Multifunctional Poly(amine-co-ester-co-orthoester) for Efficient and

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Multifunctional Poly(amine-co-ester-co-ortho ester) for Efficient and Safe Gene Delivery Junwei Zhang, Jiajia Cui, Yang Deng, Zhaozhong Jiang, and W. Mark Saltzman ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00502 • Publication Date (Web): 26 Sep 2016 Downloaded from http://pubs.acs.org on October 1, 2016

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ACS Biomaterials Science & Engineering

Multifunctional Poly(amine-co-ester-co-ortho ester) for Efficient and Safe Gene Delivery

Junwei Zhang 1, Jiajia Cui 2, Yang Deng 2, Zhaozhong Jiang 3* and W. Mark Saltzman 1,2* 1

Department of Chemical and Environmental Engineering, Yale University, 55 Prospect Street, New Haven, CT 06511, USA

2

Department of Biomedical Engineering, Yale University, 55 Prospect Street, New Haven, CT 06511, USA

3

Department of Biomedical Engineering, Molecular Innovations Center, Yale University, 600 West Campus Drive, West Haven, Connecticut 06516, USA.

*

Corresponding authors.

W. Mark Saltzman: Tel.: 203-432-4262; fax: 203-432-0030; email: [email protected]. Zhaozhong Jiang: Tel.: 203-737-3262; fax: 203-737-3289; email: [email protected].

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Abstract Cationic polymers are used for non-viral gene delivery, but current materials lack the functionality to address the multiple barriers involved in gene delivery. Here we describe the rational design and synthesis of a new family of quaterpolymers with unprecedented multifunctionality:

acid

sensitivity,

low

cationic

charge,

high

hydrophobicity,

and

biodegradability, all of which are essential for efficient and safe gene delivery. The polymers were synthesized via lipase-catalyzed polymerization of ortho ester diester, lactone, dialkyl diester, and amino diol monomers. Polymers containing ortho ester groups exhibited acidsensitive degradation at endosomal pH (4~5), facilitated efficient endosomal escape and unpackaging of the genes, and were efficient in delivering genetic materials to HEK293 cells, human glioma cells, primary mouse melanoma cells, and human umbilical vein endothelial cells (HUVECs). We also developed a highly efficient lyophilized formulation of the nanoparticles, which could be stored for a month without loss of efficiency. Key words Non-viral gene delivery, multifunctional polymer, ortho ester, lipase catalysis, DNA, siRNA


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1.

Introduction

Gene therapy with DNA is a promising approach for the treatment of cancer and other diseases 12

. However, its success is dependent on the development of efficient gene delivery vectors, since

unprotected DNA will be degraded by endogenous nucleases and has low cell uptake efficiency. An efficient DNA delivery vector must be multifunctional so that it can address the multiple barriers for gene delivery, such as gene packaging, transport through cell membrane, endosomal escape, and gene unpackaging 3. Viral vectors are highly efficient because they have evolved multiple functions to address these barriers, such as cell uptake, endosomal escape, and nuclear targeting, but they suffer from serious safety issues

3-4

. Two groups of non-viral vectors are

widely studied: cationic lipids and cationic polymers 5-8. Cationic lipids suffer from difficulties in fabrication and modification, which prevent the incorporation of versatile functionalities. In contrast, synthetic cationic polymers are promising because of their versatile chemistry. Many cationic polymers for gene transfection have been reported, such as polyethylenimine (PEI) and poly (ß-amino ester) (PBAE)

3, 9-10

. Recently, a family of poly (amine-co-ester) (PACE)

terpolymers was synthesized through lipase catalysis, which featured low cationic density and high hydrophobicity

11-12

. These polymers are among the most efficient and safe gene vectors

ever reported. However, there is still room for improvement. For example, it is potentially helpful to enhance the escape of the loaded genes from the acidic endolysosomes, which is a major limiting step for gene delivery. This is especially important for PACE because it has low nitrogen content and thus a lesser ability to take advantage of the proton sponge effect

13

. The

unpackaging of genes from PACE might also be enhanced: while high hydrophobicity can contribute to polyplex stability, it can also diminish the unpackaging of genes in cytosol and lower the transfection efficiency 3, 8. 3 ACS Paragon Plus Environment


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One way to enhance both endosomal escape and gene unpackaging is to introduce acid-sensitive groups in the polymer backbone. Once taken up by cells through endocytosis, acid-triggered breakdown of polymers in the endosome could produce intra-endosomal osmotic swelling that could rupture the endosomes and lead to escape of plasmids 14-18. The unpackaging of genes from the nanoparticles might also be enhanced after the degradation of acid-sensitive polymer, making the genes accessible for expression 14, 16, 19. Ortho ester is an ideal group to provide acid-sensitivity, because it exhibits pH-dependent hydrolysis. While it is stable in neutral and basic environments, it undergoes rapid hydrolysis in slightly acidic environment. Compared to other pH-sensitive groups, such as ester ketal

22

, and vinyl ether

23

20

, acetal

21

,

, the ortho ester bond hydrolyzes more quickly in response to mildly

acidic conditions 24. Poly (ortho esters) (POEs) were developed in the 1970s widely studied for drug and gene delivery

25-26

25-26

and have been

. They have been used to fabricate solid drug

release device, injectable gel-like materials, and micelles that could encapsulate therapeutics for many diseases, such as cancer and post-operative pain. Several of these products have been studied in clinical trials 27. POEs have also been used for gene delivery. For example, a group of cationic POEs was prepared via the copolymerization of diol, amino-diol, and diketene acetal 28. Microspheres were prepared with these materials as vectors for DNA delivery, which had low toxicity and could release genes in response to acid. However, these particles are huge (>5um), and their chemistry does not give enough flexibility to allow the fine tuning of the physicochemical properties of the polymer. In another studies, poly(ortho ester amidine) copolymers were prepared in multiple steps 29. The polymers exhibited pH-dependent hydrolysis and DNA release. However, these materials were toxic and exhibited low transfection efficiency.


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Because of the high acid sensitivity and biocompatibility of ortho ester, we chose to incorporate ortho ester groups into the main chain of PACE molecules, to make them acid-sensitive. However, chemical synthesis to incorporate ortho ester into PACE polymer is challenging because distinctly different polymerization methods are required for poly (ortho ester) and PACE. PACE was synthesized with lipase catalysis; however, the use of ortho ester groups in lipase catalysis has never been reported. To the best of our knowledge, there are no polymerization methods currently available to readily incorporate ortho ester moieties into functional polyester chains with the desirable amine and lactone structures that are essential for efficient and safe gene delivery. In this paper, we report a novel lipase-catalyzed synthetic method that allows the preparation of a new family of poly (amine-co-ester-co-ortho ester) (oPACE) polymers. The oPACEs have unprecedented multi-functionality: acid sensitivity, low cationic charge, high hydrophobicity, and biodegradability. These polymers are acid-sensitive and efficiently facilitate endosomal escape and gene unpackaging. These polymers are also highly efficient and safe in transfecting genetic materials into different cells, including difficult-to-transfect primary cells. 2.

Materials and methods

2.1.

Materials

ω-pentadecalactone (PDL), diethyl sebacate (DES), N-methyldiethanolamine (MDEA), 3,9divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane (DTSU),

ethyl glycolate, ethyl 6-hydroxy

hexanoate and methyl 10-hydroxydecanoate were purchased from Sigma Aldrich Chemical Co. and were used as received. Immobilized Candida antarctica lipase B (CALB) supported on acrylic resin (Novozym 435), potassium t-butoxide, chloroform, dichloromethane, hexane, 5 ACS Paragon Plus Environment


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pentane, diphenyl ether, tetrahydrofuran, ethylene diamine, triethylamine and chloroform-d were also obtained from Sigma Aldrich Chemical Co. The lipase catalyst was dried at 50 °C under 2.0 mmHg for 20 h prior to use. Plasmid DNA (pGL4.13) encoding the firefly luciferase (pLuc) and Luciferase Assay Buffer were obtained from Promega Co. (Madison, WI). GFP reporter gene pSicoR-GFP (pGFP) was obtained from Addgene. 2.2.

Cell culture

Human embryonic kidney 293 (HEK293) cells and U87MG cells were obtained from American Type Culture Collection (Manassas, VA) and grown at 37°C under 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin. The primary mouse melanoma cells (2697T)

30

were provided by M.

Bosenberg (Yale University), and were incubated at 37°C under 5% CO2 atmosphere in DMEM/F12 supplemented with 5% FBS, 1% NEAA, and 1% penicillin-streptomycin. Human umbilical vein endothelial cells (HUVECs) were purchased from Yale University Vascular Biology and Therapeutics core facility and cultured in M199 supplemented with 20% FBS, P/S, L-glutamine,

2.3. 1

and ECGS.

Instrument methods

H and 13C NMR spectra were recorded on Bruker AVANCE spectrometer. The chemical shifts

reported were referenced to internal tetramethylsilane (0.00 ppm) and chloroform-d was used for all NMR measurement. The number and weight average molecular weights (Mn and Mw, respectively) of polymers were measured by gel permeation chromatography (GPC) using a Waters HPLC system equipped with a model 1515 isocratic pump, a 717 plus autosampler, and a 2414 refractive index (RI) detector. Empower II GPC software was used for running the GPC 6 ACS Paragon Plus Environment

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instrument and for calculations. Columns and the RI detector were heated and maintained at 40 °C temperature during sample analysis. Chloroform was used as the eluent at a flow rate of 1.0 mL/min. Sample concentrations of 2 mg/mL and injection volumes of 100 µL were used. Polymer molecular weights were determined based on a conventional calibration curve generated by narrow polydispersity polystyrene standards from Aldrich Chemical Co. The morphology of polyplexes, which were stained with uranyl acetate, was visualized using a Zeiss EM 900 transmission electron microscope (TEM). ImageJ was used to analyze the average size of polyplexes on TEM images. Flow cytometry analysis were performed on an Attune NxT Flow Cytometer with a blue (488 nm) and a red (638 nm) laser. 2.4.

Synthesis of ortho ester diester monomers

3,9-diethylidene-2,4,8,10-tetraoxaspiro [5.5] undecane (DETOSU) was prepared through the isomerization of DTSU according to previously described method 31 (Scheme 1A). Briefly, 18 g of DTSU was mixed with 20 g of potassium t-butoxide in 100 mL ethylene diamine. The mixture was heated to 100 °C under nitrogen for 16 hours. After that, the mixture was poured into 1000 mL of water, and the product was extracted with pentane and dried with K2CO3. The product was distilled under vacuum to remove the impurities. Following the synthesis of DETOSU, ethyl glycolate (n=1), ethyl 6-hydroxyhexanoate (n=5), or methyl 10-hydroxydecanoate (n=9) was mixed with DETOSU at a 2:1 molar ratio and the reaction was carried out at room temperature in THF catalyzed by supported p-toluenesulfonic acid. After 4 hours, the catalyst was removed by filtration and the solvent was evaporated under vacuum. Three distinct ortho ester diesters (OEDEs) were obtained with quantitative yield, designated as OEDE (1), OEDE (5) and OEDE (9), respectively. All the structures were confirmed by 1H and 13C NMR.



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Scheme 1 Synthesis of (A) ortho ester diester (OEDE) monomers and (B) oPACE polymers by a twostage polymerization of OEDE (n) with PDL, DES, and MDEA. A

B

2.5.

Synthesis and purification of poly (amine-co-ester-co-ortho ester) (oPACE)

A reaction mixture containing PDL, DES, MDEA, OEDE (n=1, 5, 9), Novozym 435 catalyst (10wt% of total monomer), and diphenyl ether solvent (200 wt% versus total monomer) was prepared and purged with argon to remove air (Scheme 1B). The polymerization reactions were carried out at 90 °C in two stages: (1) in the first stage, the reaction mixtures were stirred under 1 atm of argon gas for 24 hours; (2) in the second stage, the reaction pressure was reduced to 1.6


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mmHg and the reactions were continued for a further 72 h. The polymer products were isolated and purified by precipitation in hexane to extract and remove the residual diphenyl ether solvent from the polymers. Subsequently, the products were dissolved in dichloromethane and filtered to remove catalyst particles. Evaporation of the CH2Cl2 solvent from the filtrates at 40 °C under high vacuum (1.0 mmHg) yielded the purified polymers. The control terpolymer, poly (amineco-ester) (PACE) was synthesized with a similar method using PDL, DES, and MDEA as monomers 11. 2.6.

Polymer degradation study

A weight loss assay was used to characterize the degradation of the polymers. oPACE and PACE were coated on to the bottom of glass vials, covered with 150 mM sodium acetate buffer of pH 4, pH 5, pH 6 and PBS buffer, and incubated at 37 °C for different time. After incubation, the buffer was removed carefully, and the polymers were dried. The weight and molecular weight of the residual polymer were measured. 2.7.

Polyplex preparation and characterization

To prepare DNA polyplexes at 100:1 polymer/DNA weight ratio, which was shown to be the optimized ratio in prior work 11, 4 µl of polymer solution (25 mg/ml in DMSO) was first diluted in 50 µl sodium acetate buffer (25 mM, pH = 5.6). After brief vortexing, the polymer solution was mixed with the same volume of a DNA solution in sodium acetate buffer containing 1 µg DNA and vortexed for additional 10 seconds. The polymer/DNA mixture was incubated at room temperature for 10 min before use. The size of the polyplexes was measured with dynamic laser scattering (DLS) on a Malvern Zetasizer. For the PicoGreen exclusion assay, polyplexes were prepared at different polymer/DNA weight ratio, while keeping the concentration of pDNA 9 ACS Paragon Plus Environment


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constant. All samples, as well as free pDNA solution, were analyzed with Quant-it PicoGreen dsDNA assay kit (Invitrogen). Since the PicoGreen can only bind to the free DNA, the reading reflects the concentration of the uncomplexed DNA. 2.8.

Characterization of endosomal escape efficiency of polyplexes

To measure the pH of the local environment for the DNA delivered intracellularly by different polymers, plasmid DNA (pGL4.13, or pLuc) was double-labelled with pH-sensitive FITC and pH-insensitive Cy5 using Label IT® Tracker™ Intracellular Nucleic Acid Localization Kits (Mirus Bio) 32. The day before the transfection, U87MG cells were seeded in 24-well plate at a density of 150,000 cells/well. Polyplexes were prepared with the double-labelled pDNA and PACE or oPACE9-20. Cells were treated with the polyplexes for 1 hour at 37 °C before the polyplexes were removed with PBS washing. After another hour, the cells from 7 wells were collected. Cells from 3 wells were suspended in PBS containing 2% BSA, while cells in another 4 wells were suspended in 4 intracellular pH clamping buffer (pH=4.5, 6.0, 6.8, 7.5). The cells in each vial were further washed by pelleting and resuspending in the corresponding buffer. The signal from 50,000 treated cells was analyzed according to a method previously reported 33. The fluorescence of FITC and Cy5 was excited at 488 nm and 638 nm respectively, and was detected at 530 and 670 nm, respectively. The FITC/Cy5 ratio was calculated from the median fluorescence of all the live cells. The ratio from the cells incubated with the 4 intracellular clamping buffer generated a standard curve, which was used to convert the FITC/Cy5 ratio of the other three samples into pH values. 2.9.

Acid-sensitivity of the oPACE polyplexes


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Polyplexes of PACE/DNA or oPACE/DNA were prepared at 100:1 weight ratio, and then incubated in 150 mM pH 5 sodium acetate buffer containing 5 mM EDTA at 37 °C for 4 hours. Then, the acid-treated polyplexes and fresh polyplexes were incubated with different concentrations of heparin at 37 °C for 15 mins. The amount of gene released at different heparin concentration was quantified with PicoGreen assay and normalized to the reading the same concentration of free DNA in same buffer. 2.10. In vitro transfection of DNA and siRNA For in vitro transfection, DNA polyplexes with polymer:DNA weight ratio of 100:1 were used unless otherwise noted. Cells were seeded in 24-well plates at density of 75,000 cells/well in 500 µl of medium one night before transfection. The growth medium was replaced with DMEM containing 10% FBS (without penicillin-streptomycin) and polyplexes containing 1ug of DNA was added to each well. Transfection using Lipofectamine 2000 (Invitrogen Corp.) was performed using the procedures provided by the manufacturer. The same amount of DNA was used for transfection with oPACE and Lipofectamine 2000. For luciferase gene transfection assay, pLuc plasmid was used to prepare the polyplexes. The transfection was allowed to take place for two days as done previously by others.32,34 Two days after transfection, the culture medium was removed and the cells were washed with cold PBS. Two hundred micro-liter Report Lysis Buffer (Promega) was added to each well. After a freezethaw cycle, cell lysate was collected. After a quick spin, 20 µl of cell lysate was mixed with Luciferase Assay Reagent. Luciferase expression in terms of relative light units (RLU) was measured with a GloMax® 20/20 Luminometer and normalized to the total amount of protein in the cell lysate. Total protein level was quantified using Pierce BCA protein assay kit (Pierce,



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Thermo Scientific). For GFP gene expression assay, pSicoR-GFP was used to prepare polyplexes. After 48 hours of incubation, the transfected cells in each well were harvested by trypsinization, washed with PBS and resuspended in PBS with 1% BSA. A total of 40,000 cells were analyzed with flow cytometry to detect GFP expressing cells. For siRNA knockdown, HEK293 cells stably transfected with luciferase gene (HEK293-Luc) were seeded in 24-well plates at a density of 75,000 cells/well in 500 µl of medium one night before transfection. siRNA (5'-GCUAUGAAGCGCUAUGGGCUU-3') was used to knockdown the expression of luciferase. Polyplexes of siRNA/polymer were prepared at 100:1 polymer/siRNA weight ratio with a similar method for the preparation of pDNA/polymer polyplexes. Transfection using Lipofectamine RNAiMAX (Invitrogen Corp.) was performed using the procedures provided by the manufacturer. The same concentration of siRNA (10 nM) was used in transfection with polymer or Lipofectamine RNAiMAX. Two days after transfection, the luciferase expression level was measured with similar method for luciferase pDNA transfection, and was compared to no-treatment group. 2.11. In vitro toxicity test The cytotoxicity of oPACE9-20, PACE, and PEI was studied against HEK293 and U87MG cells. The cells were seeded in 96-well plates one night before at an initial seeding density of 1.5×104 cells per well in 100 uL of DMEM. PACE/pLuc and oPACE9-20/pLuc polyplexes were prepared at 100:1 polymer:DNA weight ratio in sodium acetate buffer according to the previously described method. Polyplexes of PEI/pLuc were prepared at 3:1 polymer:DNA weight ratio with a similar method. Then, the growth medium was removed and replaced with 180 uL fresh DMEM, followed by addition of 20 uL of polyplexes solution at different concentrations so the


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final concentration of DNA was ranged from 0.15 ug/mL to 20 ug/mL. For control experiments, 20 uL sodium acetate buffer with the same amount of DNA and DMSO was added. After 48 hours incubation, the cells were assayed for metabolic activity using a standard MTT assay. 2.12.

Lyophilization and storage of the polyplexes

Polyplexes were prepared with oPACE9-20 and pLuc plasmid according to the method described above. Trehalose solutions of varying concentrations (15 mg/mL, 30 mg/mL, 60 mg/mL and 120 mg/mL in 25 mM sodium acetate buffer) was added to the polyplex suspension at a 1:1 volume ratio to get a final trehalose concentration of 7.5, 15, 30, and 60 mg/mL. The mixtures were then frozen in liquid nitrogen. After lyophilization for two days, the polyplexes were reconstructed in sodium acetate buffer and were used to treat U87MG cells. Some lyophilized polyplexes were stored in -20 °C for 7, 14 or 30 days. The gene transfection efficiency and diameter of the polyplexes were measured with the methods described above. 2.13.

Statistical analysis

All experiments were performed with three replicates unless otherwise noted. Bar graphs represents mean with standard deviation. One-way ANOVA was used to analyze the statistical significance between samples. A p values smaller than 0.05 was considered significant in the analysis. 3.

Results

3.1.

Synthesis and characterization of ortho ester diester monomers



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Here, we designed and synthesized a family of ortho ester diester (OEDE) monomers that are compatible with lipase catalysis. To prepare the OEDE monomers (Scheme 1 A), we first synthesized the diketene acetal, 3,9-diethylidene-2,4,8,10-tetraoxaspiro [5.5] undecane (DETOSU), via the isomerization of 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane (DTSU) 31. The 1H NMR resonances (Fig. S1) of the synthesized DETOSU were identical to those reported in the literature 35. Three different OEDEs were obtained with quantitative yield by the addition of Table 1 Characterization data of oPACEs Name a oPACE1-6 oPACE1-13 oPACE1-24 oPACE1-34 oPACE1-43 oPACE5-13 oPACE5-20 oPACE5-28 oPACE9-8 oPACE9-14 oPACE9-20 oPACE9-31 oPACE9-36 PACE

OEDE/DES/ OEDE/DES/ MDEA/PDL MDEA/PDL (feed molar ratio) (molar ratio) b 10/90/100/11 6/94/100/11 20/80/100/11 13/87/100/11 30/70/100/11 24/76/100/11 40/60/100/11 34/66/100/11 50/50/100/11 43/57/100/11 20/80/100/11 13/87/100/11 30/70/100/11 20/80/100/11 40/60/100/11 28/72/100/11 10/90/100/11 8/92/100/11 20/80/100/11 14/86/100/11 30/70/100/11 20/80/100/11 40/60/100/11 31/69/100/11 50/50/100/11 36/64/100/11 0/100/100/11 0/100/100/11

Mw c

PDI c

Nitrogen content (wt%)

1777 1827 1874 2169 2064 15963 13175 8767 18533 11187 9162 10092 8126 9937

1.20 1.21 1.21 1.28 1.26 2.13 2.04 2.00 2.6 2.09 1.91 1.99 1.89 2.31

4.3 4.2 4.0 3.8 3.7 4.0 3.8 3.6 4.1 3.9 3.7 3.3 3.2 4.5

a. The polymers are named as oPACEn-x, where n indicates OEDE (n) units in the copolymer, and x is the molar percentage of OEDE units vs MDEA units. b. Measured by 1H NMR spectroscopy. c. Measured by GPC with narrow polydispersity polystyrene standards.

either ethyl glycolate (n=1), ethyl 6-hydroxyhexanoate (n=5) or methyl 10-hydroxydecanoate (n=9) to DETOSU, which were named as OEDE (1), OEDE (5) and OEDE (9), respectively. The structure of the OEDE was confirmed by both 1H and

13

C NMR spectroscopy (Fig. S2). For


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example, OEDE (5) exhibited

13

C NMR resonances at 173.61 ppm due to the ester carbonyl

groups and at 111.52 ppm due to the ortho ester groups. No absorption at ~150 ppm, which would result from unreacted ketene acetal, was observed. Elemental (C & H) analysis was also used to confirm the structure (theoretical/experimental weight ratio in percentage: 9.08/10.01 and 60.88/60.62, for H and C, respectively). 3.2.

Synthesis and characterization of oPACEs

OEDEs were successfully co-polymerized with ω-pentadecalactone (PDL), diethyl sebacate (DES) and N-methyldiethanolamine (MDEA) using Candida antarctica lipase B (CALB) as catalyst (Scheme 1B). The polymerization was carried out in two-stages: a first stage under 1 atm of inert gas for 24 hours, during which monomers were polymerized into oligomers; and a second stage under 1.6 mmHg for 72 hours, during which high molecular weight polymers were formed. The structures of oPACEs were confirmed and analyzed by NMR (Fig. S3, Table 1) and GPC (Table 1). The polymer products are named as oPACEn-x, where n indicates OEDE (n) units in the polymer chain, x is the molar percentage of OEDE units vs MDEA units. For example, oPACE9-20 represents the polymer containing 20 mol% OEDE (9) relative to MDEA unites in the polymer backbone. Polymers with a range of compositions were prepared and characterized (Table 1). All the quaterpolymers have nitrogen content ranging from 3.2% to 4.3%, which is much lower than PEI (~32.6 wt% of nitrogen). The molar ratio of OEDE was varied from 8% to 36% of MDEA. oPACEs containing OEDE (5) and OEDE (9) had MW ranging from 8 kDa to 19 kDa, indicating successful polymerization. However, polymerization with the most polar OEDE (1) resulted in formation of oligomers (MW~2 kDa) only. The polydispersity of the copolymers is influenced



by the substrate size selectivity and interchain transesterification acitivity of CALB catalyst, which was discussed in details in a previous publication.36 We also observed that the polymer products contain less OEDE than the feed monomer mixtures. These observations appear to be due to the lower activity of the lipase for the highly polar OEDEs, especially OEDE (1), as the acyl binding site in the lipase CALB is more accessible to non-polar substrates 37.

B

Residual weight%

Residual weight%

A

C

D Residual weight%

Residual weight%

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100 75 50 25 0 0

50 100 150 Incubation time/hours

200

Figure 1. Acid sensitivity of oPACE. Weight loss vs time for oPACE5-20 (red) and PACE (black) upon incubation at 37 °C in (A) PBS at pH 7.4 or in sodium acetate buffer solutions at (B) pH 6.0, (C) 5.0, or (D) 4.0. The dry weight of the residual polymers is reported as a percentage of the initial polymer weight.

3.3.

Acid-sensitivity of oPACE

Degradation of oPACE in water was sensitive to pH (Fig. 1). For oPACE5-20, the rate of polymer weight loss was minimal at pH 7.4 and pH 6 during a 7-day incubation, moderate at pH 5.0, and rapid at pH 4.0. The half-life for weight loss was >168 hours at pH 7.4 and 6, ~24 hours at pH 5, and 80%) was complexed with polymer, as demonstrated by a PicoGreen exclusion assay (Fig. 2A). The size of the polyplexes decreased as the polymer:DNA weight ratio increased. At a ratio of 100:1 or higher, the diameter of the polyplexes was around 200 nm as measured by DLS (Fig. 2B). The polyplexes formed at 100:1 polymer:DNA weight ratio exhibited a well-defined spherical shape under TEM (Fig. 2C). A polymer to DNA weight ratio of 100:1 was used in all of the following studies unless otherwise noted. 3.5.

Endosomal escape of pDNA/oPACE polyplexes

To test whether the acid-sensitive oPACE could facilitate endosomal escape, we labelled pLuc plasmids with both acid-sensitive FITC and acid-insensitive Cy5 and used a previously reported


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A

B

Figure 3. oPACE facilitates endosomal escape of genes. U87MG cells were transfected with FITC-Cy5 double labelled DNA (pLuc) by PACE or oPACE9-20. (A) Standard curves between FITC/Cy5 fluorescence ratio (measured by FACS) and intracellular pH of cells. (B) The FITC/Cy5 ratio of the transfected U87MG cells was measured and converted into pH values using the standard curves. Error bars indicate standard deviation (n=3, ****: p

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