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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 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

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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. & 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; ; 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 and desiccation tolerant in nature has been 10 11 12 sought as an attractive non-toxic . 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, 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, , 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 such as (DMSO) 23 24 and 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 bacterial toxins18 and intracellular 4

5 19 20 6 trehalose synthesis through , 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 Paragon Plus Environment 4 Page 5 of 37 ACS Applied Materials & Interfaces

1 2 3 incubation time required for PP50-mediated trehalose loading is not satisfactory for cell-based 4 5 6 therapies which comprise a number of complex processes but usually have a very short time 7 8 window.22 On the other hand, the intracellular trehalose delivery efficiency (percentage of 9 10 intracellular trehalose concentration relative to total trehalose loading concentration) of PP50 was 11 12 13 limited to no more than 35%, which resulted in the use of high concentrations of extracellular 14 15 trehalose for loading.21,30 16 17 Herein, we report the development of a new approach to achieving very rapid and efficient 18 19 intracellular delivery of hydrophilic, membrane-impermeable trehalose for enhanced 20 21 22 cryopreservation of ovine erythrocytes. Inspired by naturally existing membrane proteins which 23 24 contain covalently attached long-chain fatty acids acting as membrane anchors, we have recently 25 26 reported a class of pH-responsive, membrane-anchoring, comb-like pseudopeptides with dramatic 27 28 29 endosomolytic activity for efficient cytoplasmic delivery of endocytosed materials through 30 31 endosomal escape. Through grafting hydrophobic decylamine (NDA) onto carboxylic acid groups 32 33 pendant to PLP, the comb-like PLP-NDA polymers exhibited the enhanced membrane interaction. 34 35 36 In a haemolysis model using erythrocyte membranes as a model for endosomal membranes, the 37 38 optimal PLP-NDA18 with a degree of NDA substitution at 18 mol% was non membrane-lytic at 39 40 pH 7.4 but induced nearly complete haemolysis within 20 min upon acidification to late endosomal 41

42 31 43 pH 5.5. However, delivery of membrane-impermeable molecules such as trehalose into 44 45 erythrocytes (especially ovine erythrocytes whose membranes are more fragile) without causing 46 47 significant cell damages is notably challenging due to a lack of transduction or endocytosis 48 49 pathways.21 In this paper, we investigated the effects of the side chain length, degree of substitution 50 51 52 and concentration of the comb-like pseudopeptides with different hydrophobic alkyl side chains, 53 54 the pH, temperature and time of incubation, as well as the polymer-to-cell ratio and the 55 56 57 58 59 60 ACS Paragon Plus Environment 5 ACS Applied Materials & Interfaces Page 6 of 37

1 2 3 concentration of extracellular trehalose in order to achieve very rapid and efficient loading of 4 5 6 trehalose into ovine erythrocytes for effective cryopreservation. The haemolytic activity of various 7 8 delivery formulations was minimized. The mechanism of intracellular delivery mediated by the 9 10 comb-like pseudopeptides was elucidated, and the enhanced cryopreservation of erythrocytes was 11 12 13 demonstrated. 14 15 16 17 2. Materials and methods 18 19 2.1 Materials 20 21 22 Heptylamine (HDA, C7), decylamine (NDA, C10), tetradecylamine (TDA, C14), 23 24 octadecylamine (ODA, C18), calcein, iso-phthaloyl chloride and anthrone were purchased from 25 26 Sigma-Aldrich (Dorset, UK). DMSO, N,N-dimethylformamide, 4-dimethy-laminopyridine 27 28 29 (DMAP) and sodium chloride were obtained from Fisher Scientific (Loughborough, UK). D-(+)- 30 31 Trehalose dihydrate, L-lysine methyl ester dihydrochloride, ninhydrin, triethylamine and N,N’- 32 33 dicyclohexylcarbodiimide (DCC) were purchased from Alfa Aesar (Heysham, UK). Anhydrous 34 35 36 ethanol, acetone, chloroform, hydrochloric acid, sodium hydroxide, methanol, diethyl ether, 37 38 sodium citrate dehydrate, potassium chloride, potassium dihydrogen orthophosphate and sulfuric 39 40 acid were obtained from VWR (Lutterworth, UK). Ovine erythrocytes (defibrinated, sterile), 41 42 43 which were separated from whole blood by centrifuge and contained no additives, were purchased 44 45 from TCS Biosciences (Buckingham, UK), stored in a fridge and used within three days of the 46 47 prescribed four-week shelf life. 48 49 2.2 Pseudopeptidic polymer synthesis and characterization 50 51 52 Alkyl chains, such as HDA (C7), NDA (C10), TDA (C14) and ODA (C18), were grafted onto 53

54 the backbone of the parent pseudopeptidic polymer PLP (Mn = 17.9 kDa, Mw = 35.7 kDa, 55 56 57 58 59 60 ACS Paragon Plus Environment 6 Page 7 of 37 ACS Applied Materials & Interfaces

1 2 3 polydispersity = 1.99) at various degrees of substitution via DCC/DMAP mediated amide coupling 4

5 31 6 according to the previously published method. The structures of PLP and the comb-like 7 8 pseudopeptides (Scheme 1), denoted as PLP-NDA3, PLP-NDA10, PLP-NDA18, PLP-HDA18, 9 10 PLP-TDA18 and PLP-ODA18, were confirmed using a Spectrum 100 FT-IR Spectrometer 11 12 13 (PerkinElmer, USA) and a 400 MHz NMR spectrometer (Bruker, Germany). The molecular 14 15 weight and polydispersity index (PDI) of PLP (Mw = 35.7 kDa, Mn = 17.9 kDa, PDI = 1.99) were 16 17 determined using an aqueous GPC system (Viscotek, UK), which was calibrated with polyethylene 18 19 oxide standards according to the previous publication. 23 The actual degrees of substitution of the 20 21 22 comb-like polymers, calculated as the molar percentages of the alkyl side chains relative to the 23 24 pendant carboxylic acid groups on the PLP backbone, were determined by the ratio of the integral 25 26 0.77-0.91 ppm to the integral 7.45-7.64 ppm in their 1H NMR spectra (Figure S1) and used to 27 28 29 calculate their molecular weights. The compositions of these polymers are shown in Table 1. The 30 31 FT-IR spectra (Figure S2) of the pseudopeptidic polymers in their acid forms show characteristic 32 33 peaks at 3291 (N−H str and O−H str), 1723 (C=O acid str), 1625 (amide band I), and 1530 cm−1 34 35 36 (amide band II). 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Scheme 1. Repeat unit structures of PLP, PLP-HDA, PLP-NDA, PLP-TDA and PLP-ODA. 55 56 57 58 59 60 ACS Paragon Plus Environment 7 ACS Applied Materials & Interfaces Page 8 of 37

1 2 3 Table 1. Compositions of PLP, PLP-NDA3, PLP-NDA10, PLP-NDA18, PLP-HDA18, PLP- 4 5 6 TDA18 and PLP-ODA18. 7 8 PLP PLP- PLP- PLP- PLP- PLP- PLP- 9 NDA3 NDA10 NDA18 HDA18 TDA18 ODA18 10 11 Side chain length 0 10 10 10 7 14 18 12 (carbon number) 13 14 Actual degree of 0 3 10 18 18 18 18 15 substitution (%) 16 Mn (kDa) 17.9 18.2 18.9 19.7 19.1 20.5 21.1 17 18 19 20 2.3 Trehalose loading 21 22 Trehaose were prepared by dissolving trehalose in 306 mOsm phosphate-buffered 23 24 25 saline (PBS) at various concentrations and pHs. 0.5 mL of erythrocytes were washed three times 26 27 with 1 mL of 306 mOsm PBS (pH 7.4), and then resuspended in 1 mL trehalose in the 28 29 presence or absence of the comb-like pseudopeptidic polymers. The resulting erythrocyte samples 30 31 with a final packed cell volume of 15% were incubated in a shaking water bath (120 rpm) at preset 32 33 34 temperature to allow trehalose loading. 35 36 2.4 Intracellular trehalose quantification 37 38 Anthrone reaction was applied for intracellular trehalose quantitation.21,30 After trehalose 39 40 41 loading, the erythrocyte samples were centrifuged at room temperature and washed twice with 1.5 42 43 mL PBS (pH 7.4) iso-osmotic to the incubation mixture to remove extracellular trehalose. The 44 45 erythrocytes were then mixed with 5 mL 80% (v/v) methanol and incubated in a water bath at 85 46 47 48 °C for 1 h. The samples were centrifuged at 10,500 rpm for 4 min. Their supernatants were 49 50 collected and then evaporated overnight in an oven at the preset temperature of 100 °C. Each dry 51 52 residue was dissolved in 2 mL of deionized water. 0.5 mL of the resulting trehalose solution was 53 54 55 mixed with 5 mL of 66% (v/v) sulphuric acid containing 0.05% (w/v) anthrone, followed by 56 57 58 59 60 ACS Paragon Plus Environment 8 Page 9 of 37 ACS Applied Materials & Interfaces

1 2 3 incubation at 100 °C for 15 min. The absorbance at 620 nm was measured after sample cooling. 4 5 6 Negative control (erythrocytes incubated in trehalose for less than 1 min to yield negligible 7 8 trehalose loading) was prepared to determine the anthrone absorbance of residual extracellular 9 10 trehalose after washing as well as endogenous . Intracellular trehalose concentrations were 11 12 13 obtained by comparing to a trehalose concentration versus absorbance standard curve. The 14 15 trehalose delivery efficiency was then calculated according to the following equation: 16 17 �! Delivery efficiency(%) = ×100 18 � 19 " 20 21 where, �! is the intracellular trehalose concentration after the polymer-mediated delivery and �" is 22 23 the total trehalose loading concentration. 24 25 2.5 Haemolysis assay 26 27 28 Since haemoglobin oxidation was found to be insignificant during all steps of trehalose loading 29 30 and erythrocyte cryopreservation21,30, haemolysis was evaluated by measuring the absorbance of 31 32 oxyhemoglobin through a simple spectrophotometric method, instead of measuring all forms of 33 34 32 35 haemoglobin. After trehalose loading (or after freezing and thawing), the erythrocyte samples 36 37 were centrifuged at 3,500 rpm and the absorbance of the supernatant was measured at 541 nm (one 38 39 of the oxyhemoglobin’s absorbance peaks) using a UV-Vis spectrophotometer (GENESYS 10S 40 41 42 UV−Vis, Thermo Scientific, USA). Erythrocytes before trehalose loading (or before freezing and 43 44 thawing) were lysed in deionized water to prepare positive control or resuspended in 306 mOsm 45 46 PBS buffer alone to prepare negative control. All the measured haemoglobin absorbance was 47 48 49 ensured to be in linear relationship with the number of lysed erythrocytes (Figure S3). The relative 50 51 haemolysis was calculated using the following equation: 52 53 �# – �$% Relative hemolysis (%) = ×100 54 � 55 &% 56 57 58 59 60 ACS Paragon Plus Environment 9 ACS Applied Materials & Interfaces Page 10 of 37

1 2 3 where � is sample haemoglobin absorbance induced during the experimentation (during trehalose 4 # 5 6 loading or during freezing and thawing), �&% is the positive control haemoglobin absorbance 7 8 which relates to the initial cell count and �$% is the negative control haemoglobin absorbance 9 10 which was maintained to be approximately 0.5% relative to positive control. 11 12 13 2.6 Confocal microscopy 14 15 Calcein, a membrane impermeable dye, was used as a model payload to investigate the comb- 16 17 like pseudopeptidic polymer mediated intracellular delivery. Erythrocytes were washed three times 18 19 20 with 306 mOsm PBS (pH 7.4) and incubated with 360 mM trehalose, 1 μM calcein, and 0.8 mg 21 22 mL-1 polymer (PP50 or PLP-NDA18) at a specific pH in a shaking water bath (120 rpm, 37°C) for 23 24 15 min. Negative control was prepared by incubating erythrocytes with 360 mM trehalose and 1 25 26 27 μM calcein in the absence of polymer at the same specific pH for 15 min. After incubation, the 28 29 erythrocytes were washed twice with 306 mOsm PBS (pH 7.4) and then imaged using an LSM- 30 31 510 inverted laser scanning confocal microscope (Zeiss, Germany) at 37°C. Calcein was excited 32 33 34 at 488 nm, and the emission at 535 nm was collected. 35 36 To assess the erythrocyte membrane integrity after intracellular loading, erythrocytes were 37 38 incubated with 360 mM trehalose in the presence or absence of pseudopeptidic polymer at pH 6.1 39 40 41 in a shaking water bath (120 rpm, 37°C) for 15 min, followed by washing twice with pH 7.4 PBS. 42 43 The washed erythrocytes were then resuspended in the pH 7.4 PBS buffer containing 1 μM calcein 44 45 and imaged using a laser scanning confocal microscope as described above. 46 47 2.7 Atomic force microscopy (AFM) 48 49 50 Erythrocytes were washed three times with 306 mOsm PBS (pH 7.4), incubated with 360 mM 51 52 trehalose solution in the presence or absence of 0.8 mg mL-1 pseudopeptidic polymers at pH 6.1 53 54 for 15 min, and then immobilized on a polylysine coated microscope slide. The cells were crossed 55 56 57 58 59 60 ACS Paragon Plus Environment 10 Page 11 of 37 ACS Applied Materials & Interfaces

1 2 3 linked in glutaraldehyde (1%) for 10 min, washed and then air dried.30 AFM was performed using 4 5 6 an Asylum MFP-3D microscope (Oxford Instruments Asylum Research, US) in tapping mode. 7 8 PPP-NCHR tips with resonant frequency around 320 kHz, tip radius 7 nm and spring 9 10 constant 42 N m-1 were used and tuned to a target tapping amplitude of 1– 2 V. 11 12 13 2.8 Cryopreservation 14 15 Erythrocytes (15% packed cell volume) were incubated in 360 mM trehalose solution in the 16 17 presence or absence of 0.8 mg mL-1 PLP-NDA18 at pH 6.1 at 37 °C. After 15 min of incubation, 18 19 erythrocytes were washed twice with pH 7.4 PBS buffer iso-osmotic to the incubation solution to 20 21 22 remove PLP-NDA18 and free haemoglobin and reverse membrane permeability. The supernatant 23 24 of each erythrocyte sample was then replaced by 360 mM trehalose solution at pH 7.4 yielding a 25 26 packed cell volume of approximately 15%. Negative control was prepared by incubating 27 28 29 erythrocytes with PBS (pH 6.1) alone for 15 min, followed by washing and resuspension in PBS 30 31 (pH 7.4). 1 mL of the processed erythrocytes were transferred to 2 mL polypropylene cryovials, 32 33 directly immersed in liquid (cooling rate: 300°C min-1) and then cryopreserved for 34 35 36 specific time periods. Recovery of the cryopreserved erythrocytes was accomplished by thawing 37 38 in a water bath at 37°C (thawing rate: 20°C min-1) for 15 min. Immediately after thawing, the 39 40 erythrocyte samples were centrifuged at 3,500 rpm for 3 min and the cell pellets were lysed in 41 42 43 deionized water, followed by measurement of haemoglobin absorbance at 541 nm which was 44 45 referred to the correlation curve in Figure S3 to calculate the viable cell number. The cryosurvival 46 47 rate was determined by comparing the viable cell number before and after freezing and 48 49 thawing.21,33,34 according to the following equation: 50 51 52 Viable cell number after freezing and thawing 53 Cryosurvival rate (%) = ×100 54 Viable cell number before freezing and thawing 55 56 57 58 59 60 ACS Paragon Plus Environment 11 ACS Applied Materials & Interfaces Page 12 of 37

1 2 3 2.9 Statistical analysis 4 5 6 All data points were repeated in triplicate (n = 3). Results and graphical data are presented as 7 8 mean values with standard deviation (S.D.) encompassing a 95% confidence interval. Data were 9 10 analysed for statistical significance by two-tailed Student’s t-test. 11 12 13 14 15 3. Results and Discussion 16 17 3.1. Intracellular trehalose loading by comb-like pseudopeptides 18 19 Alkyl side chains have been reported to play an important role as membrane anchors in the 20 21 35,36 22 enhancement of lipid membrane binding and insertion. Four comb-like pseudopeptides, PLP- 23 24 HDA18, PLP-NDA18, PLP-TDA18 and PLP-ODA18, were synthesized by grafting alkyl side 25 26 chains of different lengths (C7, C10, C14 and C18, respectively) onto the parent polymer at an 27 28 29 actual grafting degree of 18 mol%. The impact of alkyl side chain length on intracellular trehalose 30 31 delivery and haemolytic activity is shown in Figure 1. By slightly increasing the side chain length 32 33 from 7 to 10 carbons, intracellular trehalose loading was increased by a factor of 2.4. Other 34 35 36 researchers have reported that acylation of proteins with longer alkyl side chains can yield stronger 37 38 membrane association and deeper insertion into the lipid membrane.37 It has been noted by Mruthy 39 40 et al. that poly(propylacrylic acid) with one additional methylene unit in the pendant alkyl group 41

42 38 43 showed stronger membrane disruption than poly(α-ethylacrylic acid). Figure 1 shows that with 44 45 increasing alkyl side chain length over 10 carbons, intracellular trehalose loading was reduced 46 47 significantly. This is because an increase in alkyl chain length can remarkably enhance polymer 48 49 hydrophobicity, which if too high could cause polymer aggregation and consequently reduce 50 51 52 polymer-membrane interaction.37,39 PLP-NDA18 containing 10-carbon alkyl side chains was found 53 54 to be superior, resulting in an intracellular trehalose concentration of 177.9 ± 8.6 mM after only 55 56 57 58 59 60 ACS Paragon Plus Environment 12 Page 13 of 37 ACS Applied Materials & Interfaces

1 2 3 15 min of incubation. Intracellular trehalose concentrations between 100 and 200 mM have been 4 5 6 found to achieve favorable cryoprotection of mammalian cells including murine fibroblasts and 7 8 human oocytes and keratinocytes.15,40 In contrast, Holovati et al. have reported that the intracellular 9 10 trehalose concentration (below 15 mM) loaded by liposomes was too low to achieve improved 11

12 20 13 cryoprotection of erythrocytes. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Figure 1. Impact of alkyl side chain length at a fix degree of substitution of 18 mol% on 33 34 intracellular trehalose concentration and relative haemolysis. Erythrocytes were incubated with the 35 36 PBS buffer solution containing 360 mM trehalose and 0.8 mg mL-1 pseudopeptide for 15 min. 37 38 39 Temperature = 37°C; pH = 6.1. Mean ± S.D. (n = 3). 40 41 42 43 44 Three membrane-anchoring, comb-like pseudopeptides containing hydrophobic relatively long 45 46 NDA side chains, PLP-NDA3, PLP-NDA10 and PLP-NDA18 (actual degree of grafting at 3, 10 47 48 49 and 18 mol% respectively), were employed to facilitate intracellular delivery of membrane- 50 51 impermeable trehalose. The intracellular trehalose concentration and haemolytic activity were 52 53 shown to increase with increasing the degree of grafting with NDA side chains (Figure 2). The 54 55 56 intracellular trehalose concentration of 177.9 ± 8.6 mM induced by PLP-NDA18 was 3.9 times 57 58 59 60 ACS Paragon Plus Environment 13 ACS Applied Materials & Interfaces Page 14 of 37

1 2 3 more efficient than PLP-NDA3 after 15 min of incubation. This is attributed to our previous 4 5 6 finding that increasing the degree of grafting with NDA side chains can significantly enhance 7 8 polymer hydrophobicity, leading to considerably enhanced polymer-membrane interaction and 9 10 intracellular delivery.31 PLP-NDA18 was the most favourable for intracellular trehalose delivery, 11 12 13 with high delivery efficiency but only marginally increased haemolysis. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Figure 2. Impact of degree of grafting with NDA on trehalose loading and relative haemolysis. 33 34 Erythrocytes were incubated with the PBS buffer solution containing 360 mM trehalose and 0.8 35 36 mg mL-1 pseudopeptide for 15 min. Temperature = 37°C; pH = 6.1. Mean ± S.D. (n = 3). 37 38 39 40 41 42 3.2. Characterisation of PLP-NDA18 mediated trehalose loading 43 44 The amount of membrane-impermeable trehalose delivered intracellularly by the comb-like 45 46 pseudopeptides is a function of parameters such as polymer concentration, extracellular pH, 47 48 49 incubation temperature, and extracellular trehalose concentration. These were investigated to 50 51 optimise intracellular trehalose loading. 52 53 3.2.1. Impact of polymer concentration 54 55 56 57 58 59 60 ACS Paragon Plus Environment 14 Page 15 of 37 ACS Applied Materials & Interfaces

1 2 3 The intracellular trehalose concentration and relative haemolysis increased with increasing the 4

5 -1 6 concentration of PLP-NDA18 within the range of 0.3–0.8 mg mL at pH 7.05 (Figure 3A). The 7 8 intracellular trehalose concentration (129.5 ± 15.6 mM) achieved after 15 min of incubation with 9 10 0.8 mg mL-1 PLP-NDA18 at pH 7.05 was 1.4 times lower compared to the treatment at pH 6.1. 11 12 13 Figures S4 and S5 show that intracellular trehalose loading was enhanced with extending the 14 15 incubation time to 30 and 60 min respectively, but with concomitant increase in haemolysis. No 16 17 significant enhancement of intracellular trehalose concentration (P > 0.5) was observed with 18 19 further increasing the polymer concentration from 0.8 to 1.2 mg mL-1 (Figure 3A), indicating that 20 21 22 maximum permeability to trehalose was induced by PLP-NDA18. The concentration-dependent 23 24 trehalose loading profile is in good agreement with the reports that increasing polymer 25 26 concentration can enhance the driving force for membrane binding, leading to enhanced membrane 27 28 24,25,41 29 permeability. 30 31 3.2.2. Impact of extracellular pH 32 33 As shown in Figure 3B, significant intracellular trehalose loading was observed at pH 7.05 and 34 35 36 further enhanced with decreasing pH to 5.6 for the incubation of erythrocytes with all the three 37 38 concentrations (0.6, 0.8 and 1.2 mg mL-1) of PLP-NDA18 for 15 min. The most significant 39 40 enhancement in intracellular trehalose loading was observed upon reduction of pH from 6.5 to 6.1 41 42 43 where maximum intracellular treahlose concertation was reached. This finding is attributed to 44 45 enhanced polymer hydrophobicity resulting from side-chain modification with hydrophobic 46 47 relatively long NDA. With decreasing pH, carboxyl groups pendant to the pseudopeptides were 48 49 protonated and the hydrophobic interaction became predominant, resulting in a transition of 50 51 52 polymer conformation from expanded coils to collapsed globular structures with considerably 53 54 enhanced membrane interaction.24,25,31 55 56 57 58 59 60 ACS Paragon Plus Environment 15 ACS Applied Materials & Interfaces Page 16 of 37

1 2 3 It is worth pointing out that relative haemolysis of the ovine erythrocytes treated with trehalose 4

5 -1 6 in the presence of 0.6, 0.8 and 1.2 mg mL PLP-NDA18 within the pH range 5.6−7.05 for 15 min 7 8 were all below 20%, with negligible changes in response to pH (Figure S6). In contrast, in our 9 10 previous haemolysis model using erythrocyte membranes as a model for endosomal membranes, 11

12 -1 13 ovine erythrocytes treated with 0.5 mg mL PLP-NDA18 at pH 5.5 displayed a considerably higher 14 15 level of haemolysis at 72.8 ± 1.8% after 10 min and reached nearly complete haemolysis after 20 16 17 min through pore formation.31 This huge difference in haemolytic activity was attributed to 18 19 different polymer-to-cell ratios. The polymer-to-cell ratio in this trehalose-loading study was 20 21 22 carefully optimized to be over 40 times lower than the one used in our previous haemolysis model 23 24 for the study of endosomolytic activity. Use of the tremendously lower polymer-to-cell ratio 25 26 (readily achievable by increasing the cell density at a fixed polymer concentration) ensured 27 28 29 marginal haemolysis but still yielded favorable membrane permeabilization to allow very rapid 30 31 and efficient trehalose loading into ovine erythrocytes. This successfully addressed the major 32 33 challenge on delivery of membrane-impermeable molecules into erythrocytes lacking transduction 34 35 21 36 or endocytosis pathways without causing significant cell . Other researchers have reported 37 38 a similar observation that the content leakage from liposomes caused by melittin was reduced with 39 40 decreasing peptide-to-lipid ratio.42,43 41 42 43 3.2.3. Impact of incubation temperature 44 45 Figure 3C shows that intracellular trehalose loading and relative haemolysis were dependent 46 47 on incubation temperature. After 15 min of incubation in a water bath at 31°C, a low intracellular 48 49 trehalose concentration of 27.0 ± 5.2 mM was detected. Once increasing incubation temperature 50 51 52 to 37°C, the intracellular trehalose concentration increased by a factor of 6.5. This is likely due to 53 54 the fact that the phase behaviour and physical properties of erythrocyte membrane lipids are 55 56 57 58 59 60 ACS Paragon Plus Environment 16 Page 17 of 37 ACS Applied Materials & Interfaces

1 2 3 extremely sensitive to temperature changes.44 At physiological temperature (37°C), the cell 4 5 6 membrane is more fluid, which may contribute to the enhanced polymer-lipid interaction and the 7 8 consequent increased intracellular delivery.21,45 In addition, intracellular loading could be further 9 10 improved by the higher diffusion rate of molecules at higher temperature.21 However, increasing 11 12 13 incubation temperature from 37°C to 39°C reduced trehalose loading, which might be due to the 14 15 reduced polymer-membrane interaction caused by the formation of lipid devoid domains on the 16 17 surface once exceeding the physiological temperature.46 18 19 3.2.4. Impact of extracellular trehalose concentration 20 21 22 A higher extracellular trehalose concentration has been reported to cause a higher osmotic 23 24 imbalance across the cell membrane, which as a result can create a higher driving force to achieve 25 26 improved intracellular delivery.45 Figure 3D depicts the impact of extracellular trehalose 27 28 -1 29 concentration on intracellular trehalose loading. In the presence of 0.8 mg mL PLP-NDA18 at pH 30 31 6.1 for 60 min, intracellular trehalose loading was enhanced dramatically by a factor of 18.3 from 32 33 16.4 ± 3.2 to 300.3 ± 23.2 mM with increasing the external trehalose concentration from 50 to 360 34 35 36 mM, respectively. In contrast, Lynch et al. has reported a 2.4 times lower intracellular trehalose 37 38 concentration after 9 times longer incubation of ovine erythrocytes with PP50 pendanted with L- 39 40 phenylalanine and 360 mM extracellular trehalose.21 It is also interesting to note that the relative 41 42 43 haemolysis after 60 min of co-incubation of erythrocytes with PLP-NDA18 and trehalose within 44 45 the concentration range tested was kept below approximately 30% with limited variations. As 46 47 comparison, relative haemolysis in the presence of PP50 was decreased with increasing 48 49 extracellular trehalose concentration.21 It has been reported that PP50 and hydrophilic dextran 50 51 52 (polysaccharide of glucose) were present in a mixture with no obvious formation of complexes, 53 54 suggesting limited interaction between the two components.47 Similarly, PP50 would have limited 55 56 57 58 59 60 ACS Paragon Plus Environment 17 ACS Applied Materials & Interfaces Page 18 of 37

1 2 3 interaction with hydrophilic trehalose (disaccharide of glucose), but it was argued that surrounding 4 5 6 of PP50 by high concentrations of trehalose could inhibit its conformational change to hydrophobic 7 8 compact polymer structures at mildly acidic pH, leading to inhibition of its membrane interaction 9 10 and consequent reduction of trehalose loading and haemolysis.21 Other researchers have reported 11 12 13 similar observations on inhibition of peptide folding by high concentrations of trehalose, which 14 15 can thickly cluster around constituent amino acids and shield them from each other.48 As 16 17 comparison, such shielding effect of hydrophilic trehalose on the comb-like pseudopeptide, PLP- 18 19 NDA18, was diminished likely due to enhanced polymer hydrophobicity and its comb-like 20 21 22 structure containing hydrophobic relatively long NDA side chains. Therefore, PLP-NDA18 well 23 24 retained its membrane-permeabilizing activity in the presence of high concentrations of trehalose, 25 26 maintaining very rapid and efficient intracellular trehalose loading. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 18 Page 19 of 37 ACS Applied Materials & Interfaces

1 2 3 Figure 3. Optimization of PLP-NDA18 mediated intracellular trehalose loading. (A) Impact of 4 5 6 PLP-NDA18 concentration on intracellular trehalose concentration and relative haemolysis. 7 8 Erythrocytes were incubated with the PBS buffer containing 360 mM trehalose and different 9 10 concentrations of PLP-NDA18 for 15 min. Temperature = 37°C, pH = 7.05. (B) Impact of 11 12 13 extracellular pH on trehalose loading. Erythrocytes were incubated with the PBS buffer containing 14 15 360 mM trehalose and 0.6, 0.8 and 1.2 mg mL-1 PLP-NDA18 at various pHs at 37°C for 15 min. 16 17 (C) Impact of incubation temperature on trehalose loading and relative haemolysis. Immediately 18 19 after erythrocytes were suspended in the PBS buffer (pH 6.1) containing 360 mM trehalose and 20 21 -1 22 0.8 mg mL PLP-NDA18, the sample was quickly immersed in a temperature-controlled water 23 24 bath for 15 min at different incubation . (D) Impact of extracellular trehalose 25 26 concentration on intracellular trehalose delivery and relative haemolysis. Erythrocytes were 27 28 -1 29 incubated with the PBS buffer containing different concentrations of trehalose and 0.8 mg mL 30 31 PLP-NDA18 for 60 min. Temperature = 37°C, pH = 6.1. Mean ± S.D. (n = 3). 32 33 34 35 36 3.3. Intracellular trehalose delivery efficiency and kinetics 37 38 39 Figure 4 shows very rapid and efficient intracellular trehalose loading facilitated by 0.8 mg 40 41 mL-1 PLP-NDA18 at pH 6.1 and 37°C. Upon incubation of erythrocytes with 360 mM trehalose 42 43 alone for 15 min, the intracellular concertation (0.9 ± 0.1 mM) of the membrane-impermeable, 44 45 46 hydrophilic compound was extremely low and the calculated delivery efficiency was only 0.25 ± 47 48 0.0%. As comparison, 76.5 ± 6.2 mM trehalose (delivery efficiency 21.3 ± 1.7%) was very rapidly 49 50 loaded into erythrocytes by PLP-NDA18 after incubation for only 5 min. Intracellular trehalose 51 52 53 loading and relative haemolysis increased with increasing incubation time. When the incubation 54 55 time was extended from 15 to 60 min, the intracellular trehalose concentration achieved by PLP- 56 57 58 59 60 ACS Paragon Plus Environment 19 ACS Applied Materials & Interfaces Page 20 of 37

1 2 3 NDA18 increased from 177.9 ± 8.6 mM (delivery efficiency 49.4 ± 2.4%) to 300.3 ± 23.2 mM 4 5 6 (delivery efficiency 83.4 ± 6.4%), with the latter 334 times higher than that in the absence of PLP- 7 8 NDA18. 9 10 PP50 pendanted with L-phenylalanine was previously reported to enable enhanced intracellular 11

12 21,30,33 13 trehalose loading for effective cell cryopreservation. However, it is a considerably less 14 15 efficient trehalose loading process with a considerably lower loading rate compared to the PLP- 16 17 NDA18 mediated trehalose loading. It has been previously reported that only approximately 10 18 19 mM trehalose (delivery efficiency around 2.8%) was loaded into erythrocytes after 1.5 h of co- 20 21 21 22 incubation with PP50 , which was 30-fold less efficient than trehalose loading by the comb-like 23 24 PLP-NDA18 pendanted with hydrophobic relatively long alkyl chains after 1 h of treatment. In 25 26 order to increase the intracellular trehalose concentration to thresholds (100-200 mM) required for 27 28 15,36 29 cellular bioprotection during preservation , 9 h of treatment was usually required for PP50 to 30 31 result in an intracellular trehalose concentration at 123 ± 16 mM (delivery efficiency 34.2%).21 As 32 33 comparison, 177.9 ± 8.6 mM trehalose (delivery efficiency 49.4 ± 2.4%) was loaded into 34 35 36 erythrocytes by PLP-NDA18 after only 15 min of treatment (Figure 4), suggesting that its 37 38 intracellular delivery rate was approximately 36 times higher than PP50. Therefore, for effective 39 40 cryopreservation in this work, a short co-incubation of ovine erythrocytes with PLP-NDA18 and 41 42 43 trehalose for no more than 15 min was chosen and haemolysis was below 16.1 ± 0.7%. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 20 Page 21 of 37 ACS Applied Materials & Interfaces

1 2 3 Figure 4. (A) Time-dependent intracellular trehalose loading and relative haemolysis mediated by 4 5 6 PLP-NDA18. (B) Comparison of delivery efficiency. Erythrocytes were treated with the PBS 7 8 containing 360 mM trehalose in the absence or presence of 0.8 mg mL-1 PLP-NDA18 for specific 9 10 time periods. Temperature = 37°C; pH = 6.1. Mean ± S.D. (n = 3). 11 12 13 14 15 16 3.4. Reversibility and mechanism of membrane permeabilization 17 18 The binary mixture of trehalose and calcein, which are both membrane-impermeable, was used 19 20 to compare the intracellular delivery by the comb-like PLP-NDA18 polymer pendanted with 10- 21 22 23 carbon NDA chains and the grafted PP50 polymer pendanted with L-phenylalanine. As shown in 24 25 Figure 5A, after incubation at pH 7.4 for 15 min, no green calcein fluorescence was observed in 26 27 erythrocytes with or without polymer treatment, indicating that at physiological pH neither PLP- 28 29 30 NDA18 nor PP50 was membrane permeabilizing. After incubation at pH 6.1 for 15 min, strong 31 32 homogenous green fluorescence was only observed in the erythrocytes treated with PLP-NDA18, 33 34 whilst there was no detectable intracellular green fluorescence for the PP50-treated erythrocyte 35 36 sample. This consolidates our finding that PLP-NDA18 can mediate considerably more rapid and 37 38 39 more efficient intracellular delivery than PP50. 40 41 However, after erythrocytes were pre-treated with trehalose and PLP-NDA18 at pH 6.1 for 15 42 43 min and then washed with pH 7.4 PBS, the subsequent incubation of erythrocytes with calcein 44 45 46 alone did not result in detectable intracellular green fluorescence, which was indistinguishable 47 48 from the erythrocyte sample with pre-treatment in the absence of PLP-ND18 (Figure 5B). This 49 50 indicates that washing with pH 7.4 PBS as a quenching buffer quickly reversed the cell membrane 51 52 53 permeability and PLP-NDA18 did not cause permanent alteration of the membrane integrity. 54 55 Accordingly, the loaded trehalose became locked inside erythrocytes after the immediate post- 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Applied Materials & Interfaces Page 22 of 37

1 2 3 loading washing step (Figure 5A). This is because washing in the PBS buffer at physiological pH 4 5 6 can not only act to physically remove PLP-NDA18 from erythrocytes but also switch off its 7 8 membrane permeabilizing activity. Similar observations have been reported on the reversible 9 10 membrane permeability achieved by amphiphilic peptides43 and the PP50 polymer21. 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Figure 5. (A) Confocal microscopy images of polymer-mediated delivery into erythrocytes. 28 29 Erythrocytes were co-incubated with 360 mM trehalose and 1 μM calcein in the absence (control) 30 31 or presence of 0.8 mg mL-1 polymer (PP50 or PLP-NDA18) for 15 min; pH = 7.4 or 6.1; 32 33 34 temperature = 37°C. Erythrocytes were washed with pH 7.4 PBS before imaging. Scale bar: 2 μm. 35 36 (B) Investigation of reversibility of membrane permeability. Erythrocytes were incubated with 360 37 38 mM trehalose in the absence (control) or presence of 0.8 mg mL-1 PLP-NDA18 at pH 6.1 for 15 39 40 min, followed by washing with pH 7.4 PBS buffer and then treatment with pH 7.4 PBS containing 41 42 43 1 μM calcein before imaging. Scale bar: 4 μm. 44 45 46 47 48 AFM was applied to visualize the polymer-membrane interaction at mildly acidic pH. Figures 49 50 51 6A and S7 show surface morphologies of the erythrocyte membrane treated with PBS alone, PP50 52 53 and PLP-NDA18 at pH 6.1, respectively. Obvious membrane deformation was observed in the 54 55 erythrocytes treated with PP50 or PLP-NDA18 compared to the control. The line profiles (Figure 56 57 58 59 60 ACS Paragon Plus Environment 22 Page 23 of 37 ACS Applied Materials & Interfaces

1 2 3 6B) suggest that both PP50 and PLP-NDA18 created depressions on the erythrocyte surface. 4 5 6 Instead of gradually and uniformly thinning the lipid membrane, distinct domains with 7 8 significantly increased curvatures were formed by both amphiphilic pseudopeptides. It has been 9 10 reported that some proteins, such as epsin, endophilin and amphiphysin, can also induce a similar 11 12 13 membrane curvature change through the insertion of their amphiphilic domains into the membrane 14 15 bilayer and the reduced hydrophobicity of amino acids in such domains may impair their ability to 16 17 produce membrane curvature.49 The inserted hydrophobic protein motifs in the outer part of the 18 19 lipid membrane can serve as a “wedge”, perturbing the lipid packing and thus causing membrane 20 21 50 22 bending. In this paper, PLP-NDA18 containing relatively long alkyl side chains caused the 23 24 deeper and sharper erythrocyte surface depression (Figure 6B) and a consequent 2-fold increase 25 26 in the surface roughness as compared to PP50 with phenylalanine side chains (Figure 6C). It is 27 28 29 consistent with a report that alkyl chains play a more important role in binding of peptides with 30 31 the lipid membrane than phenylalanine.51 The stronger membrane binding and insertion of PLP- 32 33 NDA18 produced the more pronounced membrane curvature. This is in good agreement with the 34 35 36 observations reported by other researchers that several insertions into the lipid membrane held in 37 38 close proximity may result in high membrane curvatures.50,52 As a consequence, stronger 39 40 membrane stresses can be generated by higher curvatures, leading to more significant cell 41

42 50,53 43 deformation and the subsequently higher membrane permeability for improved intracellular 44 45 delivery54,55. In addition, compared to PP50, the more pronounced membrane curvature induced by 46 47 PLP-NDA18 might also result in a higher number of membrane-thinning domains, which was 48 49 demonstrated to play a key role in PP50 mediated intracellular delivery.30 Therefore, all these 50 51 52 could contribute to the tremendously more rapid and efficient intracellular trehalose delivery of 53 54 PLP-NDA18 than PP50. Compared with other membrane stress based intracellular delivery 55 56 57 58 59 60 ACS Paragon Plus Environment 23 ACS Applied Materials & Interfaces Page 24 of 37

1 2 3 technologies which rely on mechanical perturbations or shear forces provided by specifically 4

5 6 6 designed equipment , the comb-like pseudopeptides can enable efficient, controllable, cost- 7 8 effective and readily scalable intracellular delivery. Also importantly, the PLP-NDA18 induced 9 10 membrane bending and destabilization is transient and reversible (Figure 5), thus eliminating 11 12 13 safety concerns. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 6. (A) Topographic AFM micrographs and (B) line profiles of the erythrocyte membrane 46 47 -1 48 surface treated with PBS alone (control), 0.8 mg mL pseudopeptide pendanted with L- 49 50 phenylalanine (PP50), or 0.8 mg mL-1 pseudopeptide pendanted with NDA alkyl chains (PLP- 51 52 NDA18) at pH 6.1 for 15 min, temperature = 37°C. (C) Average roughness (Ra) and root mean 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 24 Page 25 of 37 ACS Applied Materials & Interfaces

1 2 3 squared roughness (RRMS) of the erythrocyte membrane surface treated with the same conditions, 4 5 6 as determined by AFM. 7 8 9 10 11 3.5. Cryopreservation of erythrocytes 12 13 A short co-incubation of ovine erythrocytes with 0.8 mg mL-1 PLP-NDA18 and 360 mM 14 15 trehalose at pH 6.1 and 37oC for only 15 min efficiently resulted in the intracellular trehalose at 16 17 18 177.9 ± 8.6 mM, reaching thresholds (100-200 mM) required for cellular bioprotection during 19 20 preservation15,36, but yielded the low haemolysis of 16.1 ± 0.7%. This has prompted an evaluation 21 22 of the trehalose method for cryopreservation of ovine erythrocytes. 23 24 25 In order to examine the effect of trehalose present both inside and outside erythrocytes on 26 27 cryoprotection, the post freeze-thaw survival rate of erythrocytes co-incubated with trehalose and 28 29 PLP-NDA18 was compared to the cryosurvival rate of erythrocytes incubated with trehalose alone. 30 31 32 Fast freezing and thawing rates have been found to be favourable for cryoprotection of erythrocytes 33 34 employing trehalose and other carbohydrates.21,56 Herein, freezing through direct immersion in 35 36 (cooling rate: 300°C min-1) followed by thawing in a water bath at 37oC (thawing 37 38 rate: 20°C min-1) was chosen as the cryopreservation protocol. 39 40 41 Figure S8 shows that cryosurvival of erythrocytes suspended in PBS alone was negligible. As 42 43 seen in Figure 7, in the presence of extracellular trehalose alone, 69.4 ± 0.4% erythrocytes 44 45 survived freeze-thaw after 1 h of cryostorage in liquid nitrogen. This is consistent with a previous 46 47 48 report that trehalose not only inhibited the formation of extracellular through 49 50 but also inhibited the formation of intracellular ice crystals via partial dehydration of 51 52 cells before freezing.57 As comparison, the cryosurvival rate of erythrocytes with both extracellular 53 54 55 and intracellular trehalose increased significantly (P < 0.001) to 90.3 ± 0.7% after cryostorage for 56 57 58 59 60 ACS Paragon Plus Environment 25 ACS Applied Materials & Interfaces Page 26 of 37

1 2 3 1 h. Almost no variation was observed in the cryosurvival rate of erythrocytes after extended 4 5 6 cryostorage in liquid nitrogen to 7 days. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Figure 7. Time-dependent cryosurvival rates of erythrocytes suspended in only 360 mM 26 27 extracellular trehalose solution yielding no significant intracellular trehalose loading, and 28 29 erythrocytes loaded with 177.9 ± 8.6 mM intracellular trehalose and suspended in 360 mM 30 31 32 extracellular trehalose. Trehalose loading was achieved by incubation of erythrocytes with 360 33 34 mM trehalose in the absence or presence of 0.8 mg mL-1 PLP-NDA18 at pH 6.1 and 37oC for 15 35 36 min. Mean ± S.D. (n = 3), ***P < 0.001. 37 38 39 40 41 Non-toxic trehalose is a more attractive bioprotectant for cryopreservation of erythrocytes 42 43 44 compared to the widely used glycerol method which requires deglycerolization after thawing to 45 46 avoid post-transfusion osmotic haemolysis.10-12,58 However, it is notably challenging to deliver 47 48 membrane-impermeable hydrophilic trehalose into mammalian cells, especially erythrocytes 49 50 21 51 lacking transduction or endocytosis pathways, in an efficient and non-invasive way. This remains 52 53 a major hurdle to utilizing trehalose as an alternative cryoprotective agent. This paper reports the 54 55 first-known cryopreservation of erythrocytes based on very rapid and efficient trehalose loading 56 57 58 59 60 ACS Paragon Plus Environment 26 Page 27 of 37 ACS Applied Materials & Interfaces

1 2 3 by pH-responsive, comb-like pseudopeptides. Intracellular trehalose threshold concentrations 4 5 6 (100–200 mM) can be achieved within no more than 15 min through reversible membrane 7 8 permeability to enable the enhanced erythrocyte cryopreservation. Further investigation into post- 9 10 thawing cell recovery, including cell morphology, deformability, osmotic fragility, protein and 11 12 13 lipid oxidation, enzymatic activities and shelf-life, remains to be carried out. This trehalose method 14 15 also has potential for preservation of nucleated cells, which currently relies on the use of toxic 16 17 dimethyl sulfoxide (DMSO) at high concentrations. Furthermore, the comb-like pseudopeptides 18 19 also have potential for intracellular delivery of other membrane-impermeable, hydrophilic 20 21 22 molecules. 23 24 25 26 4. Conclusion 27 28 29 A very rapid and efficient intracellular delivery method was developed based on reversible cell 30 31 membrane permeabilization via utilization of the pH-responsive, comb-like pseudopeptides 32 33 pendanted with relatively long alkyl chains acting as membrane anchors. The intracellular delivery 34 35 36 of membrane-impermeable trehalose was dependent on the side chain length, degree of 37 38 substitution and concentration of the polymers, the pH, temperature and time of incubation, as well 39 40 as the polymer-to-cell ratio and the concentration of extracellular trehalose. Under optimized 41 42 43 conditions, PLP-NDA18 loaded 177.9 ± 8.6 mM trehalose into erythrocytes, which resulted in 44 45 90.3 ± 0.7% erythrocyte survival after freeze-thaw, with an improvement of 20.9% in cryosurvival 46 47 over the erythrocytes suspended in only extracellular trehalose. Compare to the previously reported 48 49 PP50 pendanted with L-phenylalanine, the comb-like PLP-NDA18 tremendously shortened the 50 51 52 loading time from 9 h to 15 min, whilst achieved the higher intracellular trehalose concentration 53 54 and better erythrocyte cryosurvival. The more rapid and efficient intracellular delivery of PLP- 55 56 57 58 59 60 ACS Paragon Plus Environment 27 ACS Applied Materials & Interfaces Page 28 of 37

1 2 3 NDA18 was attributed to the more pronounced membrane curvature generated by the stronger 4 5 6 membrane insertion of the comb-like polymer with relatively long alkyl side chains. This new 7 8 method not only opens an avenue for effective cryopreservation of erythrocytes and potentially 9 10 other mammalian cells, but also provide a foundation for efficient intracellular delivery of other 11 12 13 membrane-impermeable molecules. 14 15 16 17 ASSOCIATED CONTENT 18 19 Supporting Information 20 21 1 22 H-NMR and FT-IR spectra of PLP, PLP-HDA18, PLP-NDA18, PLP-TDA18 and PLP-ODA18, 23 24 linear correlation curve between the erythrocyte concentration and haemoglobin absorbance at 541 25 26 nm, impact of PLP-NDA18 concentrations on trehalose loading and relative haemolysis after 27 28 29 treatment of erythrocytes for 30 and 60 min, pH-dependent relative haemolysis of erythrocytes 30 31 treated with trehalose and PLP-NDA18, topography, tapping amplitude and tapping phase images 32 33 of the erythrocyte membrane surface treated with PBS, PP50 or PLP-NDA18 at pH 6.1 for 15 min, 34 35 36 as well as time-dependent cryosurvival rates of erythrocytes suspended in PBS. 37 38 39 40 AUTHOR INFORMATION 41 42 Corresponding Author 43 44 45 * Tel: +44 (0)20 75942070. Fax: +44 (0)20 75945638. Email: [email protected] (R.C.) 46 47 Author Contributions 48 49 ‡S.C. and L. W. contributed equally. 50 51 All authors have read, commented on, and approved the final version of the paper. 52 53 54 Notes 55 56 A UK patent application (No. 1612150.1) related to poly (L-lysine isophthalamide) (PLP) polymers 57 58 59 60 ACS Paragon Plus Environment 28 Page 29 of 37 ACS Applied Materials & Interfaces

1 2 3 with hydrophobic pendant chains has been filed and led to a PCT application (No. WO 4 5 6 2018/011580). R.C., S.C. and L.W. are listed as inventors and declare financial interest with 7 8 AlphaCells Biotechnologies Limited, a company that may be affected by the research reported in 9 10 the enclosed paper. All other authors declare no competing interests. 11 12 13 14 15 ACKNOWLEDGMENTS 16 17 18 This work was mainly supported by the funding from the Department of Chemical Engineering 19 20 21 at Imperial College London. The Department of Chemical Engineering at Imperial College London 22 23 also provided a full Ph.D. Scholarship to S.C. and Burkett Scholarships to L.W. and J.R. In 24 25 addition, the China Scholarship Council provided J.R. a full Ph.D. Scholarship and 26 27 28 GlaxoSmithKline plc provided the funding to perform erythrocyte cryopreservation. 29 30 31 32 REFERENCES 33 34 35 (1) Yoo, J. W.; Irvine, D. J.; Discher, D. E.; Mitragotri, S. Bio-Inspired, Bioengineered and 36 37 Biomimetic Drug Delivery Carriers. Nat. Rev. Drug Discov. 2011, 10 (7), 521–535. 38 39 40 (2) Mitragotri, S.; Burke, P. A.; Langer, R. Overcoming the Challenges in Administering 41 42 Biopharmaceuticals: Formulation and Delivery Strategies. Nat. Rev. Drug Discov. 2014, 13 43 44 45 (9), 655–672. 46 47 48 (3) Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. Non- 49 50 Viral Vectors for Gene-Based Therapy. Nat. Rev. Genet. 2014, 15 (8), 541–555. 51 52 53 54 (4) Lu, Y.; Sun, W.; Gu, Z. Stimuli-Responsive Nanomaterials for Therapeutic Protein 55 56 Delivery. J. Control. Release 2014, 194, 1–19. 57 58 59 60 ACS Paragon Plus Environment 29 ACS Applied Materials & Interfaces Page 30 of 37

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1 2 3 Table of Contents Graphic 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 37