Poly l glutamic acid

Virus-free CAR transfer to primary human T cells was performed as previously described.¹⁰ (link) In brief, TRAC-targeting synthetic modified single guide RNA (100 μM; IDT) was mixed with an aqueous solution of ∼15 to 50 kDa poly(L-glutamic acid) (100 μg/μL; Sigma Aldrich)²¹ (link) and recombinant SpCas9 protein (61 μM; IDT) in a 0.96:1:0.8 volume ratio to form precomplexed ribonucleoproteins (RNPs). Two days after isolation, activated T cells were resuspended in TheraPEAK P3 Primary Cell Nucleofector Solution (Lonza; 5-10 × 10⁶ cells per 100 μL) and mixed with RNP (10.35 μL per 100 μL suspension) and CD19.CD28.CD3ζ-CAR¹⁰ (link) (Addgene; Plasmid #183473) double stranded DNA–HDR template (2.5 μl/100 μL suspension). The suspension was electroporated (in 20 μL or 100 μL electroporation vessels) in a Lonza 4D nucleofector device using the program EH-115.

Films are assembled on 10 mm diameter glass slides, squared glass microscope slides or QCM-D sensor chips previously washed with detergent and rinsed with deionized water. (PLL-PGA)_n and ([PLL-PGA]₅-PLL-NP)_n-(PLL-PGA)₂ films are assembled by subsequent dipping in 0.1 g/L Poly-L-Lysine (PLL, MW 70–150 kDa, Sigma) and Poly-L-Glutamic acid (PGA, MW 50–150 kDa, Sigma) in PBS pH 7.4, and 1 g/L carboxyl functionalized latex nanoparticles (C37261, Invitrogen), of diameter 28 ± 4 nm diluted in deionized water when necessary. Each dipping step last for 10 min followed by three 1 min buffer rinse steps. Both films are subjected to chemical cross-linking with 40 mM 1-ethyl-3-(3 [dimethylamino]propyl)carbodiimide (EDC, Sigma) and 100 mM, N-hydroxysulfosuccinimide (sulfo-NHS, Sigma) for 16h. The films containing the carboxyl functionalized latex nanoparticles (NP, Invitrogen) are incubated in tetrahydrofurane (THF) for 16h in order to remove the NP from the films and rinsed three times for 20 min in buffer, as previously described.²⁰ (link) Native (PLL-PGA)_n films are formed as described above, but without chemical EDC-NHS cross-linking.