pH-Responsive Natural Polymeric Gene Delivery Shielding System

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A pH-responsive natural polymeric gene delivery shielding system based on dynamic covalent chemistry Chang Xu, Xiuwen Guan, Lin Lin, Qing Wang, Bo Gao, Shuhua Zhang, Yanhui Li, and Huayu Tian ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00869 • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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ACS Biomaterials Science & Engineering

A pH-responsive natural polymeric gene delivery shielding system based on dynamic covalent chemistry Chang Xu,† ‡ Xiuwen Guan,‡ Lin Lin,‡ Qing Wang,† Bo Gao,† Shuhua Zhang,*,† Yanhui Li,*,† Huayu Tian,*,‡ †

Changchun University of Science and Technology, WeiXing Road 7989, Changchun 130022, China

‡

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Renmin Street 5625, Changchun 130022, China

ABSTRACT: A novel pH-responsive system based on aldehyde-bearing dextran derivatives (ODEX or FDEX) was designed to use as gene carrier shielding. Through pH sensitive Schiff base bonds between amino groups of PEI (in PEI/DNA polyplex) and aldehyde groups of dextran derivatives, PEI/DNA polyplex could be shielded and further condensed to get effectively decreasing of zeta potential with smaller size. Schiff base bonds were pH-responsive, which were relatively stable in neutral environment but in slightly acidic and acidic environments would be deformed. By using this character in this study, the PEI/DNA polyplex had been effectively shielded during circulation in body, when arrived at tumor, the slightly acid pH triggered the broken of Schiff base bonds to expose the positive PEI/DNA polyplex, and

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futher interacted with tumor cell membranes, achieving efficient gene expression. By using such characteristics, it could effevtively address the high transfection efficiency versus stability dilemma of gene carriers. FDEX/PEI/DNA nanoparticles (NPs) not only mediate higher cellular uptake and transfection efficiency in virto but also effectively accumulate in tumor with higher gene expression in vivo than the ODEX analogues. As a result, this pH-responsive system is a promising strategy for cancer therapy. KEYWORDS: pH-responsive, gene delivery, dynamic chemistry, shielding system 1. INTRODUCTION Polyethylenimine (PEI) is the most promising nonviral gene carrier1 due to its distinct “proton sponge effect”.2 Especially, PEI of molecular weight 25000 (PEI25k) has been widely regarded as the “golden standard” for gene carriers3 in vitro4 and in vivo.5 However, the positively charged PEI/DNA polyplex usually assembled or disaggregated in body,6 which is not beneficial to reach the target tissue and hinder its further use.7-9 In order to solve this problem, the shielding system was introduced. In previous studies, PEG10 or polyanions11 were chosen to shield the PEI/DNA polyplex. As the cell membrane is negatively charged, these neutral or negative charges would influence cellular uptake efficiency.12. Therefore, more reasonable shielding system should be designed. Many kinds of physiological stimuli were exploited to design effective gene delivery system, such as pH, temperature, enzyme and redox.13-15 The tumor extracellular environment has lower pH (pH ~ 6.8) than normal tissues (pH 7.2 ~ 7.4), and the pHs of endosomes are even lower (pH 5.0 ~ 5.5).16 Thus, by using variations of pH values between physiological and tumor environment, constructing pH-responsive shielding material is a common strategy. In some


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research,17-19 a pH-sensitive detachable polyethylene glycol (PEG) shielding system was designed for gene delivery. This PEGylated NPs was stable when circulating in body, and detached when it arrived at tumor, to achieve higher cellular uptake. This strategy carried out prolonged circulation and efficient tumor suppression. But PEG will induce accelerated blood clearance (ABC) phenomenon in circulation.20 Dextran, a representative natural hydrophilic polysaccharide with excellent performance such as biodegradation and good biocompatibility,21-23 is an analog of PEG.24 Compared with PEG, dextran has the possibility of avoid the risk of ABC phenomenon which is usually induced by injecting of PEGylated nanomedicine repeatedly.20 Additionally, there are abundant hydroxyl groups on the chain for chemical modification,25 and dextran after reasonable modifications was widely used as gene vectors in previous studies.26-29 So it is feasible to design a pH-responsive shielding system based on dextran for efficient gene delivery. Herein, a pH-responsive shielding system was designed for gene delivery in cancer treatment (Scheme 1). Aldehyde-modified dextrans (named as ODEX or FDEX) were used to shield PEI/DNA polyplex through pH-responsive Schiff base bonds. For ODEX shielded PEI/DNA polyplex, the formed Shift base bonds were broken at pH 5.0. So this system couldn’t possess the tumor-specific pH responsive behaviors. As comparison, for FDEX/PEI/DNA NPs, it was stable in neutral environment but would be rapidly broken in slightly acidic and acidic environments. As a result, the PEI/DNA polyplex was effectively shielded in normal tissue (pH 7.4); once accumulated around tumor (pH 6.8), the Schiff base bonds would broken and positively charged PEI/DNA polyplex exposed, then the particles would interact with the negatively charged cell membrane to achieve efficient cell uptake. The latter system was expected to have the following comprehensive advantages : 1) the synthetic routes were simple, the Schiff base bonds could be


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formed quickly and organic solvent-freely; 2) FDEX could effectively shield positive charges and compress PEI/DNA polyplex; 3) the higher cellular uptake, tumor accumulation and transfection efficiency could be achieved in acidic tumor extracellular pH (pH 6.8). In brief, this pH-responsive shielding system has great potential for further application in cancer therapy.

Scheme 1. Schematic of the pH-responsive detachable dextran shielding system. PEI/DNA polyplex was shielded by ODEX or FDEX via efficient Schiff base reaction between aldehydebearing dextrans and amino-containing PEI. The pH-responsive Schiff base bonds were stable in neutral environment but broken at the slightly acidic tumor areas. 2. RESULTS AND DISCUSSION Two kinds of aldehyde groups-bearing dextrans simplified as ODEX and FDEX were synthesized through a simple method and the synthetic routes were shown in Scheme S1


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(Supporting information, Scheme S1). The 1H NMR spectra of aldehyde group-bearing ODEX and FDEX were displayed in Figure 1A. The characteristic chemical shift of aldehyde group was at about 5.6 ppm for ODEX, and for FDEX was about 9.8 ppm. These results demonstrated that the successful synthesis of aldehyde-modified dextrans. The number of grafted aldehydes of ODEX and FDEX was unified by 1H NMR, we determined that the number of grafted aldehydes was about 18. The 1H NMR spectra of dextran derivatives mixed with PEI were also detected, the pHs of the solution were adjusted to 7.4, 6.8 and 5.0. The pH sensitive performances of ODEX/PEI and FDEX/PEI were shown in Figure 1B. When the pH was 7.4, the chemical shift of aldehyde groups for both ODEX and DEX were disappeared. For ODEX, the weak aldehyde peak appeared until the pH was adjusted to 5.0. However, the pH was 6.8 for FDEX, the aldehyde peak was reappeared (the percentage of the Schiff base bonds fractured at pH 6.8 and 5.0 were about 66.67 % and 82.83 %, respectively). These phenomena illustrated two vital messages: 1) the aldehyde groups of dextran derivatives could react with the amino groups of PEI via Schiff base reaction; 2) the Schiff base bonds were fractured in slightly acid pH for FDEX, it declared that FDEX would detach in the tumor site (pH ~ 6.8).


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Figure 1. (A) 1H NMR spectra of aldehyde group-bearing ODEX and FDEX. (B) 1H NMR spectra of reactions between aldehyde groups-bearing ODEX, FDEX and PEI at pH 7.4, 6.8 and 5.0. The characteristic chemical shift of aldehyde groups are noted in black frame to display the pH sensitivity of the Schiff base bonds.


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The zeta potential and particle size of ODEX/PEI/DNA NPs and FDEX/PEI/DNA NPs with different mass ratios and pH values (pH 7.4 and pH 6.8) were investigated. The results were shown in Figure 2, and the TEM observation of polyplexes was shown in Figure S1. As we can see, with increasing the amount of ODEX or FDEX, the zeta potential of delivery system was decreased (Figure 2A, C), which indicated that PEI/DNA polyplex could be effectively shielded by ODEX or FDEX with various effects. For ODEX/PEI/DNA NPs, there was no obvious change between two pH values; but for FDEX/PEI/DNA NPs, there was a significant difference. Due to the cleavage of the Schiff base bonds at pH 6.8, more positive charges were exposed, as a result, the zeta potential was much higher than pH 7.4. In addition, the aldehydes of FDEX were more active due to the stronger electron-deficient ability and less steric hindrance, which made FDEX interact with PEI much stronger. Meanwhile, dextran as a macromolecule, it had a shielding effect on PEI/DNA polyplex, so the potential at pH 6.8 shown a slight downward. With the mass ratio increased, the particle size of ODEX/PEI/DNA NPs was largely reduced (Figure 2B), which illustrated that the ODEX had the ability to compress PEI/DNA polyplex. Due to the amino groups of the PEI to be protonated at acidic environment, which made the PEI/DNA polyplex much tighter, so the particle size at pH 6.8 was slightly smaller than pH 7.4. The particle size was reduced when the PEI/DNA polyplex were shielded by FDEX (Figure 2D) compared with ODEX. The PEI/DNA polyplex was effectively compressed with the addition of the FDEX shielding system. Another phenomenon was that the particle size at pH 6.8 was slightly larger than pH 7.4. It was owing to the FDEX shielding system detached from pH 7.4 to pH 6.8, caused the PEI/DNA polyplex slightly larger. At the same time, agarose gel retardation


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assay was carried out to evaluate the pDNA binding ability (Figure S2), the results meant that both the ODEX/PEI/DNA and FDEX/PEI/DNA NPs could tightly retard the pDNA.

Figure 2. Zeta potential and particle size of ODEX/PEI/DNA NPs (A, B) and FDEX/PEI/DNA NPs (C, D) with various mass ratios at pH 7.4 and pH 6.8. In vitro transfection experiments for ODEX/PEI/DNA NPs and FDEX/PEI/DNA NPs were performed using HeLa cells at pH 7.4 and 6.8 with various mass ratios[(0, 2.5, 5, 7.5, 10, 15):2.5:1]. As shown in Figure 3A, the transfection efficiency had no obvious difference between pH 7.4 and 6.8 for ODEX/PEI/DNA NPs. Owing to this slightly acidic environment was not enough to make the ODEX shielding system detach. On the contrary, the transfection efficiency of FDEX/PEI/DNA NPs was greatly improved when pH decreased from 7.4 to 6.8 (Figure 3B).


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This phenomenon was due to the detachment of FDEX from NPs, exposuring more positive charges and resulting in higher transfection efficiency. It was worth mentioning that the Schiff base bonds formed by FDEX were more suitable for the slightly acidic environment of the tumor. The above analyses showed that the zeta potential and particle size were influenced by pH values, and based on the results in this study, smaller size and higher positively charged surface were beneficial to improve transfection efficiency. In the meanwhile, FDEX/PEI/DNA NPs in the slightly acidic environment around the tumor could be a promising gene carrier. The FDEX:PEI:DNA NPs with a mass ratio of 10:2.5:1 demonstrated the biggest differences between two values (The transfection efficiency at pH 6.8 was 2.5 times of the level at pH 7.4), what’s more, it displayed the highest transfection efficiency, so, 10:2.5:1 was selected the optimal ratio for

the

following

experiments.

Meanwhile,

the

stability of

ODEX/PEI/DNA

and

FDEX/PEI/DNA NPs in PBS and culture medium was provided (Figure S3), which indicated the ranking stability of ODEX and FDEX shielded NPs.

Figure 3. Transfection efficiency of ODEX/PEI/DNA NPs (A) and FDEX/PEI/DNA NPs (B) with different mass ratios at pH 7.4 and 6.8, in HeLa cells. The data are represented as a mean ± SD (n = 3; *P

pH-Responsive Natural Polymeric Gene Delivery Shielding System

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