Polyplex Micelle with pH-Responsive PEG Detachment and

Subscriber access provided by UNIV OF ESSEX

Article

Polyplex Micelle with pH-Responsive PEG Detachment and Functional Tetraphenylene Incorporation to Promote Systemic Gene Expression Zhu Jiang, Qixian Chen, Xi Yang, Xiyi Chen, Zhen Li, De-E Liu, Wei Li, Yingjie Lei, and Hui Gao Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00557 • Publication Date (Web): 29 Sep 2017 Downloaded from http://pubs.acs.org on October 1, 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.

Bioconjugate Chemistry 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 31

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

Bioconjugate Chemistry

Polyplex Micelle with pH-Responsive PEG Detachment and Functional Tetraphenylene Incorporation to Promote Systemic Gene Expression Zhu Jiang,a Qixian Chen,b,* Xi Yang,c Xiyi Chen,d Zhen Li,e De-E Liu,a Wei Li,a Yingjie Lei,a Hui Gao a,*

a

School of Chemistry and Chemical Engineering, Tianjin Key Laboratory of Organic

Solar Cells and Photochemical Conversion, Tianjin University of Technology, Tianjin 300384 (P. R. China) E-mail: [email protected]

b

School of Life Science and Biotechnology, Dalian University of Technology, No. 2

Linggong Road, Dalian 116024 (P. R. China) E-mail: [email protected]

c

Department of Neurosurgery, South Campus, Renji Hospital, Shanghai Jiao Tong

University School of Medicine, Shanghai 200127 (P. R. China)

d

School of Public Health, Dalian Medical University, No. 9 West Section Lvshun

South Road, Dalian 116044 (P. R. China)

e

College of Pharmacy, Dalian Medical University, No. 9 West Section Lvshun South

Road, Dalian 116044 (P. R. China)

ACS Paragon Plus Environment


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 31

ABSTRACT: Tetraphenylene (TPE), characterized to be a lipophilic and aggregation-induced-emissive fluorophore, was attempted to incorporate into an electrostatic

self-assembled

polyethyleneimine-poly(ethylene

glycol)

(PEI-PEG)/plasmid DNA (pDNA) complexed micelle. The hydrophobic character of TPE appeared to drive higher degree condensation of the pDNA payload, which consequently resulted in not only strengthened colloidal stability of the constructed polyplex micelle but also improved biocompatibility by virtue of the elevated PEG crowdedness owing to the TPE-induced collapse-down of pDNA. These beneficial consequences potentially permitted a larger number of polyplex micelles internalized into the cells. PEG segments were designed to enable selective detachment from polyplex micelle in acidic milieu, e.g. the tumor microenvironment, and intracellular endosome compartment, based on the strategic arrangement of acid-responsive cleavable linkage between PEG and PEI. Upon PEG detachment, the exposure of cationic PEI/TPE polyplex was allowed for direct interaction with the cell membrane, endosome membrane and charged intracellular species, thus promoting cell internalization, endosome escape and the release of the pDNA payload. Of note, this association of cationic PEI/TPE polyplex with the endosome membrane could be further facilitated with the aid of lipophilic TPE, thereby eliciting pronounced destabilization potency to the endosome membrane and exerting endosome escape function. Eventually, the proposed system ensemble of these facile strategies, including responsive PEG detachment and functional TPE incorporation, was validated to provide efficient gene expression in the targeted tumors with appreciable safety profile via systemic administration.


Page 3 of 31

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


INTRODUCTION The intriguing potential of gene therapy in restoring the expression of the desirable functional proteins has spurred intensive researches in manufacture of gene transport systems for pursuit of the therapeutic outcomes.1-3 Pertaining to the concerns, particularly for the fatal immunogenicity of viral vectors as gene transport systems,4 synthetic delivery vehicles were stressed for development to endow the critical functionalities to circumvent the string of extracellular and intracellular predefined biological barriers. Cationic

gene carriers

(e.g.

polycations,

cationic

lipids)5

which

could

electrostatically complex with opposite-charged anionic plasmid DNA (pDNA) have emerged as a tempting modality of gene transport systems in view of their ease in DNA encapsulation, wide availability in chemistry engineering6 and low cost in scale-up manufacture.7 Among the diverse cationic transfection compounds, polyethyleneimine gained immense popularity due to its important functionalities, particularly its intriguing affinity with cell membrane for entry into cells8 and its destabilizing potency to endosome membrane for retrieving the DNA payload from endosome entrapment.9, 10 Nevertheless, the cationic surface of PEI-based polyplex also has the propensity of electrostatically interacting with the charged biological structures (e.g. distorting and destabilizing the structure of plasma membrane), consequently conducing to the poor biocompatibility and unfavorable cytotoxicity.11, 12

A valid strategy to address these drawbacks is manufacture of a spatial shell [e.g. poly(ethylene glycol): PEG] surrounding the cationic polyplex, which has been documented to afford markedly improved biocompatibility and safety profile.13, 14 Yet, the cell internalization and endosome escape potency of PEI were also diminished due to the spatial presence of biocompatible PEG shell for the minimized reaction with these biological membranes.15, 16 To overcome this dilemma of PEGylation, we attempted a PEG detachment scheme by introducing a pH-responsive cleavable Schiff-base linkage between PEI and PEG segments (Scheme 1). Following this scheme, the exposure of cationic PEI polyplex



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

and thus its association to the cell membrane and further endosome membrane to execute internalization and destabilization to the endosome membrane was encouraged as a consequence of the selective breakage of Schiff-base linkage in the acidic tumor tissues and intracellular endosome compartment. To further facilitate the association affinity of PEI polyplex with the biological membranes, we strived to supplement a lipophilic TPE component into the polyplex based on electrostatic association of anionic tetraphenylene (TPE) and cationic PEI polyplex with the aim of amplifying the cell internalization activity and endosome membrane destabilization potency and thus gene transfection activity. Noteworthy was the hydrophobic character of TPE, which is assumed to increase the interfacial energy of inner PEI polyplex and outer aqueous medium.17-19 A higher degree of pDNA condensation could be readily anticipated to minimize the induced unfavorable interfacial energy, consequently accounting for an ultimate formation with improved colloidal stabilities and subsequent promoted internalization into the cells. Moreover, the PEG crowdedness of polyplex micelle post TPE incorporation is also anticipated to elevate upon the TPE-induced collapse-down of pDNA and squeeze of the tether PEG segments, which potentially conduced to improved biocompatibility and safety profile.

Scheme 1. Schematic illustration of PEI-PEG/TPE/DNA polyplex micelle formulation and the journey of gene transportation.

RESULTS AND DISCUSSION


Page 4 of 31

Page 5 of 31

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


Polymer synthesis and characterizations. The starting material of PEI (25 kDa) was used to react with PEG by the following synthetic Scheme 1. In brief, aldehyde moiety was functionalized at the terminus of poly(ethylene glycol) methyl ether to obtain

PEG-CHO

through

esterification

between

carboxyl

group

from

p-formylbenzoic acid and the terminal hydroxyl group of PEG (2 kDa). Furthermore, the aldehyde-terminal functionalized PEG was transferred to react with the primary amine from PEI to yield PEI-PEG (Fig. S1), characterized to possess a pH-responsive cleavable Schiff-base linkage. The yielded PEI-PEG copolymer was characterized by 1H-NMR measurement. The characteristic peaks of PEG (δ = 3.7 ppm) and PEI (δ: 2.10 ～ 3.10 ppm) were confirmed (Fig. S2). Meanwhile, an up-field shift of the protons assigned to the benzene unit indicated the graft reaction of PEG-CHO (benzene adjacent to CHO) and PEI. Specifically, the appearance of peak at δ 8.05 ppm affirmed the formation of Schiff-base (-HC=N-) linkages.20 Disappearance of the aldehyde peak (δ = 10.07 ppm) in the 1H-NMR spectrum of PEI-PEG also validated the reaction between PEG-CHO and PEI. The grafting ratio of PEG-CHO to PEI was calculated to be approximate 9.6 PEG segments per PEI based on 1H-NMR spectrum of PEI-PEG. Meanwhile, the GPC characterizations for the starting polymer of PEI and the yielded copolymer of PEI-PEG confirmed the formation of high molecule weight product of PEI-PEG (Table S1). Moreover, FT-IR spectroscopy analysis also confirmed the production of PEI-PEG copolymer. Note that the peak at 1114 cm-1 represented the ether bond from PEG segments, aldehyde stretching bond appeared at 1722 cm-1, and the peak at 3420 cm-1 could be assigned to the bending vibration of N-H belonging to PEI21 (Fig. S3). The disappearance of aldehyde signal was observed, meanwhile the emergence of peak at 1634 cm-1 validated the formation of Schiff-base linkage between PEG and PEI.22 On the other hand, the functional component TPE-(COOH)4 (abbreviated as TPE hereafter) was synthesized according to the procedures reported previously,23 and its detailed characterizations were described in Fig. S2 and S3.



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 31

To obtain a direct evidence of TPE being incorporated into the polycationic PEI-PEG, fluorescence emission was recorded for the reaction solution of TPE (constant concentration) and PEI-PEG solution (varied concentrations). In respect to the distinctive aggregation-induced emission character of TPE, progressive rise in fluorescence emissive intensity of TPE along a rising concentration of PEI-PEG (Fig. 1)

suggested

the

successful

incorporation

of

TPE

molecules

into

the

polycationic-based formation. Most likely, the anionic charges of carboxyl groups from TPE induced electrostatic association with the cationic PEI to result in complexed formation. Consequently, the restricted motion of TPE due to polyion complexation conduced to a pronounced jump in fluorescence emission of TPE as a result of its unique aggregation-induced emission behaviors.24, 25

Fig. 1 Fluorescence emissive spectrum of TPE solution incubated with varying concentrations of PEI-PEG in PBS (pH 7.4).

Polyplex preparation and characterizations. The synthesized copolymer PEI-PEG was used to prepare polyplex micelle based on electrostatic complexation by mixing with plasmid DNA (pDNA) solution in PBS buffer (10 mM, pH 7.4) under vortex at N/P ratios of 3, 6 and 10 (defined as the molar ratio of amine groups from PEI to the phosphate groups from pDNA). The electrostatic complexation was confirmed by gel


Page 7 of 31

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


electrophoresis (Fig. S4). Clearly, pDNA was subjected to complete electrostatic neutralization in all the formulations (PEI, PEI-PEG, and PEI-PEG/TPE) at all N/P ratios of 3, 6 and 10, as evidenced by pDNA being static in the electrophoresis field. In consistency,

SEM captured nanoscaled DNA-condensed structures as ellipsoids with average major-axis length of 454 nm and minor-axis length of 155 nm (Fig. 2A). To explore the impact of TPE incorporation on the structure of polyplex micelle from PEI-PEG, SEM measurement was carried out for polyplex micelle of PEI-PEG prior to and post TPE incorporation. As for the polyplex micelle post TPE incorporation, SEM captured markedly higher degree condensed structures of ellipsoids with average major-axis length of approximate 109 nm and average minor-axis length of approximate 73 nm (Fig. 2B). This observation was distinctive from the original PEI-PEG polyplex micelle. This promoted condensation should be attributable to the hydrophobic residues of TPE. The association of anionic TPE to the cationic PEI polyplex mounted the interfacial energy between the inner PEI polyplex and outer aqueous medium. Accordingly, the hydrophobic character of TPE could serve as an additional drive force to stimulate a higher degree condensation of pDNA.26, 27

Fig. 2 SEM morphologies of PEI-PEG/pDNA polyplex micelles prior to TPE incorporation (A) and post TPE incorporation (B). Scale bar: 200 nm.

“Stealth” property of PEGylation. It can be speculated that the incorporation of the hydrophobic TPE into polyplex micelle can promote its colloidal stabilities. This is important for the electrostatic-based self-assembly formulation given that there are plenty of charged biological species and structures in the biological milieu, which potentially cause the structure dissociation of the polyplex micelle. Moreover, the



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 31

prompted condensation due to TPE incorporation is postulated to result in a high-degree of PEGylation crowdedness, which induced collapse-down of the pDNA payload. Hence, PEGylation was consequently advocated for the polyplex micelle to afford improved biocompatibility and stealthy behavior.28,

29

To verify this

speculation, the size derivations of PEI-PEG/pDNA polyplex micelle and PEI/pDNA polyplex in presence of BSA were monitored by dynamic light scattering (DLS) measurement. Regarding to the possible interference of polyplexes self-aggregation, the aforementioned samples (PEI-PEG/pDNA polyplex micelle and PEI/pDNA polyplex) were incubated in BSA-free buffer solution as the standard, which exhibited limited increase in DLS size. Upon exposure to BSA, the size of PEI/pDNA polyplex was subjected to marked increase (approximately two-fold in diameter), whereas the size derivation of PEI-PEG/pDNA polyplex micelle was limited despite extended incubation (Fig. 3), suggesting the benefit of PEGylation in reducing non-specific protein adhesion. Furthermore, the resistance of PEGylated structure prior to and post TPE incorporation to BSA adsorption was quantitatively estimated (Table 1). In agreement with our speculations, the control sample of PEI-based polyplex was subjected to intensive protein adsorption. Reduced BSA adsorption was observed for the PEI-PEG-based polyplex micelle. Of note, minimal BSA adsorption was confirmed for PEI-PEG/TPE, indicating the formation of condensed PEG shell upon incorporation of hydrophobic TPE. To this end, both PEGylation and TPE have been validated to be important in pursuit of the biocompatibility (Fig. S5) and stealthy functionality.


Page 9 of 31

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


Fig. 3 Size derivations of PEI/DNA polyplex and PEI-PEG/DNA polyplex micelle in presence and absence of BSA by DLS measurement. Data represent mean ± S.D. (n = 3).

Table 1. Resistance of diverse polyplex formations to BSA adsorption. Sample

Quantification of BSA adsorption to the structure (mg/mg)*

PEI

9.27±0.49

PEI-PEG

4.02±0.12

PEI-PEG/TPE

0.41±0.08

*

Data represent mean ± S.D. (n=3).

Stability evaluation of the polyplex micelle. The resistance of polyplex micelle against exchange reaction with anionic heparin was evaluated. Here, the polyplex micelle prior to and post TPE incorporation were incubated with heparin at varying concentrations, and dissociation behaviors of the polyplex micelle to release pDNA through polyion exchange reaction were determined by gel electrophoresis of the reaction solution. Apparent dissociation to release DNA was observed to start from heparin concentration of 20 µg/mL for the sample of polyplex micelle prior to TPE incorporation (Fig. 4A). Yet, the polyplex micelle with functional components of TPE showed markedly improved resistance to dissociation even at heparin concentration



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

exceeding 30 µg/mL (Fig. 4B). These results approved the functional role of the TPE incorporation in promoting colloidal stabilities and advocating PEGylation stealthy function for reducing non-specific interactions of anionic biological species.

Fig. 4 Resistance of polyplex micelles against the exchange reaction of anionic heparin at varying concentrations (µg/mL). (A): PEI-PEG/pDNA polyplex micelle prior to TPE incorporation (PEI-PEG/pDNA) and (B): PEI-PEG/pDNA polyplex micelle post TPE incorporation (PEI-PEG/TPE/pDNA).

pH-responsive cleavage of the Schiff-base linkage. Although the improved structural stabilities are postulated to permit a larger number of polyplex micelle internalized into the cells, PEGylated polyplex micelles were subjected to poor cell internalization due to the reduced affinity to the cell membrane and inferior activity to exert endosomal escape, and ultimately low transfection efficiency.30-32 To address these drawbacks, polyplex micelles have to afford adequate facilities to internalize into the cells and retrieve polyplex micelle from endosome entrapment. Recent investigations suggested that the polyplex micelles constructed from cationic species could manage to internalize into cells via electrostatic interaction with cell membrane and escape from endosome by means of endosome membrane destabilization process.33, 34 Apparently, the improved PEGylation crowdedness as a result of TPE incorporation should lower the interactive affinity of cationic PEI and anionic biological membranes. To address this drawback, we attempted a pH-responsive cleavable linkage between PEI and PEG segments, where the linkage of Schiff-base was subjected to selective hydrolysis reaction in the acidic milieu. Specifically, once approaching mild acidic tumor microenvironment (pH～6.8), PEG shell of polyplex


Page 10 of 31

Page 11 of 31

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


micelle is assumed to be susceptible to detachment, thereby facilitating the cell internalization. What is more, the residual tethered PEG chains was subjected to complete detachment in strong acidic late endosome/lysosome (pH～5.0) so as for exposure of the cationic PEI,

thereby accelerating endosome

membrane

destabilization. Moreover, the lipophilic TPE could further facilitate the interactive affinity of the polyplex with endosome membrane. To verify the facile pH-responsive cleavage of the Schiff-base linkage, 1H-NMR spectra was recorded for the polymer of PEI-PEG at pH 7.4, pH 6.8 and pH 5.0. The resembled 1H-NMR (Fig. 5A) spectrum of PEI-PEG at pH 7.4 to its original spectrum verified its molecular structure stability in physiological condition. On the contrary, pertaining to the 1H-NMR of PEI-PEG at pH 6.8 and pH 5.0 (Fig. 5B and 5C), the emergence of the aldehyde residue at 10.07 ppm together with the transformation of the broad peak between 7.5 and 8.25 ppm into two individual doublets approved the hydrolysis of Schiff-base linkage into original aldehyde and primary amine at acidic milieu. Meanwhile, zeta potential measurement for the polyplex micelle at pH 7.4, 6.8 and 5.0 (Table 2) observed a pronounced jump at the surface net charge responsive to the decrease in pH of incubation milieu, indicating the readily PEG detachment behavior under incubation in acidic environment. Following the PEG detachment in the acidic milieu, it is readily to anticipate dissociation of the residue PEI/pDNA polyplex to release the pDNA payload since polyplex formulation without PEGylation is more susceptible to exchange reaction with the intracellular charged species. To verify it, we employed an EtBr assay to trace the dissociation behavior of PEI-PEG/pDNA polyplex micelle at pH 7.4 and pH 5.0, wherein dextran sulfate was used as a modal intracellular charged molecule to study the potential dissociation behaviors of the tested PEI-PEG/pDNA polyplex micelle. The S/P ratio was defined as the molar ratio between the sulfur from dextran sulfate and the phosphate from pDNA. Note that pronounced jump in fluorescence emission of EtBr could achieve when EtBr binds to pDNA. Hence, EtBr could serve a useful probe to distinguish complexed pDNA (complexation hinders the bind of EtBr and pDNA) and released pDNA. In Fig. 6, facilitated dissociation of polyplex micelle



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

was observed at pH 5.0, as opposed to reluctant release of pDNA from polyplex micelle despite a high concentration of competing dextran sulfate at pH 7.4. To this end, the strategic arrange of acid-liable PEGylation detachment validated its important role in facilitate the release of the pDNA payload in the acidic intracellular compartment, which should endow substantial benefit for the subsequent transcription activity.

Fig. 5 1H-NMR spectra of PEI-PEG: (A) in D2O at pH 7.4, adjusted by adding NaOD; (B) in D2O at pH 6.8, adjusted using DCl; (C) in D2O at pH 5.0, adjusted using DCl.

Table 2. Zeta potential of PEI-PEG/TPE/pDNA polyplex micelles at pH 7.4, 6.8 and 5.0.* pH

zeta potential (mV)

7.4

9.27±0.57

6.8

13.6±1.39

5.0

21.9±1.49

*

Data represent mean ± S.D. (n=3).


Page 12 of 31

Page 13 of 31

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


Fig. 6 The dissociation behaviors of PEI-PEG/pDNA polyplex micelle estimated by an EtBr assay. Data represent mean ± S.D. (n = 3).

CLSM observation of intracellular distribution of polyplex micelles. To verify the functional role of pH responsive linkage and TPE incorporation in promoting cell transfection, the cell internalization profile and endosome escape ability of PEI/pDNA and PEI-PEG/TPE/pDNA were evaluated. Similar pattern of the red dots were observed to distribute inside the cells treated with PEI-PEG/TPE/Cy5-DNA polyplex micelle and PEI/Cy5-DNA polyplex (Fig. 7). This result is consistent with our speculation that the strengthened colloidal stability by virtue of TPE incorporation could overcome the reluctant cell internalization of PEGylated formation. To our interests, as compared to the high endosome escape efficiency from commercial transfection agent of PEI (colocalization ratio of late endosome/lysosome and Cy5-pDNA: 57%) (Fig. 7A), exceedingly appreciable endosome escape activity for PEI-PEG/TPE (21%) was observed (Fig. 7B). This result validated our strategies of arranging acidic-liable detachment of PEG shell and lipophilic component of TPE in pursuit of endosome escape activity. Another noteworthy was the intracellular distribution of lipophilic component of TPE, aside from being colocalizing with polyplex micelle, was found to abundant in colocalization of membrane structure (e.g. nucleus membrane, as evidenced by TPE encompassing the contour of the nucleus) (Fig. 7). This important observation indicated the functional role of lipophilic TPE in



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

facilitating association of polyplex micelles with the plasma membrane (e.g. endosome membrane),35-37 thereby encouraging the subsequent endosome membrane destabilization activity by cationic PEI. To this end, the results verified the functional detachment scheme of PEG shell and lipophilic TPE incorporation for cell internalization and destabilization reaction to the endosome membrane as a worthy strategy to promote transfection. Transfection efficiency. The in vitro transfection efficiency of all PEI derivatives-based gene delivery formulations at different N/P ratios was evaluated in A549 and HeLa cell lines (Fig. 8). For all tested formulations, the gene expression level increased with increasing N/P ratio in both cells. This is consistent with the previous research that high N/P ratio is favorable in construction of stable polyplex formation and promotion of cell internalization.33,34 Remarkably, PEI-PEG copolymer-based polyplex micelle mediated comparable transfection efficiency to PEI, and even higher transfection efficiency was observed at relative low N/P ratios in HeLa cells. Specifically, the transfection efficiency mediated by PEI-PEG/TPE polyplex micelle exhibited approximately 3.3 times higher than PEI-PEG polyplex at N/P ratio of 6 in HeLa cells. This is particularly intriguing in light that the reduced dosage of cationic materials for therapeutic is crucial in terms of safety concerns. To this end, the obtained results validated the strategic pH-responsive PEG detachment and functional TPE components as a facile and valid approach to promote the transfection efficiency of PEGylated cationic gene delivery systems.


Page 14 of 31

Page 15 of 31

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


Fig. 7 CLSM observation of intracellular distribution of PEI-based polyplex (A) and PEI-PEG/TPE-based polyplex micelle (B) in A549 cells. Endosomes were stained by Lyso Tracker Green (green), pDNA was labeled by Cy5 (red). Merged-1 indicated the merged image of green channel and red channel, and Merged-2 indicated the merged image of blue channel, green channel and red channel. The colocalization degree of late endosome/lysosome and Cy5-pDNA was calculated according to the formula: colocalization degree (100%) = the number of yellow pixels/the total number of yellow and red pixels × 100%, where yellow indicates the pDNA that is present in the compartment of late endosome/lysosome.

Fig. 8 Transfection efficiencies of diverse PEI derivatives-based gene delivery formulation at varying N/P ratios in A549 cells (A) and HeLa cells (B). PEI (Mw: 25 kDa, branched) was used as the control. Results are expressed as mean ± S.D. (n = 4). Asterisk (*) indicates significant differences (*p ＜ 0.05).

Systemic gene expression and toxicity profile. The reporter gene ‘CAG-Luc’ was loaded into PEI, PEI-PEG and PEI-PEG/TPE based gene delivery formulations. The aforementioned

solutions

were

intravenously

administered

into

the

A549

tumor-bearing mice via the tail vein. The mice were euthanized at 48 h post injection. The CAG-Luc expression in the tumors was evaluated quantitatively in the lysis solution of the tumors. As shown in Fig. 9, markedly gene expression could be observed for the samples of PEI-PEG and PEI-PEG/TPE, particularly for the sample of PEI-PEG/TPE, which should be attributable to its appreciable colloidal stability



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

and efficient cell transfection activity.

Fig. 9 Systemic gene expression of CAG-Luc in PEI, PEI-PEG and PEI-PEG/TPE. Asterisk (*) indicates significant differences (*p ＜ 0.05). Data represent mean ± S.D. (n = 6).

On the other hand, the toxic profile of the aforementioned PEI, PEI-PEG and PEI-PEG/TPE based gene delivery systems was assessed by a lactic dehydrogenase (LDH) assay. Note that LDH is an intracellular protein abundantly inside almost all lines of cells. Hence, the LDH level in the blood could be utilized as an important index to assess the overall systemic toxicity of the tested samples. To this respect, we attempted to quantify the LDH level of in the blood 2 h and 24 h post intravenous administration of the aforementioned PEI, PEI-PEG and PEI-PEG/TPE based gene delivery formulations. As shown in Fig. 10, PEI-based sample exhibited severe toxicity, as evidenced by the high level of LDH. This is consistent with the previous study that non-PEGylated cationic system tends to interact with the biological structures, particularly exerting drastic potency in destabilization of cellular membrane, eventfully resulting in liberation of intracellular LDH into the blood. In contrast, negligible toxicity in terms of LDH level in the bloodstream was confirmed for the PEGylated formulation (PEI-PEG and PEI-PEG/TPE based gene delivery systems). These results validated the safety profile and efficiency of our constructed


Page 16 of 31

Page 17 of 31

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


systems (PEI-PEG/TPE) for translation into practical applications.

Fig. 10 Systemic toxicity of PEI, PEI-PEG and PEI-PEG/TPE-based gene delivery formulations. Asterisk (*) indicates significant differences (*p ＜ 0.05). Data represent mean ± S.D. (n = 3).

CONCLUSION We strived to incorporate a lipophilic TPE molecule into a PEGylated polymeric gene delivery system composed of PEI-PEG. The TPE with hydrophobic character could serve as a functional component in strengthening the colloidal stability of the incorporated polyplex micelle and improving the biocompatibility and stealthy function due to TPE-induced collapse-down of pDNA payload. The enhanced stabilities and biocompatibility resulted in a larger number of polyplex micelles internalized into the cells. Of note, the PEG segments were schemed to undergo selective detachment from polyplex micelle by virtue of acid-liable cleavable Schiffbase linkage, which entitled the direct exposure of cationic PEI/pDNA polyplex to interact with the cell membrane and endosome membrane. The association to endosome membrane could be further pronounced with the aid of lipophilic TPE incorporation, thereby conferring marked destabilization potency to the endosome membrane and exerting endosome escape activity. Eventually, the constructed formulation characterized with pH-responsive PEG detachment strategy and



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 31

functional TPE components was determined to provide efficient gene expression in the culture cells and the targeted tumors with appreciable safety profile.

EXPERIMENTAL SECTION Materials.

Poly

(ethylene

glycol)

methyl

ether

(mPEG2000),

4,

4’-dimethoxybenzophenone and brorontribromide (BBr3) were purchased from Chemical Technology Co. Ltd. (Shanghai, China). p-Formylbenzoic acid, heparin sodium salt and dextran sulfate were obtained from Aladdin Bio-Chem Technology Co. Ltd. (Shanghai, China). (Dimethyl amino) pyridine was purchased from Alfa Aesar (Shanghai, China). Polyethylenimine [Mw: 25 kDa, branched] was purchased from Sigma-Aldrich (Shanghai, China). Luciferase assay kit was from Promega (Madison, USA). BCA Protein Assay Reagent Kit was purchased from Pierce (Madison, USA). Cell Counting Kit-8 (CCK-8) was employed in cytotoxicity assay (Dojindo Laboratories, Kumamoto, Japan). All other reagents were obtained from Tianjin Chemical Reagent Co. (Tianjin, China). Synthesis of mPEG-CHO. Methoxy poly (ethylene glycol) benzaldehyde (mPEG-CHO) was synthesized according to the procedure as described in the previous report.38 In brief, mPEG2000 (2.5 g, 1.25 mmol), p-formylbenzoic acid (1.5 g, 10 mmol), DCC (2.1 g, 10.18 mmol) and DMAP (0.16 g, 1.31 mmol) were dispersed in 75 mL of anhydrous dichloromethane (DCM). Molecular sieves were supplemented in the reaction solution. After 24 h reaction under stirring at room temperature, the reaction solution was transferred for filtration. The filtrate was collected and evaporated as possible to remove the solvent. The crude product was suspended in 25 mL double-distilled water, followed by stirring for 30 min. The above solution was transferred for filtration to collect the filtrate. Following subsequent extraction with DCM, the organic phase was collected and treated with Na2SO4 overnight. The suspension was filtered through the filter paper and precipitate in chilled diethyl ether to yield the ultimate product of mPEG-CHO (Yield: 1.2 g, 46.0%). Synthesis of PEI-PEG. PEI-PEG was obtained according to the procedure as


Page 19 of 31

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


described in the previous report with minor modifications.39 In brief, the synthesized mPEG-CHO (129 mg, 0.06 mol) in 6 mL anhydrous DMSO was slowly added into PEI25K (60 mg, 0.12 mmol) by dropwise over 5 min. The reaction was conducted for 48 h under stirring at 25 °C. Subsequently, the reaction solution was transferred to dialysis for purification (Spectra/Por RC, MWCO 8-14 kDa) against deionized water for 5 days before lyophilization (Yield: 76.0 mg, 40.2%). Preparation of polyplex. The N/P ratio was defined as the molar ratio of nitrogen from polymers and phosphorus from pDNA. To prepare PEI-PEG/TPE/pDNA polyplex at varying N/P ratios, PEI-PEG was dissolved in PBS (pH 7.4) and sterilized by filtration throughout a 0.22 µm membrane, followed by addition of TPE solution to the above solution, then vortex mixing with pDNA (50 µg/mL). Zeta potential measurement and 1H-NMR for insight on hydrolysis of PEI-PEG. The zeta potential of polyplex was determined on a Zetasizer Nano ZS90 instrument (Malvern Instruments, Southborough, MA). To simulate the physiological condition and the acidic tumor tissue and endosome milieu, incubation of polyplex PEI-PEG/TPE/pDNA at pH 7.4, pH 6.8 and pH 5.0 was conducted for the measurement. In brief, 500 µL of PEI-PEG/TPE/pDNA polyplex (at N/P ratio of 10) was prepared in PBS 7.4. The pH of polyplex solution was adjusted to 6.8 or 5.0 and transferred to zeta potential measurement. 1H-NMR measurement was performed for PEI-PEG at pH 7.4, pH 6.8 and pH 5.0 to gain the evidence of the hydrolysis of Schiff-base linkage between PEG segment and PEI segment. PEI-PEG (5 mg) was dissolved in D2O, adjusted by DCl to pH 6.8 or pH 5.0 or adjusted by NaOD to pH 7.4. The solution was incubated for 3 h at 37 °C prior to 1H-NMR measurement. EtBr Assay. The dissociation behavior of the electrostatically complexed PEI-PEG/pDNA formation was estimated based on an EtBr assay. The PEI-PEG/pDNA polyplex micelle solution (pDNA concentration: 50 µg/mL) was prepared at N/P ratio of 6 according to the aforementioned procedure in 2 mM HEPES buffer (pH 7.4). Aiming for PEG detachment, the pH of the prepared polyplex micelle solution was adjusted to 5.0 with 100 mM acetate acid buffer (pH 5.0). On the other hand, dextran sulfate was dissolved into distilled water at 50 mg/mL as the stock



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

solution. The PEI-PEG/pDNA polyplex micelle solutions at pH 7.4 or pH 5.0 were mixed with dextran sulfate at varying concentrations and EtBr solution at constant concentration, followed by 6 h incubation under dark, wherein the pDNA and EtBr in the reaction solution was adjusted to be 15 µg/mL and 25 µg/mL, respectively. Note that, the naked pDNA was used as the standard control. The fluorescence intensity of each reaction solution was measured at λem = 590 nm (λex = 510 nm) at 25 °C using a spectrofluorometer (FP-6500, JASCO, Tokyo, Japan). All data represent the mean of three independent measurements. Size derivations of polyplex in presence of serum albumin. The size derivations of polyplex in presence of serum albumin was investigated by Dynamic Light Scattering (DLS) (Zetasizer, Nano ZS90, Malvernt, UK). In brief, 400 µL PEI/pDNA and PEI-PEG/pDNA (at N/P ratio of 10) was supplemented with bovine serum albumin (BSA) solution (the final BSA concentration: 0.25 mg/mL). At varying incubation time post BSA supplementation, the average diameter of reaction solution was recorded by DLS at 25 °C. Given the possible interference of complexes self-aggregation, the above two samples were incubated in BSA-free buffer solution as control. Quantification of resistance to BSA absorbance. BSA aqueous solution (2 mg/mL) was added into PEI, PEI-PEG and PEI-PEG/TPE solution with equal volume, followed by incubation under shaking for 30 min at 37 °C. Centrifugation of the reaction solution was conducted at 10000 rpm for 5 min, the supernatant was collected. BSA concentration in the supernatant was determined by a UV-vis spectrophotometer (TU-1900, Persee, Beijing, China) at 280 nm. Accordingly, the BSA adsorption on the polymer surface was calculated according to the following formula: q = (C0 –C1)V/m Where C0 and C1 stands for initial BSA concentration of the reaction solution and BSA concentration in the collected supernatant, respectively; V stands for the total volume of the reaction solution, and m stands for the weight of polymer. Fluorescence emission of TPE. To 1 mL of TPE (31.95 µg/mL) in PBS (pH 7.4), the


Page 20 of 31

Page 21 of 31

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


solution of PEI-PEG was added gradually to obtain a series of samples (polymer concentration ranged from 0 to 0.0332 µg/mL). The resulting mixture was allowed for 60 s reaction in the dark. The fluorescence spectra of the solution were recorded by a fluorescence spectrophotometer (F-7000, Hitachi, Tokyo, Japan). The excitation wavelength was set as 330 nm, and emission fluorescence was monitored in a range of 350 nm to 640 nm. Confocal Laser Scanning Microscopy. Confocal laser scanning microscopy (CLSM, Zeiss LSM510, Oberkochen, Germany) was performed to explore insight into the internalization and subcellular localization of polyplex. A549 cells were plated onto a 35 mm glass bottom dish at a density of 105 cells, incubated in 400 µL RPIM 1640 medium supplemented with 10% FBS for 24 h. The medium was exchanged with 1 mL of free-serum medium at pH 6.8, followed by addition of 150 µL polyplex (labeled with Cy5). After 3 h culture in humidified atmosphere of 5% CO2 at 37 °C, the cells were washed with PBS, followed by 21 h post-incubation in 1 mL of fresh media. Then, cells were washed with PBS and treated with Lyso Tracker Green DND-26 (YEASEN, Shanghai, China) to stain lysosome. The cells were subjected to fixation with 75% alcohol prior to CLSM observation. The observation was carried out by a LSM510 (Nikon 108, Japan), in which the excitation wavelength was set at 488 nm for Lyso Tracker Green, 633 nm for Cy5 and 402 nm for TPE. The colocalization degree of late endosome/lysosome and Cy5-pDNA was calculated according to the formula: Colocalization degree (100%) = the number of yellow pixels/the total number of yellow and red pixels × 100%.

In vitro gene transfection. A549 cells or Hela cells were seeded in a 24-well plate at a density of 4 × 104 cells per well in 400 µL medium. After 24 h incubation, the culture medium was changed to pH 6.8, followed by addition of 40 µL polyplex solution containing 1µg pDNA per well, where PEI25K/pDNA was included as a control. After 48 h incubation, the cells were washed twice with PBS and treated with 100 µL cell lysis reagent. To test the luciferase activity, a commercial kit (Promega Co., Cergy Pontoise, France) and a luminometer (FLX800, Bio Tek, Winooski, VT) were applied according to the standard protocol provided by the manufacturer. Protein



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

concentration in the cell lysate was quantified by BCA protein assay kit (Pierce, Thermo Fisher Scientific, Waltham, MA). The transfection efficiency was expressed as relative light units per milligram protein (RLU/mg protein). All data were represented by a mean and standard deviation from four samples. Gene expression in tumors via systemic route. Diverse polyplex micelles containing CAG-Luc (10 µg in 200 µL of 10 mM HEPES containing 150 mM NaCl) were intravenously administered into the A549 tumor-bearing mice via the tail vein. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University. The mice were euthanized at 48 h post injection, and the xenografted tumors were excised and homogenized in cell lysis buffer. The Luc expression was measured for 10 s from the photoluminescence intensity using Mithras LB 940 (Berthold Technologies, UK). The data were normalized against the weight of the tumor tissues (n = 6). Systemic toxicity. Diverse polyplex micelles (PEI/pDNA, PEI-PEG/pDNA and PEI-PEG/TPE/pDNA) at N/P ratio of 6 were intravenously administrated into the tail vein of Balb/c mice (female, 6-week-old) at pDNA dosage of 20 µg/mouse. The blood was collected at 2 h and 24 h post-administration from inferior vena cava to determine the plasma level of lactate dehydrogenase (LDH) to assess the overall systemic toxicity of the administered samples with DRI- CHEM 7000i (Fuji Film, Tokyo, Japan).

Statistical analysis. Significant differences in transfection efficiency and cell viability between different polyplex groups were performed using a Student’s t-test.

ASSOCIATED CONTENT


Page 22 of 31

Page 23 of 31

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


Supporting Information Synthetic Scheme, characterization of TPE derivatives and polymers, GPC, morphology of polyplex, agarose gel electrophoresis, particle size and zeta potential measurements, cell culture, cytotoxicity assays and 1H-NMR spectra.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] ORCID Qixian Chen: 0000-0002-3091-671X Hui Gao: 0000-0002-5009-9999 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research was funded by National Natural Science Foundation of China (No. 21374079 ， 21674080), and 131 talents program of Tianjin, the Science and Technology Correspondent Project of Tianjin (16JCTPJC49800). Q. C. acknowledge the funding support from the Fundamental Research Funds for the Central Universities.

ABBREVIATIONS TPE: tetraphenylene, teracarboxylphenylene PEG: poly (ethylene glycol) 1

H-NMR: 1H-nuclear magnetic resonance

FT-IR: fourier transform infrared spectroscopy PBS: phosphate buffered saline SEM: scanning electron microscope DLS: dynamic laser light scattering



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 24 of 31

BSA: bovine serum albumin CLSM: confocal laser scanning microscopy GPC: gel permeation chromatography EtBr: ethidium bromide LDH: lactate dehydrogenase CCK-8: cell counting kit-8 OD: optical density

REFERENCES (1) Yue, D., Cheng, G., He, Y., Nie, Y., Jiang, Q., Cai, X., and Gu, Z. (2014) Influence of reduction-sensitive diselenide bonds and disulfide bonds on oligoethylenimine conjugates for gene delivery. J. Mater. Chem. B 2, 7210-7221. (2) Li, C., Yang, Y.-W., Liang, Z.-X., Wu, G.-L., and Gao, H. (2013) Post-modification

of

poly(glycidyl

methacrylate)s

with

alkyl

amine

and

isothiocyanate for effective pDNA delivery. Polym. Chem. 4, 4366-4374. (3) Veiman, K. L., Kunnapuu, K., Lehto, T., Kiisholts, K., Parn, K., Langel, U., and Kurrikoff, K. (2015) PEG shielded MMP sensitive CPPs for efficient and tumor specific gene delivery in vivo. J. Control. Release 209, 238-247. (4) Cai, X., Jin, R., Wang, J., Yue, D., Jiang, Q., Wu, Y., and Gu, Z. (2016) Bioreducible fluorinated peptide dendrimers capable of circumventing various physiological barriers for highly efficient and safe gene delivery. ACS Appl. Mater. Inter. 8, 5821-5832. (5) Liu, X., Xiang, J., Zhu, D., Jiang, L., Zhou, Z., Tang, J., Liu, X., Huang, Y., and Shen, Y. (2016) Fusogenic reactive oxygen species triggered charge-reversal vector


Page 25 of 31

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


for effective gene delivery. Adv. Mater. 28, 1743-1752. (6) Liang, Z., Wu, X., Yang, Y.-W., Li, C., Wu, G., and Gao, H. (2013) Quaternized amino poly(glycerol-methacrylate)s for enhanced pDNA delivery. Polym. Chem. 4, 3514-3523. (7) Zhang, J., Wang, Z., Lin, W., and Chen, S. (2014) Gene transfection in complex media using PCBMAEE-PCBMA copolymer with both hydrolytic and zwitterionic blocks. Biomaterials 35, 7909-7918. (8) Li, J., Yu, X., Wang, Y., Yuan, Y., Xiao, H., Cheng, D., and Shuai, X. (2014) A reduction and pH dual-sensitive polymeric vector for long-circulating and tumor-targeted siRNA delivery. Adv. Mater. 26, 8217-8224. (9) Nam, K., Jung, S., Nam, J. P., and Kim, S. W. (2015) Poly(ethylenimine) conjugated bioreducible dendrimer for efficient gene delivery. J. Control. Release 220, 447-455. (10) Gao, H., Takemoto, H., Chen, Q., Naito, M., Uchida, H., Liu, X., Miyata, K., and Kataoka, K. (2015) Regulated protonation of polyaspartamide derivatives bearing repeated aminoethylene side chains for efficient intracellular siRNA delivery with minimal cytotoxicity. Chem. Commun. 51, 3158-3161. (11) Zhou, Z., Shen, Y., Tang, J., Jin, E., Ma, X., Sun, Q., Zhang, B., Van Kirk, E. A., and Murdoch, W. J. (2011) Linear polyethyleneimine-based charge-reversal nanoparticles for nuclear-targeted drug delivery. J. Mater. Chem. 21, 19114-19123. (12) Yang, B., Jia, H., Wang, X., Chen, S., Zhang, X., Zhuo, R., and Feng, J. (2014) Self-assembled vehicle construction via boronic acid coupling and host-guest



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 26 of 31

interaction for serum-tolerant DNA transport and pH-responsive drug delivery. Adv. Healthcare Mater. 3, 596-608. (13) Feng, T., Dong, X., Tian, H., Hon-Wah Lam, M., Liang, H., Wei, Y., and Chen, X. (2014) PEGylated poly(aspartate-g-OEI) copolymers for effective and prolonged gene transfection. J. Mater. Chem. B 2, 2725-2732. (14) Wang, Y., Chen, P., and Shen, J. (2007) A facile entrapment approach to construct PEGylated polyplexes for improving stability in physiological condition. Colloids and Surfaces B: Biointerfaces 58, 188-196. (15) Hatakeyama, H., Akita, H., and Harashima, H. (2011) A multifunctional envelope type nano device (MEND) for gene delivery to tumours based on the EPR effect: a strategy for overcoming the PEG dilemma. Adv. Drug Deliv. Rev. 63, 152-160. (16) Li, J., Ge, Z., and Liu, S. (2013) PEG-sheddable polyplex micelles as smart gene carriers based on MMP-cleavable peptide-linked block copolymers. Chem. Commun. 49, 6974-6976. (17) Dehshahri, A., Oskuee, R. K., Shier, W. T., Hatefi, A., and Ramezani, M. (2009) Gene

transfer

efficiency

of

high

primary

amine

content,

hydrophobic,

alkyl-oligoamine derivatives of polyethylenimine. Biomaterials 30, 4187-4194. (18) Mussi, S. V., and Torchilin, V. P. (2013) Recent trends in the use of lipidic nanoparticles as Pharmaceutical Carriers for Cancer Therapy and Diagnostics. J. Mater. Chem. B 1, 5201-5209. (19) Teo, P. Y., Yang, C., Hedrick, J. L., Engler, A. C., Coady, D. J.,


Page 27 of 31

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


Ghaem-Maghami, S., George, A. J., and Yang, Y. Y. (2013) Hydrophobic modification of low molecular weight polyethylenimine for improved gene transfection. Biomaterials 34, 7971-7979. (20) Guo, P., Gu, W., Chen, Q., Lu, H., Han, X., Li, W., and Gao, H. (2015) Dual functionalized amino poly(glycerol methacrylate) with guanidine and schiff-base linked imidazole for enhanced gene transfection and minimized cytotoxicity. J. Mater. Chem. B 3, 6911-6918. (21) Gao, L., Fei, J., Zhao, J., Cui, W., Cui, Y., and Li, J. (2012) pH- and redox-responsive polysaccharide-based microcapsules with autofluorescence for biomedical applications. Chem. Eur. J. 18, 3185-3192. (22) Shi, B., Zhang, H., Dai, S., Du, X., Bi, J., and Qiao, S. Z. (2014) Intracellular microenvironment responsive polymers: a multiple-stage transport platform for high-performance gene delivery. Small 10, 871-877. (23) Han, X., Liu, D. E., Wang, T., Lu, H., Ma, J., Chen, Q., and Gao, H. (2015) Aggregation-induced-emissive molecule incorporated into polymeric nanoparticulate as FRET donor for observing doxorubicin delivery. ACS Appl. Mater. Inter. 7, 23760-23766. (24) Yuan, Y., Zhang, C.-J., Xu, S., and Liu, B. (2016) A self-reporting AIE probe with a built-in singlet oxygen sensor for targeted photodynamic ablation of cancer cells. Chem. Sci. 7, 1862-1866. (25) Jiang, B.-P., Tan, X., Shen, X.-C., Lei, W.-Q., Liang, W.-Q., Ji, S.-C., and Liang, H. (2016) One-step fabrication of a multifunctional aggregation-induced emission



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

nanoaggregate for targeted cell imaging and enzyme-triggered cancer chemotherapy. ACS Macro Lett. 5, 450-454. (26) Han, X., Chen, Q., Lu, H., Ma, J., and Gao, H. (2015) Probe intracellular trafficking of a polymeric DNA delivery vehicle by functionalization with an aggregation-induced emissive tetraphenylethene derivative. ACS Appl. Mater. Inter. 7, 28494-28501. (27) Han, X., Chen, Q., Lu, H., Guo, P., Li, W., Wu, G., Ma, J., and Gao, H. (2016) Incorporation of an aggregation-induced-emissive tetraphenylethene derivative into cationic gene delivery vehicles manifested the nuclear translocation of uncomplexed DNA. Chem. Commun. 52, 3907-3910. (28) Gu, J., Cheng, W.-P., Liu, J., Lo, S.-Y., Smith, D., Qu, X., and Yang, Z. (2008) pH-triggered reversible “stealth” polycationic micelles. Biomacromolecules 9, 255-262. (29) Oupicky, D., Ogris, M., Howard, K. A., Dash, P. R., Ulbrich, K., and Seymour, L. W. (2002) Importance of lateral and steric stabilization of polyelectrolyte gene delivery vectors for extended systemic circulation. Mol. Ther. 5, 463-472. (30) Zhang, M.-H., Gu, Z.-P., Zhang, X., and Fan, M.-M. (2015) pH-sensitive ternary nanoparticles for nonviral gene delivery. RSC Adv. 5, 44291-44298. (31) Chen, S., Rong, L., Lei, Q., Cao, P.-X., Qin, S.-Y., Zheng, D.-W., Jia, H.-Z., Zhu, J.-Y., Cheng, S.-X., Zhuo, R.-X., et al. (2016) A surface charge-switchable and folate modified system for co-delivery of proapoptosis peptide and p53 plasmid in cancer therapy. Biomaterials 77, 149-163.


Page 28 of 31

Page 29 of 31

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


(32) Ke, W., Zha, Z., Mukerabigwi, J. F., Chen, W., Wang, Y., He, C., and Ge, Z. (2017)

Matrix

metalloproteinase-responsive

multifunctional

peptide-linked

amphiphilic block copolymers for intelligent systemic anticancer drug delivery. Bioconjugate Chem. 28, 2190-2198. (33) Chen, Q., Osada, K., Ge, Z., Uchida, S., Tockary, T. A., Dirisala, A., Matsui, A., Toh, K., Takeda, K. M., Liu, X., et al. (2017) Polyplex micelle installing intracellular self-processing functionalities without free catiomers for safe and efficient systemic gene therapy through tumor vasculature targeting. Biomaterials 113, 253-265. (34) Chen, Q., Osada, K., Ishii, T., Oba, M., Uchida, S., Tockary, T. A., Endo, T., Ge, Z., Kinoh, H., Kano, M. R., et al. (2012) Homo-catiomer integration into PEGylated polyplex micelle from block-catiomer for systemic anti-angiogenic gene therapy for fibrotic pancreatic tumors. Biomaterials 33, 4722-4730. (35) Zha, Z., Li, J., and Ge, Z. (2015) Endosomal-escape polymers based on multicomponent reaction-synthesized monomers integrating alkyl and imidazolyl moieties for efficient gene delivery. ACS Macro Lett. 4, 1123-1127. (36) Yi, H. X., Wu, J., Du, Y. Z., Hu, Y. W., Yuan, H., You, J., and Hu, F. Q. (2015) Effect of anionic PEGylated polypeptide on gene transfection mediated by glycolipid conjugate micelles. Mol. Pharm. 12, 1072-1083. (37) Yan, J., Du, Y. Z., Chen, F. Y., You, J., Yuan, H., and Hu, F. Q. (2013) Effect of proteins with different isoelectric points on the gene transfection efficiency mediated by stearic acid grafted chitosan oligosaccharide micelles. Mol. Pharm. 10, 2568-2577. (38) Wang X.-G., Dong Z.-Y., Cheng, H., Wan S.-S., Chen W.-H., Zou, M.-Z., Huo,



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

J.-W., Deng, H.-X., and Zhang, X.-Z. (2015) A multifunctional metal-organic framework based tumor targeting drug delivery system for cancer therapy. Nanoscale 7, 16061-16070. (39) Xiao, D., Jia, H. Z., Zhang, J., Liu, C. W., Zhuo, R. X., and Zhang, X. Z. (2014) A dual-responsive mesoporous silica nanoparticle for tumor-triggered targeting drug delivery. Small 10, 591-598.


Page 30 of 31

Page 31 of 31

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

Polyplex

Micelle

with

pH-Responsive

PEG

Detachment

Tetraphenylene Incorporation to Promote Systemic Gene Expression


and

Functional

Polyplex Micelle with pH-Responsive PEG Detachment and

Recommend Documents