Intracellular Delivery Platform for âRecalcitrantâ Cells: When Polymeric

Subscriber access provided by MacEwan University Libraries

Letter

An Intracellular delivery Platform for ‘Recalcitrant’ Cells: When Polymeric Carrier Marries Photoporation Jingxian Wu, Hui Xue, Zhonglin Lyu, Zhenhua Li, Yangcui Qu, Yajun Xu, Lei Wang, Qian Yu, and Hong Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06201 • Publication Date (Web): 20 Jun 2017 Downloaded from http://pubs.acs.org on June 22, 2017

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 free 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 accessible to all readers and 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.

ACS Applied Materials & Interfaces 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 25

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 Applied Materials & Interfaces

An Intracellular Delivery Platform for ‘Recalcitrant’ Cells: When Polymeric Carrier Marries Photoporation Jingxian Wua, Hui Xuea, Zhonglin Lyua, Zhenhua Lia, Yangcui Qua, Yajun Xub, Lei Wanga*, Qian Yua*, Hong Chena a

State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials,

College of Chemistry, Chemical Engineering and Materials Science, Soochow University, 199 Ren'ai Road, Suzhou, 215123, P. R. China b

Jiangsu Biosurf biotech co. Ltd, 218 Xinghu Street, Suzhou,215123, P. R. China

KEYWORDS: intracellular delivery, polyethylenimine, gold nanoparticle layer, photoporation, gene transfection.

ABSTRACT: The intracellular delivery of exogenous macromolecules is of great interest for both fundamental biological research and clinical applications. Although traditional delivery systems based on either carrier mediation or membrane disruption have some advantages, however, they are generally limited with respect to delivery efficiency and cytotoxicity. Herein, a collaborative intracellular delivery platform with excellent comprehensive performance is developed using polyethylenimine of low molecular weight (LPEI) as a gene carrier in

ACS Paragon Plus Environment

1


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 25

conjunction with a gold nanoparticle layer (GNPL) acting as a photoporation agent. In this system the LPEI protects the plasmid DNA (pDNA) to avoid possible nuclease degradation, and the GNPL improves the delivery efficiency of the LPEI/pDNA complex to the cells. The collaboration of LPEI and GNPL is shown to give significantly higher transfection efficiencies for hard-to-transfect cells (88.5 ± 9.2% for mouse embryonic fibroblasts, 94.0 ± 6.3% for human umbilical vein endothelial cells) compared to existing techniques without compromising cell viability.

The intracellular delivery of exogenous macromolecules is of great interest for both fundamental biological research and clinical applications.1 In particular the efficient delivery of plasmid DNA (pDNA) (also referred to gene transfection) is rapidly becoming a key element of gene therapy and regenerative medicine, allowing the manipulation or “engineering” of cellular behavior and function.2 Success in intracellular delivery of macromolecules is highly dependent on having a safe and efficient delivery system.3 Carrier-mediated delivery is beneficial since the carrier can package the macromolecule and protect it from degradation by endonucleases.4 Compared to viral carriers that generally have concerns with respect to immunogenicity and carcinogenicity, non-viral carriers are of great interest because of their non-immunogenicity, low cost, ease of large scale production, and relative safety.5 Polyethylenimine (PEI) is a representative synthetic non-viral gene carriers with superior transfection efficiency due to its unique “proton sponge effect” for endosomal escape of the gene payload.6 The commercially available 25 kDa branched PEI in particular has been widely considered as the “gold standard” carrier for gene delivery.7 Unfortunately, such high molecular weight (MW) PEI (HPEI) is generally cytotoxic, thus limiting its potential for clinical application, while very low MW PEI


2

Page 3 of 25

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


(LPEI, MW < 2 kDa) is acceptable with respect to cytotoxicity, but has low transfer efficiency. To overcome these limitations, considerable efforts have been made to prepare novel PEI derivatives containing multiple polymer chains such as by grafting large quantity of LPEI to hydrophobic8 and inorganic substrates9, or by crosslinking LPEI via biodegradable linkers.10 However, complex synthesis procedures requiring significant development and optimization are needed to prepare such PEI derivatives. Moreover, transfection efficiencies using these derivatives have generally been investigated for easy-to-transfect established cell lines (HeLa, A549, COS-7)11-13, while efficiencies for hard-to-transfect primary cell lines have rarely been reported. In addition to the use of carriers, membrane disruption is a promising alternative approach for intracellular delivery.14 Membrane disruption is based on physical techniques such as microinjection, electroporation and photoporation to disrupt the plasma membrane and deliver the macromolecular cargo directly to the cytoplasm.15-16 Without using carriers, membranedisruption-mediated systems provide the opportunity to deliver almost any kind of molecule rapidly to a wide range of cell types.3 Recently, we have developed a disruption method based on contact of cells with a laser-activated gold nanoparticle layer (GNPL) which disrupts the cell membrane by a “photoporation” effect.17 Following irradiation, the GNPL converts the absorbed laser energy to heat, leading to enhanced membrane permeability of cells cultured on the surface of GNPL, and thus to facilitated entry of macromolecules into the cytosol. Although this method outperforms the leading commercial reagent, Lipofectamine 2000, in the transfection of hard-totransfect primary cell lines, the efficiency is still not as good as that for easy-to-transfect cell lines. For this platform and all other membrane-disruption-mediated systems, the pDNA is not protected and thus is at risk of degradation by endonucleases in the extracellular space, resulting


3


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 25

in a shortened half-life of the pDNA. This effect has been reported to be a “bottleneck” with respect to transfection efficiency.18 In the work, we developed a novel intracellular delivery platform by integration of LPEI as a carrier with laser-activated GNPL as a membrane disruption agent to deliver pDNA to cells (Scheme 1). The pDNA is protected by LPEI, and disruption of the plasma membrane by the GNPL facilitates delivery of the LPEI/pDNA complex into cytosol. It is shown that the use of LPEI in collaboration with GNPL leads to relatively high transfection efficiency for even hardto-transfect cells, while cell viability is maintained at a high level.

Scheme 1. Schematic of a collaborative intracellular delivery platform based on LPEI as carrier and GNPL as disruption agent. LPEI/pDNA complex is formed by electrostatic interactions. Targeted cells are cultured on the GNPL, and then exposed to laser irradiation to enhance the permeability of the cell membrane, thereby assisting the diffusion of LPEI/pDNA complex from the surrounding medium into the cell.

A carrier must have the ability to condense and load pDNA such that it is protected from the effects of plasma constituents. Since the complexation of anionic pDNA with cationic LPEI occurs via electrostatic interactions, the molar ratio of nitrogen in LPEI to phosphorus in pDNA


4

Page 5 of 25

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


(N/P ratio) is an important variable in the process of forming the complex.6 To investigate the effect of the N/P ratio on complex formation, agarose gel electrophoresis was performed (Figure 1a). It was found that the electrophoretic mobility of the pDNA was completely suppressed at N/P ratios above 10. The resulting LPEI/pDNA complexes formed spherical nanoparticles as observed by transmission electron microscopy (TEM, Figure S1, SI). The size and zeta potential of the nanoparticles prepared with different N/P ratios were measured using a Zetasizer (Figure 1b). With increasing N/P ratio from 10 to 60, the particle size decreased from 259 to 133 nm while the zeta potential increased from 27.2 to 35.5 mV, suggesting that the pDNA was effectively “packaged” by LPEI and that the resulting positively charged moiety bound readily to the negative cell membrane for enhanced endocytosis.19


5


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 25

Figure 1. (a) Agarose gel electrophoresis. (b) Hydrodynamic diameter and zeta potential of LPEI/pDNA complexes formed at various N/P ratios. (c) Relative cell viability (HUVECs). (d) Number of GFP-positive HUVECs (expressing green fluorescence) grown on GNPL surface 48 h after laser irradiation to deliver LPEI/pDNA complexes with various N/P ratios. Data are presented as mean ± SD (n = 6), 0.01 < *p < 0.05, ***p < 0.001.

The in vitro cytotoxicity and gene transfection efficiency of these complexes in contact with GNPL under laser irradiation were evaluated using pDNA encoding green fluorescent protein (GFP) as a reporter gene and human umbilical vein endothelial cells (HUVECs) as a model hardto-transfect cell line. Based on preliminary experiments, it was found that the surface roughness of the GNPL affected the transfection efficiency. Accordingly, we used the optimized roughness condition for this study (see Figures S2 and S3, SI, for details). The cytotoxicity and transfection efficiency were assessed using the CCK-8 assay and by counting GFP-positive cells using fluorescence microscopy, respectively. As shown in Figure 1c, cell viability was well maintained (≈ 80%) at N/P between 10 and 20 but decreased sharply at N/P between 40 and 60, suggesting that high positive charge may result in the aggregation of complexes outside the cell membrane leading to cell necrosis.20 With respect to transfection efficiency, the complex formed at N/P = 20 gave the best performance. At this ratio 94.0 ± 6.3% of HUVECs showed a green fluorescence signal (Figure 1d and Figure S4, SI), much higher than reported for methods such as electroporation or using nanoparticles as carriers.21-22 Taken together, the LPEI/pDNA complex with N/P = 20 gave the best overall performance, with low cytotoxicity and high transfection efficiency, and thus it was chosen for further study.


6

Page 7 of 25

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


To investigate the delivery mechanism and the relative contributions of LPEI and GNPL, transfection efficiency and cytotoxicity were compared with two controls: LPEI/pDNA without laser irradiation and GNPL with laser irradiation without LPEI complexation. In addition, 25 kDa HPEI was included for comparison as it is the widely accepted “gold standard” for gene delivery.7 To minimize cytotoxicity the lowest N/P ratio of 3 was used for HPEI/pDNA complex formation. Although the HPEI/pDNA complex gave a relatively high transfection efficiency (75.1 ± 18.6%, Figure 2), it should be noted that the viability of HUVECs after transfection decreased to less than 5% over 48 h, indicating that HPEI is cytotoxic especially for the fragile primary cell lines.20 In contrast, the viability of HUVECs on GNPL 48 h after laser irradiation was well maintained (Figure 2d), suggesting negligible cytotoxicity of GNPL. On the other hand, without either LPEI complexation or laser irradiation, the transfection efficiency for HUVECs decreased significantly (p < 0.001) based on the number of GFP positive cells and the fluorescence intensity. While for the easy-to-transfect HeLa cells, the laser-irradiated GNPL alone without LPEI complexation was found to be efficient enough to yield ~100% transfection (Figure S5, SI). In contrast, with combined LPEI complexation and laser irradiation, the transfection efficiency was higher than for HPEI (p < 0.05) and cell viability was well maintained. The improved transfection efficiency of this collaborative system was further verified using a plasmid DNA pRL-CMV encoding Renilla luciferase reporter gene (Figure S6, SI). These data suggest that our platform combines the respective advantages of carrier mediation and membrane disruption: i.e. the LPEI condenses the pDNA and protects it from endosomal degradation, while the laser-irradiated GNPL disrupts the plasma membrane and facilitates delivery of LPEI/pDNA to the cytosol. Endothelial cells are considered as hard-totransfect due to their strong self-protective properties and relatively low spreading tendency.23


7


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 25

Efficient pDNA delivery to endothelial cells thus remains challenging. In pursuit of improved efficiency several modified PEI have been developed. These include PEI conjugated to peptides having specific recognition for endothelial cells24, and PEI modified with zwitterionic polymers.25 However, the highest transfection efficiency achieved is of the order of 30%, i.e. much lower than for the collaborative platform reported here. In addition, our method is relatively simple compared to chemical modification.

Figure 2. Transfection of HUVECs with pDNA under different conditions. (I) Using HPEI/pDNA complex (HPEI, N/P ratio = 3); (II) using LPEI/pDNA complex (LPEI, N/P ratio = 20); (III) using GNPL with laser irradiation; (IV) using LPEI/pDNA complex with GNPL with laser irradiation. (a) Typical confocal microscopy images showing the expression of GFP in HUVECs after transfection. Cell nuclei were stained with DAPI (blue fluorescence), and cells


8

Page 9 of 25

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


expressing green fluorescence are GFP-positive cells. Scale bar, 100 µm. (b) Fluorescence intensity. (c) GFP-positive cell number. (d) Cell viability 48 h after gene transfection. Data are presented as mean ± SD (n = 6), *0.01< p < 0.05, ***p < 0.001.

One of the advantages of our GNPL-based photoporation method is its broad applicability across diverse cell types.3 In addition to HUVECs, we investigated the delivery of pDNA to mouse embryonic fibroblasts (mEFs) which are widely employed in the preparation of induced pluripotent stem cells and are also hard-to-transfect.26 The LPEI/pDNA complex was formed using the same N/P ratio and delivered to mEFs under the same conditions as described above for HUVECs. Gene expression and cell viability were evaluated as shown in Figure 3. Similarly to HUVECs, our system gave high transfection efficiency (88.5 ± 9.2%) and high cell viability (99.8 ± 4.1%) for mEFs. Efficient intracellular delivery to mEFs has previously been challenging. For example, the transfection efficiencies of mEFs with pDNA using pDNA/magnetic nanoparticles and optimized electroporation were reported as ~11%27 and ~40%28, respectively. Furthermore, for these methods either sophisticated equipment or complex modification processes are required. In contrast, our system provides a highly efficient and relatively simple approach for intracellular delivery, especially for hard-to-transfect cell lines that are difficult to treat using more traditional methods.


9


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 25

Figure 3. Transfection of mEFs with pDNA under different conditions. (I) Using HPEI/pDNA complex (HPEI, N/P ratio = 3); (II) using LPEI/pDNA complex (LPEI, N/P ratio = 20); (III) using GNPL with laser irradiation; (IV) using LPEI/pDNA complex together with GNPL with laser irradiation. (a) Typical confocal microscopy images showing the expression of GFP in HUVECs after transfection. Cell nuclei were stained by DAPI (blue fluorescence) and cells expressing green fluorescence are GFP-positive cells. Scale bar, 100 µm. (b) Fluorescence intensity. (c) GFP-positive cell number. (d) Cell viability 48 h after gene transfection. Data are presented as mean ± SD (n = 6), *0.01< p < 0.05, ***p < 0.001.


10

Page 11 of 25

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


Finally, we used our platform to deliver functional pDNA to illustrate its potential for endothelialization of artificial blood vessels and vascular stents.22 For this purpose ZNF580 gene, which codes for a zinc finger protein, was chosen. This protein plays a critical role in alleviating atherosclerosis and has been shown to promote the proliferation and migration of endothelial cells.29 The plasmid ZNF580 (pZNF580) was complexed with LPEI and delivered to HUVECs as described above. The transfection efficiency was measured using real-time reverse transcription quantitative polymerase chain reaction (RT-PCR) at the level of ZNF580 gene transcription. The data in Figure 4a show that 48 h after transfection, the relative ZNF580 microRNA (mRNA) content was ~1.3 times higher than that of the negative control group. Moreover, transfection with pZNF580 resulted in a significant increase in both initial attachment and long-term proliferation of HUVECs (Figure S7, SI). In particular, after 48 h culture the density of adherent pZNF580 transfected cells was about twice that of non-transfected cells (Figure 4b). These data suggest that our transfection method can effectively deliver pZNF580 to HUVECs such that proliferation for revascularization purposes may be enhanced.

Figure 4. (a) Relative mRNA expression level of HUVECs measured by RT-PCR 48 h after transfection with pZNF580 using the LPEI/GNPL delivery system. (b) Comparison of numbers


11


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 25

of HUVECs cultured on the surface of GNPL with and without transfection with pZNF580. Data are presented as mean ± SD (n = 6), 0.01 < *p < 0.05.

In summary, we have developed an effective platform for intracellular delivery using LPEI as carrier and laser-irradiated GNPL as photoporation agent for cell membrane disruption. Using this collaborative platform, high efficiency delivery of pDNA to two hard-to-transfect cell lines, HUVECs and mEFs, was achieved without compromising cell viability. HUVECs transfected with the functional gene ZNF580 using our platform may have potential for endothelialization of blood contacting devices. Considering efficacy and convenience, we believe that our method has potential in many areas of biological research and in clinical applications.

ASSOCIATED CONTENT Additional text with details on materials, cell culture, preparation and characterization of gold nanoparticle layer (GNPL), preparation and characterization of pGFP/LPEI complexes, transfection of HeLa cells with pGFP via GNPL of varying roughness, transfection of HeLa cells, HUVECs, and mEFs with pGFP by LPEI/GNPL system, transfection of HUVECs with pRL-CMV or pZNF580 by LPEI/GNPL systems, cell counting assay and statistical analysis; seven figures showing characterization of pGFP/LPEI complexes and GNPL surfaces, transfection of HeLa cells with pGFP via GNPL of varying roughness, comparison of transfection efficiency between HeLa cells and HUVECs, and transfection of HUVECs with pGFP, pRL-CMV, or pZNF580 by LPEI/GNPL system. This material is available free of charge via the Internet at http://pubs.acs.org.


12

Page 13 of 25


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 Paragon Plus Environment

13


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 25

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources National Natural Science Foundation of China (21404076, 21334004 and 21674074), the Natural Science Foundation of Jiangsu Province (BK20140316).

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21404076, 21334004 and 21674074), the Natural Science Foundation of Jiangsu Province (BK20140316). The authors thank Prof. John L. Brash for helpful discussions, Mr. Shuaibing Jiang for technical assistance with scanning electron microscopy (SEM) and Mr. Changming Hu for technical assistance with transmission electron microscopy (TEM).

REFERENCES 1.

Brannon-Peppas, L.; Blanchette, J. O., Nanoparticle and Targeted Systems for Cancer

Therapy. Adv. Drug Delivery Rev. 2012, 64, 206-212.


14

Page 15 of 25

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


2.

Naldini, L., Gene Therapy Returns to Centre Stage. Nature 2015, 526, 351-360.

3.

Stewart, M. P.; Sharei, A.; Ding, X.; Sahay, G.; Langer, R.; Jensen, K. F., In Vitro and Ex

Vivo Strategies for Intracellular Delivery. Nature 2016, 538, 183-192. 4.

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. 5.

Mintzer, M. A.; Simanek, E. E., Nonviral Vectors for Gene Delivery. Chem. Rev. 2008,

109, 259-302. 6.

Yue, Y.; Jin, F.; Deng, R.; Cai, J.; Dai, Z.; Lin, M. C. M.; Kung, H.-F.; Mattebjerg, M.

A.; Andresen, T. L.; Wu, C., Revisit Complexation Between DNA and Polyethylenimine — Effect of Length of Free Polycationic Chains on Gene Transfection. J. Controlled Release. 2011, 152, 143-151. 7.

Kircheis, R.; Wightman, L.; Wagner, E., Design and Gene Delivery Activity of Modified

Polyethylenimines. Adv. Drug Delivery Rev. 2001, 53, 341-358. 8.

Teo, P. Y.; Yang, C.; Hedrick, J. L.; Engler, A. C.; Coady, D. J.; Ghaem-Maghami, S.;

George, A. J. T.; Yang, Y. Y., Hydrophobic Modification of Low Molecular Weight Polyethylenimine for Improved Gene Transfection. Biomaterials 2013, 34, 7971-7979. 9.

Tian, H.; Guo, Z.; Chen, J.; Lin, L.; Xia, J.; Dong, X.; Chen, X., PEI Conjugated Gold

Nanoparticles: Efficient Gene Carriers with Visible Fluorescence. Adv. Healthcare Mater. 2012, 1, 337-341.


15


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 25

10. Peng, Q.; Hu, C.; Cheng, J.; Zhong, Z.; Zhuo, R., Influence of Disulfide Density and Molecular Weight on Disulfide Cross-Linked Polyethylenimine as Gene Vectors. Bioconjugate Chem. 2009, 20, 340-346. 11. Yamada, H.; Loretz, B.; Lehr, C.-M., Design of Starch-Graft-PEI Polymers: an Effective and Biodegradable Gene Delivery Platform. Biomacromolecules 2014, 15, 1753-1761. 12. Yang, C.; Cheng, W.; Teo, P. Y.; Engler, A. C.; Coady, D. J.; Hedrick, J. L.; Yang, Y. Y., Mitigated Cytotoxicity and Tremendously Enhanced Gene Transfection Efficiency of PEI through Facile One-Step Carbamate Modification. Adv. Healthcare Mater. 2013, 2, 1304-1308. 13. Feng, L.; Zhang, S.; Liu, Z., Graphene Based Gene Transfection. Nanoscale 2011, 3, 1252-1257. 14. Ding, X.; Stewart, M. P.; Sharei, A.; Weaver, J. C.; Langer, R. S.; Jensen, K. F., HighThroughput Nuclear Delivery and Rapid Expression of DNA via Mechanical and Electrical CellMembrane Disruption. Nat. Biomed. Eng. 2017, 1, 0039. 15. Gurtovenko, A. A.; Anwar, J.; Vattulainen, I., Defect-Mediated Trafficking across Cell Membranes: Insights from in Silico Modeling. Chem. Rev. 2010, 110, 6077-6103. 16. Meacham, J. M.; Durvasula, K.; Degertekin, F. L.; Fedorov, A. G., Physical Methods for Intracellular Delivery: Practical Aspects from Laboratory Use to Industrial-Scale Processing. J. Lab. Autom. 2014, 19, 1-18. 17. Lyu, Z.; Zhou, F.; Liu, Q.; Xue, H.; Yu, Q.; Chen, H., A Universal Platform for Macromolecular Delivery into Cells Using Gold Nanoparticle Layers via the Photoporation Effect. Adv. Funct. Mater. 2016, 26, 5787-5795.


16

Page 17 of 25

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


18. Mansouri, S.; Lavigne, P.; Corsi, K.; Benderdour, M.; Beaumont, E.; Fernandes, J. C., Chitosan-DNA Nanoparticles as Non-Viral Vectors in Gene Therapy: Strategies to Improve Transfection Efficacy. Eur. J. Pharm. Biopharm. 2004, 57, 1-8. 19. Kievit, F. M.; Veiseh, O.; Bhattarai, N.; Fang, C.; Gunn, J. W.; Lee, D.; Ellenbogen, R. G.; Olson, J. M.; Zhang, M., PEI-PEG-Chitosan Copolymer Coated Iron Oxide Nanoparticles for Safe Gene Delivery: Synthesis, Complexation, and Transfection. Adv. Funct. Mater. 2009, 19, 2244-2251. 20. Hall, A.; Lachelt, U.; Bartek, J.; Wagner, E.; Moghimi, S. M., Polyplex Evolution: Understanding Biology, Optimizing Performance. Mol. Ther. 2017, 25, 1-15. 21. Yang, J.; Li, Q.; Yang, X.; Feng, Y.; Ren, X.; Shi, C.; Zhang, W., Multitargeting Gene Delivery Systems for Enhancing the Transfection of Endothelial Cells. Macromol. Rapid Commun. 2016, 37, 1926-1931. 22. Yang, J.; Feng, Y.; Zhang, L., Biodegradable Carrier/Gene Complexes to Mediate the Transfection and Proliferation of Human Vascular Endothelial Cells. Polym. Adv. Technol. 2015, 26, 1370-1377. 23. Doerks, T.; Copley, R. R.; Schultz, J.; Ponting, C. P.; Bork, P., Systematic Identification of Novel Protein Domain Families Associated with Nuclear Functions. Genome Res. 2002, 12, 47-56. 24. Hao, X.; Li, Q.; Lv, J.; Yu, L.; Ren, X.; Zhang, L.; Feng, Y.; Zhang, W., CREDVWLinked Polymeric Micelles as a Targeting Gene Transfer Vector for Selective Transfection and Proliferation of Endothelial Cells. ACS Appl. Mater. Interfaces 2015, 7, 12128-12140.


17


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 25

25. Yang, J.; Hao, X.; Li, Q.; Akpanyung, M.; Nejjari, A.; Neve, A. L.; Ren, X.; Guo, J.; Feng, Y.; Shi, C.; Zhang, W., CAGW Peptide- and PEG-Modified Gene Carrier for Selective Gene Delivery and Promotion of Angiogenesis in HUVECs in Vivo. ACS Appl. Mater. Interfaces 2017, 9, 4485-4497 26. Zhu, K.; Li, J.; Lai, H.; Yang, C.; Guo, C.; Wang, C., Reprogramming Fibroblasts to Pluripotency Using Arginine-Terminated Polyamidoamine Nanoparticles Based Non-Viral Gene Delivery System. Int. J. Nanomed. 2014, 9, 5837-5847. 27. Fouriki, A.; Dobson, J., Nanomagnetic Gene Transfection for Non-Viral Gene Delivery in NIH 3T3 Mouse Embryonic Fibroblasts. Materials 2013, 6, 255-264. 28. Okita, K.; Nakagawa M.; Hyenjong, H.; Ichisaka T.; Yamanaka S., Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors. Science 2008, 322, 949-953. 29. Feng, Y.; Guo, M.; Liu, W.; Hao, X.; Lu, W.; Ren, X.; Shi, C.; Zhang, W., Co-SelfAssembly of Cationic Microparticles to Deliver pEGFP-ZNF580 for Promoting the Transfection and Migration of Endothelial Cells. Int. J. Nanomed. 2017, 12, 137-149.


18

Page 19 of 25

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 Graphic


19


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

TOC 119x65mm (300 x 300 DPI)


Page 20 of 25

Page 21 of 25

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


Scheme 1 150x82mm (300 x 300 DPI)



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

Fig1 150x120mm (300 x 300 DPI)


Page 22 of 25

Page 23 of 25

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


Fig2 150x116mm (300 x 300 DPI)



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

Fig3 150x121mm (300 x 300 DPI)


Page 24 of 25

Page 25 of 25

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


Fig4 170x63mm (300 x 300 DPI)


Intracellular Delivery Platform for âRecalcitrantâ Cells: When Polymeric

Recommend Documents

Intracellular Delivery Platform for âRecalcitrantâ Cells: When Polymeric