), or microfluidic-based cell squeezing (Sharei et al. 2013). content assays in multiwell plates and cell microarrays (Ziauddin Alternative techniques enhance endocytosis by packing cargo and Sabatini 2001; Erfle et al. 2007, 2008). into nanoparticles or nanocapsules (Slowing et al. 2007; Yan et al. 2010), complexing with cell-penetrating peptides (CPPs) (Morris et al. 2001; Ramakrishna et al. 2014), fusing with super- Results charged proteins (Thompson et al. 2012), or induced transduction by osmocytosis and propanebetaine (iTOP) (D’Astolfo et al. 2015). Optimization of the method However, these approaches bear a number of limitations such as In the beginning, we tested how varying concentrations of trans- the need for special equipment, tedious translational fusion, con- fection reagent and protein influence the efficiency of solid-phase jugation to a carrier molecule, or low post-transfection cell viabil- reverse transfection and, as a result, cellular response. Based on ity. Microinjection does not have these pitfalls but is applicable those results, we introduced a transfection “optimization matrix” only in low throughput. Viral vector and plasmid-based delivery comprising four different concentrations of transfection reagent is limited to nucleic acids, packing of large cargo is challenging, and two concentrations of the corresponding protein, resulting and integration into the host genome may occur in an uncon- in eight different conditions (Supplemental Table S1). The transfec- trolled manner (Ramakrishna et al. 2014; Wang et al. 2016). tion reagents Lipofectamine RNAiMAX and Lipofectamine 3000 Therefore, more convenient, versatile, scalable, and less detrimen- showed the best performance in HeLa and HEK293T cells. At least tal delivery methods of large cargo are urgently needed for basic re- three replicates were performed for each experiment, and a total search and therapeutic applications. of approximately 1400–4000 cells per replicate were counted. We Lipofection is based on cationic lipids that interact with neg- applied the matrix for analyzing the transfection efficiency and lo- atively charged nucleic acids. It became one of the most popular calization of a small protein, namely green fluorescent protein ways for cellular delivery of nucleic acids due to its applicability (GFP), with a size of 27 kDa. In agreement with the literature to many cell types and up to 100-fold higher efficiency in compar- (Llopis et al. 1998), the intracellular-delivered GFP was localizing ison to other chemical transfection methods (Kaestner et al. 2015). to the cytoplasm and nucleus (Fig. 1A). The highest transfection ef- Recently, even less charged and chemically diverse macro- ficiency, of more than 73% with the expected localization, was ob- molecules, like proteins, were transfected by lipofection. To in- tained by using 0.5 µL Lipofectamine RNAiMAX and 1 µg GFP, crease the efficiency of membrane transfer, the proteins were whereas other reaction compositions either reduced the fused to negatively charged carriers (e.g., supernegative GFP) be- © 2017 Bulkescher et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it Corresponding author: [email protected] is available under a Creative Commons License (Attribution-NonCommercial Article published online before print. Article, supplemental material, and publi- 4.0 International), as described at http://creativecommons.org/licenses/ cation date are at http://www.genome.org/cgi/doi/10.1101/gr.215103.116. by-nc/4.0/. 27:1–7 Published by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/17; www.genome.org Genome Research 1 www.genome.org Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Bulkescher et al. Figure 1. Comparison of protein localization after solid-phase reverse antibody transfection and immunofluorescent staining in HeLa cells. (A) The in- tracellular distribution of transfected GFP. The upper images show the localization of transfected anti-GM130 (B), anti-alpha tubulin (C), and anti-KIF11 (D) antibodies. The lower images show the results of the IF staining using the same antibodies. (E) Transfection efficiency of antibodies and GFP. Bars rep- resent average transfection efficiency derived from six independent replicas with every antibody shown and 14 independent replicas with GFP; error bars represent the standard deviations. GFP, anti-KIF11, and anti-alpha tubulin antibodies were transfected with Lipofectamine RNAiMAX, using reaction con- dition I for GFP and condition VIII for antibodies. Anti-GM130 antibody was transfected with Lipofectamine 3000 using condition VI (Supplemental Table S1). The transfections were performed in 384-well plates. Nuclei were counterstained with Hoechst 33342 dye. (Scale bar) 20 µm. transfection efficiency and/or induced excessive accumulation of anti-KIF11 and anti-alpha tubulin antibodies as well as for GFP, GFP in punctate structures (Supplemental Fig. S1). Then we tested whereas Lipofectamine 3000 was more suitable for anti-GM130 an- solid-phase reverse transfection of antibodies as an example case tibody delivery (Supplemental Methods). for large proteins (an average size of ∼150 kDa). We initially tested Observation of the subcellular localizations of the transfected the efficiency of antibodies to enter living cells via solid-phase re- antibodies showed different localization patterns, which resem- verse transfection. For this, we used several antibodies that work bled the localizations obtained by IF (Fig. 1B–D; Supplemental well in conventional immunofluorescent staining (IF) in fixed cells Fig. S2). All tested antibodies and GFP also accumulated in punc- and target proteins localizing to diverse subcellular organelles. One tate intracellular structures but at a varying degree (Fig. 1). As the of them was a fluorescently labeled antibody recognizing Golgin intracellular delivery of proteins is expected to exploit endocytic subfamily A member 2 (GOLGA2; further in the text GM130), a ma- pathways, the structures likely present various populations of en- trix protein of the Golgi complex. We also included the molecular dosomes and lysosomes. Indeed, we could detect a partial colocal- motor kinesin family member 11 (KIF11) and an anti-alpha tubulin ization of the transfected anti-KIF11 antibody and early antibody. Following the transfection with the antibodies and incu- endosomes (Supplemental Fig. S3). A thorough spatial and tempo- bation for 2–4 h, cells were detached from the coated surface by ral analysis of distribution of the transfected protein through the trypsinization and replated on a mock-coated surface to remove endocytic pathway was out of scope of this study. At any rate, the excess of the antibody that did not enter the cells. HeLa cells when optimizing the transfection conditions, we considered not were fixed after an additional 4 h of incubation and
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