Subscriber access provided by UNIV OF TEXAS DALLAS
Article
Rapidly visualizing the membrane affinity of gene vectors using polydiacetylene-based allochroic vesicles Jing-Wen Wang, Feng Zheng, Huan Chen, Ya Ding, and Xing-Hua Xia ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00102 • Publication Date (Web): 14 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019
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. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. 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 20 1 2 3 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 Sensors
Rapidly visualizing the membrane affinity of gene vectors using polydiacetylene-based allochroic vesicles Jing-Wen Wang †,, Feng Zheng †,, Huan Chen ‡, Ya Ding †,*, Xing-Hua Xia§
†
Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China
Pharmaceutical University, Nanjing 210009, China ‡
Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical
University, Nanjing 210009, China §
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical
Engineering, Nanjing University, Nanjing 210023, China
The first two authors contributed equally to this work.
*
Corresponding author: E-mail:
[email protected] (Y. Ding)
Abstract. The high-throughput screening of chemically active substances has aroused widespread interest in recent years. The screening of drug carriers is usually ignored, although they interact directly with physiological barriers and target cells, and determine the fate and efficacy of drugs in vivo. In this work, a series of polydiacetylene (PDA) vesicles (ca. 550 nm) that simulate the cell membrane are constructed to detect the membrane affinity of gene vectors. The surface potentials of vesicles are adjusted by changing the phospholipid composition using different charged compounds. All vesicles show the rapid color changes upon the addition of gene vectors by the naked eye within less than 5 min. The optimized 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-PDA vesicles display the most sensitive discoloration response to the commercially available gene vectors, including Lipofectamine 2000, EntransterTM-H4000, and polyethylenimine. The logarithm of transfection efficiency for green fluorescent protein plasmid (pGFP) mediated by these three vectors in L02 and HepG2 cells demonstrate an excellent linear correlation with the logarithm of membrane affinity (log Kb) of the gene vectors detected by DMPC-PDA vesicles. This rapid visualization ACS Paragon Plus Environment
ACS Sensors 1 2 3 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
Page 2 of 20
method not only allows the in vitro membrane affinity prediction of gene vectors that greatly contributes to the gene transfection efficiency, but also offers a universal strategy for the potential high-throughput screening of various carrier materials featuring high cell affinity. Keywords: allochroic polydiacetylene vesicles, gene vector, membrane affinity, transfection efficiency, high-throughput screening
In gene therapy, the effective transfection of nucleic acids relies on the role of gene vectors.1 These vectors protect nucleic acids against degradation by nucleases in the circulation and import the genetic material into target cells as a gene drug to treat disease.2 Since the first introduction of exogenous DNA into animal cells in 1979 by calcium phosphate transfection technology,3 various nonviral vectors, such as cationic liposomes,4 polylysine (PLL),5 and polyethyleneimine (PEI),6 have been employed as alternatives to overcome the risks of viral vectors, e.g., random insertion sites, cytopathic effects, and mutagenesis.7,8 An efficient gene vector should meet the desirable capacity to compact the target gene, introduce it into the desired host cell, and release the genetic material, allowing it to integrate into the genome.911
During the gene transfection process, the affinity of gene vectors to the cell membrane is a critical
parameter that is closely related to their transfection efficiency. Traditional evaluation methods of gene transfection efficiency in terms of fluorescence intensity generated by a green fluorescent protein (GFP)-encoding plasmid12,13 are inefficient, complex, and cell-type dependent. In addition, this reporter gene method is time consuming and difficult to implement in high-throughput screening for gene vectors.14 Thus, developing a method for the rapid visualization of the affinity between cell membranes and gene vectors will facilitate the in vitro screening of numerous synthetic nonviral
ACS Paragon Plus Environment
Page 3 of 20 1 2 3 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 Sensors
vectors having potential high transfection capability. The polydiacetylene (PDA) polymer is an interesting series of amphiphilic polymers, which is formed by 1, 4 addition of the diacetylenic monomers, initiated by UV irradiation (Figure 1A).15 The resulting polymer is intensely colored, typically a deep blue. If the R groups of the diacetylenic monomers are designed to impart amphiphilic character to the molecule, the reactive monomers can be self-assembled into thin films or vesicles, forming chromic molecular assemblies. Since no chemical initiators or catalysts are required for the polymerization process, the polymers are not contaminated with impurities, and consequently, purification steps are not required.16 In response to heat
(thermochromism),17
organic
solvents
(solvatochromism),18
mechanical
stress
(mechanochromism),19 and ligand-receptor interactions (affinochromism),20-24 a color change of PDA vesicles occurs from blue (max~ 630 nm) to red (max~ 490 nm) due to the disturbance and/or change of an ordered membrane structure of polymeric vesicles.15 Although much simple and crude, PDA vesicles are analogous to the cell membrane in that molecular recognition is directly linked to signal transduction within a single supramolecular assembly. Thus, PDA-based vesicles were used as optical sensors to detect various analytes, such as microorganisms,25,26 proteins,27-29 and membranedisrupting events in cells.30,31 In our previous work, PDA vesicles were employed to detect the membrane affinity to several chemical drugs with both hydrophobicity and a positive charge, such as tetracaine hydrochloride, acebutolol hydrochloride, and propranolol hydrochloride.25,32 The logarithm of affinity constant (Kb) of drug molecules exhibited excellent linear correlation with the logarithm of the liposome/water partition coefficient (Km) or the n-octanol/water partition coefficient (Koct). Tetracaine hydrochloride had the highest Kb value among all the tested drugs, reflecting its highest affinity among all test drug molecules towards PDA vesicles that mimic cell membranes. However, in the medicine field, nucleic ACS Paragon Plus Environment
ACS Sensors 1 2 3 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
Page 4 of 20
acids are commonly encapsulated in various vectors to improve their transfection efficiency. Therefore, the vectors are what the cells really see after DNA entering the body and determine the biological fate of these nucleic acids in vivo. To date, there have been no reports on the evaluation of cell membrane affinity to gene vectors, although the in vitro screening of them is also very important for achieving desirable gene therapy. In this work, PDA vesicles were constructed and their surface potentials were adjusted by adding different charged phospholipids, e.g., 1, 2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DMPG), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and stearamide (SA). Firstly, DMPC and DA monomer at a molar ratio of 2: 3 were used to obtain DMPC-PDA vesicles. To adjust the surface potentials of vesicles, different moles of DMPG or SA were added to replace parts of DMPC (Figure 1A). These vesicles maintained the discoloration ability of PDA and were used as optical sensors to detect the membrane affinity of commercially available gene vectors, including Lipofectamine 2000 (Lipo), EntransterTM-H4000 (Entr), and polyethylenimine (PEI). Upon the addition of gene vectors, the rapid color change of the vesicles can be observed by the naked eye in less than 5 min (Figure 1B). The DMPC-PDA vesicles composed of DMPC and DA monomer at a molar ratio of 2: 3 showed the most sensitive color change in response to different vectors. The Kb values were calculated based on the ultraviolet absorption of vesicles. For the optimized DMPC-PDA vesicles that showed the most sensitivity to vectors, Lipo exhibited a highest membrane affinity. In addition, it was also found that the transfection efficiency of green fluorescent protein plasmid (pGFP) mediated by these three vectors exhibits an excellent linear correlation with their membrane affinities detected by DMPC-PDA vesicles. Therefore, the color change in DMPC-PDA vesicles allows the rapid visualization of the membrane affinity of gene vectors, which might be a robust tool for screening nonvirial gene vectors and even the desirable biomaterials for drug delivery. ACS Paragon Plus Environment
Page 5 of 20 1 2 3 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 Sensors
Figure 1. Schematic illustration of (A) the structure of nanovesicle composed by DA, PDA, DMPC, and/or DMPG/SA and their chemical structures, (B) the fast visualization method to detect the membrane affinity of gene vectors using PDA nanovesicles, comparing to (C) a traditional cell transfection method.
EXPERIMENTAL SECTION Materials. DMPC and DMPG were purchased from AVT Pharmaceutical Co., Ltd. (Shanghai, China). SA was obtained from TCI Development Co., Ltd. (Shanghai, China). 10, 12Pentacosadiynoic acid (98%, DA) was provided by Alfa Aesar (USA). Lipo and Entr were supplied ACS Paragon Plus Environment
ACS Sensors 1 2 3 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
Page 6 of 20
from Invitrogen (USA) and Engreen (New Zealand), respectively. PEI (molecular weight of 25,000 Da) was purchased from PolyScience (USA). All aqueous solutions were prepared using deionized water (>18 M, PURELAB Classic Corp., USA). Characterizations. Ultraviolet-visible (UV-vis) spectra were collected with a UV3600 spectrophotometer (Shimadzu, Japan). The UV-vis absorbance of all samples was measured with a microplate reader (POLARstar Omega, BMG LABTECH, Germany). The hydrodynamic diameter and zeta potential of the vesicles were detected by a Zetasizer 3000HS instrument (Malvern Instruments, Malvern, UK) using a 633-nm He-Ne laser at 25 °C. The reporter gene expression, green fluorescent protein (GFP), is quantified by flow cytometry (Accuri C6, BD, USA) with a ex of 475 nm and em of 509 nm. Preparation of PDA vesicles. PDA vesicles were synthesized using a film dispersion method, as previously described.30 DMPC, DMPG, and SA were added to adjust the surface potential of the PDA vesicles. Briefly, DMPC and DA monomer at a molar ratio of 2: 3 were dissolved in a roundbottomed flask containing a chloroform-ethanol mixed solution with a volume ratio of 1: 1. After the removal of the organic solvent under vacuum, a thin lipid film was formed, and then 25 mL of deionized water was added into the flask to obtain a suspension containing 1.0 mM phospholipid. The suspension was sonicated at 70 °C for 5 min at a power of 125 W and became milky pink in color. The mixture was cooled overnight at 4 °C to obtain DMPC-DA vesicles. To realize the intermolecular crosslinking of DA, the stirred solution was irradiated at room temperature using a 254 nm ultraviolet light at 7 lumens for 10 min to obtain blue polymerized DMPC-PDA vesicles, denoted hereafter as DMPC. The solution was stored at 4 °C prior to use. To change the surface potentials of vesicles, different moles of negatively charged DMPG or
ACS Paragon Plus Environment
Page 7 of 20 1 2 3 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 Sensors
positively charged SA were added in the preparation solutions to replace parts of DMPC. Using the similar preparation method described above, DMPG-DMPC (5: 95)-PDA, SA-DMPC (5: 95)-PDA, and SA-DMPC (10: 90)-PDA vesicles (all in molar ratios) were obtained, denoted hereafter as DMPG, SA-5 and SA-10, respectively. Membrane affinity assay. Four different PDA vesicles were added to 96-well plates (15 μL per well), and then 5, 10, 15, and 20 μL of Lipo, Entr or PEI solution (1 mg/mL) was added at room temperature. The solution in each well was diluted to 150 μL with phosphate-buffered saline (PBS). The color change in each well can be observed in 5 min. To obtain stable ultraviolet absorption signals, the vesicle solution was equilibrated for 30 min after each gene vector addition before the UV-vis absorbance was recorded at 540 nm and 650 nm in each well. All experiments were repeated thrice. The percent conversion from the blue phase to the red phase at the addition of a given gene vector was characterized by the colorimetric response (CR)33 based on the following equations:34 CR = (PB0 – PBf) / PB0 100%
(1)
PB0 or f = Ablue / (Ablue + Ared)
(2)
where A is the absorbance at the wavelength of either the blue (at 650 nm) or the red form (at 540 nm) in nanovesicles, and PB0 and PBf are the initial and final percent blue before and after adding the test gene vectors into vesicles, respectively. The linear regression equation of the 1/CR and reciprocal of the vector concentration (1/C) was obtained from the following equation:34 1/CR = 1/(KbCRmax) 1/C + 1/CRmax (3) where Kb is calculated from the slope and the intercept and is used to represent the membrane affinity constant of the gene vector.
ACS Paragon Plus Environment
ACS Sensors 1 2 3 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
Page 8 of 20
Preparation of pGFP complexes and their loading efficiencies. The pGFP was purified from the transformed Escherichia coli using the Endo-Free Maxi Plasmid Kit (Tiangen Biotech Co., Ltd., Beijing) according to the manufacturer’s protocol. Then, different amounts of Lipo, Entr, and PEI were mixed with 3 μg of pGFP in 300 μL of serum-free medium at a mass ratio (pGFP/Lipo, pGFP/Entr or pGFP/PEI) of 1: 2, 1: 3, 1: 4, and 1: 5. The mixture was incubated at room temperature for 5 min prior to use. To determine the DNA loading efficiency of gene vectors, 200 μL of the pGFP complex solution was mixed with an equivalent volume of isopropanol, and the mixture was shaken for 15 seconds using a vortex oscillator. Free DNA was extracted by 400 μL of phenol/chloroform/isopropanol (25: 24: 1), and the optical density (O.D.) value of the DNA at 260 nm was measured with a UV-vis spectrophotometer. Finally, the binding degree of pGFP with the vector was expressed as the following equation:35 Loading rate = (AD - AND) / (AD - AB) 100%
(4)
where AD is the O.D. value after release of the DNA from the complex using isopropanol, AND is the O.D. value of the DNA complex, and AB is the O.D. value of the vector solution. In vitro cell transfection of pGFP. L02 cells and HepG2 cells were separately seeded in 6-well plates at a density of 80%. After 24 h, 250 μL of the Lipo-pGFP, Entr-pGFP, or PEI-pGFP complex solutions with different C/P ratios prepared above was added to each well and diluted with 250 μL of fresh serum-free medium. After incubation at 37 °C for 48 h, the expression of GFP was observed by confocal laser scanning microscopy (CLSM) and detected by flow cytometry.
RESULTS AND DISCUSSION Preparation and characterization of PDA vesicles. The PDA vesicles were prepared as we
ACS Paragon Plus Environment
Page 9 of 20 1 2 3 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 Sensors
previously reported.33 To regulate the surface potentials of PDA vesicles, the phospholipid composition is adjusted using DMPG and SA instead of DMPC (Figure 1A). PDA vesicles with different zeta potentials, i.e., DMPC, DMPG, SA-5, and SA-10, were obtained using the film dispersion method and demonstrated different shades of blue (Figure 2A). The UV-vis spectra of four PDA vesicles were displayed in Figure 2B. The maximum absorption wavelength of these vesicles was located at 650 nm. This value is consistent with our previous studies,34 indicating that the change in phospholipid composition has little effect on the absorbance of PDA vesicles. In addition, the phospholipid composition change did not affect the size of vesicles significantly. The hydrodynamic diameters of PDA vesicles were similar, e.g. 556.3 ± 11.1, 576.2 ± 3.8, 532.9 ± 24.5, and 567.5 ± 54.1 nm for DMPC, DMPG, SA-5, and SA-10, respectively (Figure 2C). However, the zeta potentials were obviously different. The zeta potentials of DMPC, DMPG, SA-5, and SA-10 were determined to be -32.13 ± 0.42, -6.05 ± 2.01, 2.50 ± 0.82, and 7.38 ± 0.28 mV, respectively (Figure 2D). These data show that the addition of negatively charged DMPG and positively charged SA can effectively adjust the surface potential of PDA vesicles to imitate the cell membrane surface of different cell lines, including normal and tumor cells. 25, 36-38
Figure 2. (A) Photograph, (B) UV-vis spectra, (C) hydrodynamic diameters, and (D) zeta potentials of DMPC, DMPG, SA-5, and SA-10 vesicles. Data points represent mean ± SD (n = 3).
ACS Paragon Plus Environment
ACS Sensors 1 2 3 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
Page 10 of 20
Membrane affinity assay of gene vectors by PDA vesicles. To evaluate the assay ability of PDA vesicles with different phospholipid compositions, three commercially available gene vectors, Lipo, Entr, and PEI, were employed to observe their affinity to PDA vesicles. Keeping the concentration of PDA vesicles constant (0.1 mM) but changing the concentration of gene vectors, the color of the mixed solutions changed from the initial blue to a purple to pink color in 5 min (Figure 3A). This biochromism can be explained by (1) multipoint interactions of the functional group of gene vectors with the PDA-vesicle surface, disrupting the ordered membrane structure, and/or (2) insertion of gene vector hydrophobic domains into the PDA membrane.15,39 These disturbances led to a blue shift of the maximum ultraviolet absorption from 650 nm to 540 nm (Figure 3B).
Figure 3. Membrane affinity assay of gene vectors using different PDA vesicles. (A) Photograph of color change for DMPC, DMPG, SA-5, and SA-10 solutions (0.1 mM, in PBS at pH 8.0) after adding Lipo, PEI, and Entr solutions (60 mg/L in PBS at pH 8.0) to each well at room temperature for 30 min. (B) Absorption spectral change of a typical DMPC vesicle solution with the addition of Lipo (60 mg/L in PBS at pH 8.0). (C) CR values of DMPC,
ACS Paragon Plus Environment
Page 11 of 20 1 2 3 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 Sensors
DMPG, SA-5, and SA-10 vesicles with the addition of different gene vectors, e.g. Lipo, Entr, and PEI (64 mg/L in PBS at pH 8.0). (D) Kb values of Lipo, Entr, and PEI against DMPC, DMPG, SA-5, and SA-10 vesicles obtained from Figure S2. Data points represent mean ± SD (n = 3).
According to equation (1), CR values reflected the degree of blue shift in the maximum ultraviolet absorption of vesicles upon the addition of different concentrations of Lipo, Entr, and PEI (Figure S1). It is interesting that, although DMPC vesicles have only slight negative zeta potential closest to most mammalian cells (-6.05 ± 2.01 mV in Figure 2D), they exhibited a relatively higher CR value against various test vectors (Figure S1A). For example, the sensitive response of DMPC compared to other three vesicles among three test vectors at the concentration of 64 mg/L was showed in Figure 3C. However, the addition of any charged substances, whether positive or negative, would weaken the disturbance of the PDA such as DMPG, SA-5, and SA-10 (Figure S1, B-D). Thus, DMPC vesicle might be a promising sensor for simulating mammalian cells and screening different gene vectors. Subsequently, Kb values of gene vectors representing their membrane affinity towards PDA vesicles were also calculated by the double-reciprocal curve of CR based on equation (3) (Figures S2). From the data in Figure 3D, we found that, among three vectors, Lipo displayed the highest affinity to almost all vesicles, although zeta potentials of Entr (30.31 ± 0.86 mV) and PEI (39.61 ± 2.89 mV) were higher than that of Lipo (4.15 ± 1.72 mV) (Figure S3). This high affinity could be due to the similar lipid composition of Lipo and PDA vesicles compared with the cationic polymers of PEI or Entr. It is noteworthy that, in the cases of Entr and PEI, the higher the zeta potential of the cationic polymer, the lower the membrane affinity with PDA vesicles. This phenomenon was consistent with our previous reports in the affinity detection of local anesthetic and lipid-soluble drugs with PDA vesicles.25,34 The affinity of a carrier with the cell membrane was not only determined by
ACS Paragon Plus Environment
ACS Sensors 1 2 3 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
Page 12 of 20
the electric charge of the carrier materials, but also greatly influenced by their lipophilicity of hydrophobic domains. From the above results, it is hypothesized that Lipo would show a desirable gene transfection due to its high membrane affinity and lipid-like structure at the cellular level. In vitro gene transfection efficiency. To confirm our hypothesis, pGFP transfection efficiency of Lipo, Entr, and PEI was detected in the human normal liver (L02) and hepatocellular carcinoma (HepG2) cell lines. According to the manufacturer’s recommendations, for optimal transfection efficiency, DNA complexes of Lipo, Entr, and PEI with the vector: DNA mass ratio of 1: 2, 1: 3, 1: 4, and 1: 5 were prepared for cell transfection. As shown in Table S1, the DNA loading efficiency of Lipo, Entr and PEI was 82-85% for all mass ratios, indicating no significant difference in DNA loading capacity between Lipo, Entr, and PEI. And then, the transfection efficiency of Lipo, Entr, and PEI was detected in L02 and HepG2 cells by the reporter gene method, e.g., the proportion of cells expressing GFP. Figure 4A showed the representative CLSM images of cells after 48 h of incubation with the pGFP complexes. Green fluorescence arises from the expression of GFP. The intracellular fluorescence intensity of GFP was measured by flow cytometry (Figure 4B). Of special note, the same pGFP complexes showed different transfection efficiency in L02 and HepG2 cells, even under the same conditions. The tumor cell is more difficult to be transfected, which might be due to differences in the composition and structure of the cell membrane between these two cell lines. Lipo-pGFP at a mass ratio of 1: 3 showed the highest transfection rate in both L02 and HepG2 cells (62.10% and 25.33%, respectively), while the maximum transfection rate of Entr-pGFP was 56.30% (1: 4) in L02 cells and 22.27% (1: 3) in HepG2 cells and the maximum transfection rate of PEI-pGFP was 45.87% (1: 4) in L02 cells and 20.00% (1: 2) in HepG2 cells. It is obvious that the expression of GFP mediated by Lipo, Entr, and PEI are in a descending order in either L02 or HepG2 cells. This trend coincides with the trend of Kb ACS Paragon Plus Environment
Page 13 of 20 1 2 3 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 Sensors
values for these three materials. When the double logarithmic curve of transfection efficiency of the three gene vectors as a function of their membrane affinity (Kb) towards DMPC vesicles was plotted (Figure 4D), it is interestingly found that the log (transfection efficiency) versus log (Kb) exhibited an excellent linear correlation with R2 = 0.9984 in L02 cells (a) and R2 = 0.9548 in HepG2 cells (b). These findings provide important insights into the possible rapid visualization of PDA vesicles, especially the optimized DMPC vesicles, for simulating the cell membrane to screen not only the chemical drugs but also their carriers.
ACS Paragon Plus Environment
ACS Sensors 1 2 3 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
Page 14 of 20
Figure 4. (A) Typical CLSM images of L02 and HepG2 cells used for determining the transfection efficiency of Lipo, Entr and PEI after 48 h of incubation with Lipo-pGFP (mass ratio 1: 3), Entr-pGFP (mass ratio 1: 3) and PEIpGFP (mass ratio 1: 3). The scale bar is 200 m (upper panels) and 40 m (below panels). (B) Plasmid gene transfection results determined by flow cytometry in (a) L02 and (b) HepG2 cells when transfected with Lipo-, Entr-, or PEI-pGFP complex vector at a mass ratio of 1: 2, 1: 3, 1: 4, and 1: 5, respectively. Data represent the mean ± SD (n = 3). (C) Comparison of vector membrane affinity to the DMPC vesicle system (Kb) and the highest transfection efficiency for vectors in different cell lines, correlation between log (transfection efficiency) and log (Kb) in (a) L02 cells and (b) HepG2 cells.
CONCLUSION
ACS Paragon Plus Environment
Page 15 of 20 1 2 3 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 Sensors
In summary, we have established a rapid visualization method based on the discoloration of a series of PDA vesicles for detecting the membrane affinity of gene vectors. Using commercial Lipo, Entr and PEI as model gene vectors, the color changes and Kb values that reflect the affinity between vesicles and vectors can be quickly detected. After properly adjusting the surface potential of the PDA vesicles by changing the composition of the vesicle structure, DMPC-PDA vesicles showed the most sensitive response to the tested vectors. It was proved that the membrane affinity detected by PDA vesicles and GFP expression in L02 and HepG2 cells mediated by gene vectors were closely related. It demonstrated that the PDA vesicle sensor could be a universal strategy to the potential high-throughput screening of carrier materials for efficient drug delivery.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: The CR value-concentrations curves and the corresponding double reciprocal curves of Lipo, Entr, and PEI after interacting with DMPC, DMPG, SA-5, and SA-10 vesicles for 30 min. Zeta potentials of Lipo, Entr, and PEI solution at a concentration of 60 mg/L.
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected] ORCID Xing-Hua Xia: 0000-0001-9831-4048 ACS Paragon Plus Environment
ACS Sensors 1 2 3 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
Page 16 of 20
Ya Ding: 0000-0001-6214-5641 Author Contributions
These authors contributed equally to this work.
Notes The authors declare no competing financial interest.
ACKOWLEDGEMENTS Many thanks to Prof. Yong Yang, China Pharmaceutical University, who provided the Escherichia coli DH5-α cells transformed with pGFP. This work was supported by grants from the National Natural Science Foundation of China (30900337, 31470916, 31870946), the Funding of Double Firstrate discipline construction (CPU2018GF07), and the Open Project Program of MOE Key Laboratory of Drug Quality Control and Pharmacovigilance (DQCP2015MS01).
REFERENCES (1) Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 2014, 15, 541-555. (2) Li, W.; Szoka, F. C. Jr. Lipid-based nanoparticles for nucleic acid delivery. Pharm. Res. 2007, 24, 438-449. (3) Mulligan, R. C.; Howard, B. H.; Berg, P. Synthesis of rabbit beta-globin in cultured monkey kidney cells following infection with a SV40 beta-globin recombinant genome. Nature 1979, 277, 108-114. (4) Fraley, R.; Subramani, S.; Berg, P.; Papahadjopoulos, D. Introduction of liposome-encapsulated SV40 DNA into cells. J. Biol. Chem. 1980, 255, 10431-10435. (5) Remy, J. S.; Kichler, A.; Mordvinov, V.; Schuber, F.; Behr, J. P. Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: a stage toward artificial viruses. Proc. Natl. Acad. Sci. 1995, 92, 1744-1748. (6) Kircheis, R.; Kichler, A.; Wallner, G.; Kursa, M.; Ogris, M.; Felzmann, T.; Buchberger, M.; Wagner, E. Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Ther. 1997, 4, 409-418.
ACS Paragon Plus Environment
Page 17 of 20 1 2 3 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 Sensors
(7) Jones, G. D.; Langsjoen, A.; Neumann, S. M. M. C.; Zomlefer, J. The polymerization of ethylenimine. J. Org. Chem. 1944, 09, 125-147. (8) Keles, E.; Song, Y.; Du, D.; Dong, W. J.; Lin, Y. Recent progress in nanomaterials for gene delivery applications. Biomater. Sci. 2016, 4, 1291-1309. (9) Wasungu, L.; Hoekstra, D. Cationic lipids, lipoplexes and intracellular delivery of genes. J. Control. Release 2006, 116, 255-264. (10) Godbey, W. T.; Wu, K. K.; Mikos, A. G. Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc. Natl. Acad. Sci. USA 1999, 96, 51775181. (11) Breunig, M.; Lungwitz, U.; Liebl, R.; Fontanari, C.; Klar, J.; Kurtz, A.; Blunk, T.; Goepferich, A. Gene delivery with low molecular weight linear polyethylenimines. J. Gene. Med. 2005, 7, 12871298. (12) Kumar, P.; Wu, H.; Mcbride, J. L.; Jung, K. E.; Kim, M. H.; Davidson, B. L.; Lee, S. K.; Shankar, P.; Manjunath, N. Transvascular delivery of small interfering RNA to the central nervous system. Nature 2007, 448, 39-43. (13) Priller, J.; Flügel, A.; Wehner, T.; Boentert, M.; Haas, C. A.; Prinz, M.; Fernándezklett, F.; Prass, K.; Bechmann, I.; de Boer, B. A.; Frotscher, M.; Kreutzberg, G. W.; Persons, D. A.; Dimaql, U. Targeting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment. Nat. Med. 2001, 7, 1356-1361. (14) van Gaal, E. V.; van Eijk, R.; Oosting, R. S.; Kok, R. J.; Hennink, W. E.; Crommelin, D. J.; Mastrobattista, E. How to screen non-viral gene delivery systems in vitro? J. Control. Release 2011, 154, 218-232. (15) Okada, S.; Peng, S.; Spevak, W.; Charych, D. Color and chromism of polydiacetylene vesicles. Acc. Chem. Res. 1998, 31, 229-239. (16) Sun X.; Chen T.; Huang S.; Li L.; Peng H. Chromatic polydiacetylene with novel sensitivity. Chem. Soc. Rev. 2010, 39, 4244-4257 (17) Müller, H.; Eckhardt, C. J.; Chance, R. R.; Baughman, R. H. Optical and electrical properties of a polydiacetylene crystal: poly(5,7-dodecadiyne-1,12-diol-bis phenylurethane). Chem. Phys. Lett. 1977, 50, 22-25. (18) Nava, A. D.; Thakur, M.; Tonelli, A. E. Carbon-13 NMR structural studies of a soluble polydiacetylene, poly(4BCMU). Macromolecules 1990, 23, 3055-3063. (19) Tomioka, Y.; Tanaka, N.; Imazeki, S. Effects of side-group interactions on pressure-induced chromism of polydiacetylene monolayer at a gas-water interface. Thin Solid Films 1989, 179, 27-31. (20) Charych, D. H.; Nagy, J. O.; Spevaki, W.; Bednarski, M. D. Direct colorimetric detection of a receptor-ligand interaction by a polymerized bilayer assembly. Science 1993, 261, 585-588. (21) Wang, C.; Ma, Z. Colorimetric detection of oligonucleotides using a polydiacetylene vesicle ACS Paragon Plus Environment
ACS Sensors 1 2 3 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
Page 18 of 20
sensor. Anal. Bioanal. Chem. 2005, 382, 1708-1710. (22) Jung, Y. K.; Kim, T. W.; Kim, J.; Kim, J. M.; Park, H. G. Universal colorimetric detection of nucleic acids based on polydiacetylene (PDA) liposomes. Adv. Funct. Mater. 2008, 18, 701-708. (23) Cheng, Q.; Stevens, R. C. Coupling of an induced fit enzyme to polydiacetylene thin films: Colorimetric detection of glucose. Adv. Mater. 1997, 9, 481-483. (24) Kolusheva, S.; Shahal, T.; Jelinek, R. Cation-selective color sensors composed of ionophore−phospholipid−polydiacetylene mixed vesicles. J. Am. Chem. Soc. 2000, 122, 776-780. (25) Wei, M.; Liu, J.; Xia, Y.; Feng, F.; Liu, W.; Zheng, F. A polydiacetylene-based fluorescence assay for the measurement of lipid membrane affinity. RSC Adv. 2015, 5, 66420-66425. (26) Pindzola, B. A.; Nguyen, A. T.; Reppy, M. A. Antibody-functionalized polydiacetylene coatings on nanoporous membranes for microorganism detection. Chem. Commun （ Camb ） . 2006, 8, 906908. (27) Ma, Z.; Li, J.; Liu, M.; Cao, J.; Zou, Z.; Tu, J.; Jiang, L. Colorimetric detection of escherichia coli by polydiacetylene vesicles functionalized with glycolipid. J. Am. Chem. Soc. 1998, 120, 1267812679. (28) Jung, Y. K.; Kim, T. W.; Park, H. G.; Soh, H. T. Specific colorimetric detection of proteins using bidentate aptamer ‐ conjugated polydiacetylene (PDA) liposomes. Adv. Funct. Mater. 2010, 20, 3092-3097. (29) Assali, M.; Leal, M. P.; Fernández, I.; Khiar, N. Synthesis and non-covalent functionalization of carbon nanotubes rings: new nanomaterials with lectin affinity. Nanotechnology 2013, 24, 085604. (30) Deng, J.; Sheng, Z.; Zhou, K.; Duan, M.; Yu, C. Y.; Jiang, L. Construction of effective receptor for recognition of avian influenza H5N1 protein HA1 by assembly of monohead glycolipids on polydiacetylene vesicle surface. Bioconjugate Chem. 2009, 20, 533-537. (31) Orynbayeva, Z.; Kolusheva, S.; Livneh, E.; Lichtenshtein, A.; Nathan, I.; Jelinek, R. Visualization of membrane processes in living cells by surface-attached chromatic polymer patches. Angew. Chem. Int. Ed. 2005, 44, 1092-1096. (32) Shtelman, E.; Tomer, A.; Kolusheva, S.; Jelinek, R. Imaging membrane processes in erythrocyte ghosts by surface fusion of a chromatic polymer. Anal. Biochem. 2006, 348, 151-153. (33) Kolusheva, S.; Boyer, L.; Jelinek, R. A colorimetric assay for rapid screening of antimicrobial peptides. Nat. Biotechnol. 2000, 18, 225-227. (34) Zheng, F.; Wu, Z.; Chen, Y. A quantitative method for the measurement of membrane affinity by polydiacetylene-based colorimetric assay. Anal. Biochem. 2012, 420, 171-176. (35) Schnierle, B S.; Groner, B. Retroviral targeted delivery. Gene Ther. 1996, 3, 1069-1073. (36) Zhang, Y.; Yang, M.; Portney, N. G.; Cui, D.; Budak, G.; Ozbay, E.; Ozkan, M.; Ozkan, C. S. Zeta potential: a surface electrical characteristic to probe the interaction of nanoparticles with normal and cancer human breast epithelial cells. Biomed. Microdevices 2008, 10, 321-328. ACS Paragon Plus Environment
Page 19 of 20 1 2 3 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 Sensors
(37) Bondar, O. V.; Saifullina, D. V.; Shakhmaeva, I. I.; Mavlyutova, I. I.; Abdullin, T. I. Monitoring of the zeta potential of human cells upon reduction in their viability and interaction with polymers. Acta Naturae 2012, 4, 78-81. (38) Redmann, K.; Jenssen, H. L.; Köhler, H. J. Experimental and functional changes in transmembrane potential and zeta potential of single cultured cells. Exp. Cell Res. 1974, 87, 281289. (39) Ahn, D. J.; Kim, J. M. Fluorogenic polydiacetylene supramolecules: immobilization, micropatterning, and application to label-free chemosensors. Acc. Chem. Res. 2008, 41, 805-816.
ACS Paragon Plus Environment
ACS Sensors 1 2 3 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
Table of Contents (TOC)
ACS Paragon Plus Environment
Page 20 of 20
