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This paper presents the development of two novel non\u2010viral vectors by cross\u2010linking glycerol molecules with low molecular weight polyethylenimine (PEI). The vectors, namely, HG\u2010PEI (45 mol% glycerol content) and LG\u2010PEI (9 mol% glycerol content) have apparently similar DNA binding, DNA unpacking and cellular uptake abilities but differ in buffering capacity. The cellular uptake and subsequent transfection efficiency of LG\u2010PEI is superior to commercially available PEI 25\u2009k. Interestingly, although the cellular uptake of HG\u2010PEI is higher than that of PEI 25\u2009k, the transgene expression by HG\u2010PEI\u2010mediated transfection is very low. Inhibitor and co\u2010localization studies demonstrate the mechanism of endocytosis and formation of endosomes prone to lysosomal lysis of HG\u2010PEI polyplexes as a consequence of its weak buffering capacity. Importantly, when the lysosomal lysis is inhibited, the transgene expression of HG\u2010PEI\u2010mediated transfection increases by 9\u2010fold of its initial capacity which is comparable to the transfection efficiency of PEI 25\u2009k. These results indicated that the buffering capacity of the polymers primarily impacts endosomal escape and subsequent transfection efficiency. Furthermore, this study highlights the significance of cross\u2010linkers in optimizing the buffering capacity when designing polymers for gene delivery.<jats:inline-graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"graphic\/mabi201400463-gra-0001.png\" xlink:title=\"mabi201400463-gra-0001\"\/><\/jats:p><\/jats:sec>","DOI":"10.1002\/mabi.201400463","type":"journal-article","created":{"date-parts":[[2015,1,8]],"date-time":"2015-01-08T13:22:40Z","timestamp":1420723360000},"page":"622-635","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":56,"title":["Tuning the Buffering Capacity of Polyethylenimine with Glycerol Molecules for Efficient Gene Delivery: Staying In or Out of the Endosomes"],"prefix":"10.1002","volume":"15","author":[{"given":"Bijay","family":"Singh","sequence":"first","affiliation":[{"name":"Department of Agricultural Biotechnology Research Institute for Agriculture and Life Sciences, Seoul National University Seoul 151\u2010921 Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sushila","family":"Maharjan","sequence":"additional","affiliation":[{"name":"Department of Agricultural Biotechnology Research Institute for Agriculture and Life Sciences, Seoul National University Seoul 151\u2010921 Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tae\u2010Eun","family":"Park","sequence":"additional","affiliation":[{"name":"Department of Agricultural Biotechnology Research Institute for Agriculture and Life Sciences, Seoul National University Seoul 151\u2010921 Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tao","family":"Jiang","sequence":"additional","affiliation":[{"name":"Department of Agricultural Biotechnology Research Institute for Agriculture and Life Sciences, Seoul National University Seoul 151\u2010921 Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sang\u2010Kee","family":"Kang","sequence":"additional","affiliation":[{"name":"Institute of Green\u2010Bio Science and Technology Seoul National University Kangwon\u2010Do 232\u2010916 Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yun\u2010Jaie","family":"Choi","sequence":"additional","affiliation":[{"name":"Department of Agricultural Biotechnology Research Institute for Agriculture and Life Sciences, Seoul National University Seoul 151\u2010921 Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chong\u2010Su","family":"Cho","sequence":"additional","affiliation":[{"name":"Department of Agricultural Biotechnology Research Institute for Agriculture and Life Sciences, Seoul National University Seoul 151\u2010921 Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2015,1,8]]},"reference":[{"key":"e_1_2_6_1_1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.gt.3302592"},{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.98.3.1200"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.1211\/002235703765951311"},{"key":"e_1_2_6_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/nn3049494"},{"key":"e_1_2_6_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/jgm.696"},{"key":"e_1_2_6_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0169-409X(97)00109-9"},{"key":"e_1_2_6_7_1","doi-asserted-by":"publisher","DOI":"10.1006\/bioo.1994.1025"},{"key":"e_1_2_6_8_1","first-page":"1074","volume":"3","author":"Boussif O.","year":"1996","journal-title":"Gene Ther."},{"key":"e_1_2_6_9_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.92.16.7297"},{"key":"e_1_2_6_10_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.192581499"},{"key":"e_1_2_6_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrd1775"},{"key":"e_1_2_6_12_1","doi-asserted-by":"publisher","DOI":"10.1021\/ar7002336"},{"key":"e_1_2_6_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2004.11.015"},{"key":"e_1_2_6_14_1","doi-asserted-by":"publisher","DOI":"10.1002\/jgm.1252"},{"key":"e_1_2_6_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biomaterials.2012.06.037"},{"key":"e_1_2_6_16_1","doi-asserted-by":"publisher","DOI":"10.1021\/mp300072r"},{"key":"e_1_2_6_17_1","volume":"10","author":"Lee W. 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