Subscriber access provided by Imperial College London | Library Biological and Medical Applications of Materials and Interfaces Comb-Like Pseudopeptides Enable Very Rapid and Efficient Intracellular Trehalose Delivery for Enhanced Cryopreservation of Erythrocytes Siyuan Chen, Liwei Wu, Jie Ren, Victoria L. Bemmer, Richard Zajicek, and Rongjun Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.0c03260 • Publication Date (Web): 04 Jun 2020 Downloaded from pubs.acs.org on June 13, 2020 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. 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ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts. is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties. Page 1 of 37 ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 Comb-Like Pseudopeptides Enable Very Rapid and 8 9 10 11 Efficient Intracellular Trehalose Delivery for 12 13 14 15 16 Enhanced Cryopreservation of Erythrocytes 17 18 19 20 Siyuan Chen a‡, Liwei Wu a‡, Jie Ren a, Victoria Bemmer b, Richard Zajicek c, Rongjun Chen a* 21 22 23 a. 24 Department of Chemical Engineering, Imperial College London, South Kensington Campus, 25 26 London, SW7 2AZ, United Kingdom 27 28 29 b. Department of Materials, Imperial College London, South Kensington Campus, London, SW7 30 31 32 2AZ, United Kingdom 33 34 35 c. Cell & Gene Therapy Platform CMC, Platform Technology & Sciences, GlaxoSmithKline plc 36 37 R&D, Gunnels Wood, Stevenage, Hertfordshire SG1 2NY, United Kingdom 38 39 40 41 *Corresponding author: [email protected] 42 43 ‡ 44 Equal contribution to this study. 45 46 47 48 49 50 KEYWORDS: pH-responsive; pseudopeptide; comb-like polymer; cryopreservation; blood; 51 52 trehalose 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 1 ACS Applied Materials & Interfaces Page 2 of 37 1 2 3 ABSTRACT 4 5 6 7 Cell cryopreservation plays a key role in development of reproducible and cost-effective cell-based 8 9 therapies. Trehalose accumulated in freezing and desiccation tolerant organisms in nature has been 10 11 12 sought as an attractive non-toxic cryoprotectant. Herein, we report a co-incubation method for very 13 14 rapid and efficient delivery of membrane-impermeable trehalose into ovine erythrocytes through 15 16 reversible membrane permeabilization using pH-responsive, comb-like pseudopeptides. The 17 18 19 pseudopeptidic polymers containing relatively long alkyl side chains were synthesized to mimic 20 21 membrane-anchoring fusogenic proteins. The intracellular trehalose delivery efficiency was 22 23 optimized by manipulating the side chain length, degree of substitution and concentration of the 24 25 26 pseudopeptides with different hydrophobic alkyl side chains, the pH, temperature and time of 27 28 incubation, as well as the polymer-to-cell ratio and the concentration of extracellular trehalose. 29 30 Treatment of erythrocytes with the comb-like pseudopeptides for only 15 min yielded an 31 32 33 intracellular trehalose concentration of 177.9 ± 8.6 mM, which resulted in 90.3 ± 0.7% survival 34 35 after freeze-thaw. The very rapid and efficient delivery was found to be attributed to the reversible, 36 37 pronounced membrane curvature change as a result of strong membrane insertion of the comb-like 38 39 pseudopeptides. The pseudopeptides can enable efficient intracellular delivery of not only 40 41 42 trehalose for improved cell cryopreservation, but also other membrane-impermeable cargos. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 2 Page 3 of 37 ACS Applied Materials & Interfaces 1 2 3 1. Introduction 4 5 6 It remains a major challenge to delivering hydrophilic, membrane-impermeable molecules into 7 8 the cell interior.1 Clinical applications of polar biomolecules, such as nucleic acids, recombinant 9 10 therapeutic proteins, enzymes, monoclonal antibodies and saccharides, can be impaired by 11 12 2–5 13 inefficient intracellular delivery. Therefore, there is an urgent need to develop delivery vehicles, 14 15 which may facilitate efficient intracellular delivery of hydrophilic, membrane-impermeable 16 17 molecules to their target sites without causing cytotoxicity.6 18 19 Cryopreservation of therapeutic cells is crucial for the development of reproducible and cost- 20 21 22 effective cell-based therapies. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) 23 24 and glycerol are commonly used for cell cryopreservation.7 In order to ensure a steady blood 25 26 supply especially for rare blood groups, erythrocytes are usually cryopreserved with glycerol to 27 28 8,9 29 prolong its shelf-life. However, deglycerolization must be performed after thawing to avoid post- 30 31 transfusion osmotic haemolysis, which is not only labor-intensive and technically demanding, but 32 33 also results in considerably reduced cell viability by 15-20%.10,11 Therefore, there is increasing 34 35 36 interest in developing an alternative method for improved erythrocyte cryopreservation. Trehalose, 37 38 a hydrophilic non-reducing disaccharide of glucose, accumulates in a range of freezing and 39 40 desiccation tolerant organisms in nature and has attracted tremendous attention in the field of cell 41 42 12–14 43 preservation. 44 45 Trehalose is required to be present both inside and outside a cell to ensure optimum 46 47 bioprotection during preservation.15 Unfortunately, mammalian cells are incapable of synthesizing 48 49 or importing trehalose.14 Efficient intracellular delivery of trehalose is challenging due to its 50 51 52 membrane-impermeable and hydrophilic nature. Several approaches have been investigated to 53 54 achieve intracellular trehalose localization, including physical methods such as microinjection16 55 56 57 58 59 60 ACS Paragon Plus Environment 3 ACS Applied Materials & Interfaces Page 4 of 37 1 2 3 and electroporation17, biological methods such as mutant bacterial toxins18 and intracellular 4 5 19 20 6 trehalose synthesis through genetic engineering , and chemical methods such as liposomes . 7 8 However, these methods are limited due to either lack of scalability (e.g. microinjection), or 9 10 insufficient intracellular trehalose for desired bioprotection (e.g. liposomes)21. Besides, some 11 12 13 invasive approaches (e.g. electroporation, mutant bacterial toxins) may affect membrane integrity 14 15 and have safety concerns.22 16 17 This study employs amphiphilic synthetic biopolymers to enhance membrane permeability in 18 19 order to improve intracellular trehalose delivery without causing obvious cell damage. A class of 20 21 22 biodegradable, pH-responsive pseudopeptides has been recently developed to mimic the pH- 23 24 responsive, membrane-permeabilizing activity of fusogenic viral peptides. The biomimetic 25 26 pseudopeptidic polymers have been synthesized by grafting hydrophobic natural amino acids, such 27 28 29 as L-phenylalanine, onto the pendant carboxylic acid groups of an anionic, metabolite-derived 30 31 polyamide, poly(L-lysine isophthalamide) (PLP).23–25 Unlike pathogenic viruses or viral peptides 32 33 which have safety concerns and production difficulties, these pseudopeptidic polycarboxylates are 34 35 36 safer and easy-to-synthesize at large scale. The grafted pseudopeptides displayed a pH-responsive 37 38 coil-to-globule conformational change, which has been shown to trigger membrane destabilization 39 40 at a value approaching physiological pH.24–26 They have been successfully employed for 41 42 43 intracellular delivery of small molecules, therapeutic protein and siRNA to cells, spheroids and 44 45 mouse models.24,27–29 The grafted biopolymers have also been used for intracellular trehalose 46 47 loading for cell cryopreservation. In particular, PP50 (molar percentage of L-phenylalanine at 50% 48 49 relative to the pendant carboxylic acid groups on the PLP backbone) was shown to load trehalose 50 51 52 into erythrocytes, reaching intracellular trehalose threshold concentrations (100–200 mM) 53 54 required for bioprotection of mammalian cells in approximately 9 hours.21,30 However, the long 55 56 57 58 59 60 ACS