Size Dual-Rebound Gene

Subscriber access provided by UNIV NEW ORLEANS

Communication

Ultrasensitive pH Triggered Charge/Size Dual-Rebound Gene Delivery System Xiuwen Guan, Zhaopei Guo, Lin Lin, Jie Chen, Huayu Tian, and Xuesi Chen Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.6b02536 • Publication Date (Web): 19 Sep 2016 Downloaded from http://pubs.acs.org on September 27, 2016

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.

Nano Letters is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

Ultrasensitive pH Triggered Charge/Size Dual-Rebound Gene Delivery System Xiuwen Guan,†,‡,§ Zhaopei Guo,†,§ Lin Lin,† Jie Chen,† Huayu Tian,*,† and Xuesi Chen*,† †

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, Changchun 130022, P. R. China ‡

University of Chinese Academy of Sciences, Beijing 100049, P.R. China

ABSTRACT: A facile strategy is developed to construct an ultrasensitive pH triggered charge/size dual-rebound gene delivery system for efficient tumor treatment. Therapeutic gene is complexed by polyethylenimine (PEI) and poly-L-glutamate (PLG), further in situ tightened by aldehyde modified polyethylene glycol (PEG) via Schiff base reaction. The generated Schiff base bonds are stable in neutral pH but cleavable in tumor extracellular pH. This gene delivery system possesses following favorable properties: (1) the tunable gene delivery system is constructed by chemical bench-free "green" and fast process which is favored by clinician, (2) PEG crosslinking shields the surface positive charges and tightens the complex particles, leading to decreased cytotoxicity, improved stability and prolonged circulation, (3) PEG shielding can be rapidly peeled off by acidic pH as soon as arriving tumors, (4) dual charge/size ultrasensitively rebounding to higher positive potential and bigger size enhances tumor cell uptake efficiency. A series of experiments both in vitro and in vivo are carried out to investigate this gene delivery system in detail. An anti-angiogenesis therapeutic gene is carried for the treatment of CT26 tumors in mice, achieving superior anti-tumor efficacy which is well proved by sufficient biological evidences. The system has great potentials for cancer therapy in the future.

KEYWORDS: pH triggered, charge/size dual-rebound, PEG shielding, gene delivery, cancer therapy

1

ACS Paragon Plus Environment

Nano Letters

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

Gene therapy is a powerful and promising approach for cancer therapy.1 Up to now, various polycationic gene delivery systems have been widely developed because of their good safety performance and easy functionalization.2-4 Generally, the gene could be loaded by the positively charged polycationic gene carriers through electrostatic interactions and finally formed complex nanoparticles (NPs) with surplus positive charges on the NPs.5 Various biological barriers should be overcomed before the NPs successfully accomplished the gene transfection in targeted cells.6,7 There were different requirements for the properties of NPs in different phases during the complicated delivery process. In most cases, the requirements were contradictory.8 For example, low positive charges or negative charges were favourable for the NPs to maintain stability and decrease non-specific adsorptions during circulation, however, high positive charges were required for high cellular uptake efficiency when the NPs attached to the tumor cells.9-11 On the other hand, circulatory system and tumor cells also had different preference for the size of the NPs, smaller size was helpful for long circulation, properly increasing size was benefit to cellular uptake according to the reported works.12,13 In another case, PEGylation could realize long circulation for NPs, but it also hindered the tumor cell uptake.14-16 Meanwhile, the lower cell uptake for normal cells in circulation was better, but higher tumor cell uptake was eagerly needed for NPs internalization. The existence of so many dilemmas has greatly challenged the development of efficient gene delivery systems. Different strategies were attempted to coordinate the dilemmas by many research groups.17-19 In Wang's study, a pH-responsive charge-conversional nanogel was developed. The nanogel was negatively charged at physiological pH and positively charged in the acidic tumor extracellular pH. The charge conversion increased the cell uptake and promoted drug release, which led to remarkable killing effect on tumor cells.20 A dendritic lipopeptide based delivery system with charge-tunable shielding was reported by Gu's group,21 the system had negatively charged surface at normal pH with low protein interactions and prolonged circulation, and further tuned to positively charged at tumor extracellular pH, the system had achieved effective tumor suppression. A novel size changeable nanocarrier was designed by Zhou's gruop,22 the micelle had a small size in physiological conditions, when accumulated at tumor tissues, the size increased in response to the acidic pH, and then the micelles were internalized by the tumor cells. Furthermore, after the micelles escaped from lysosomes, they 2


Page 2 of 20

Page 3 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

became much smaller under the cleavage of the glutathione (GSH), and finally entered cell nuclei and released the cargo. In Wang's research,23 a tumor acidity-sensitive bridged copolymer was designed for overcoming the conflict of PEGylation, the PEG corona was stable during circulation, but could be detached at tumor sites for facilitating the cellular uptake, the system had achieved prolonged circulation and enhanced tumor inhibition activity. In another research of Wang's group,24 a tumor-pH-labile polymeric NP was reported, the PEGylated NPs had decreased zeta potential and lower cell uptake in circulatory system. When arrived at tumor site, the PEG layer of the NPs was detached and the zeta potential increased, both of them could promote the cellular uptake and finally improve the in vivo anti-tumor efficiency. But the above strategies focused only one or two dilemmas, and also they had to undergo complicated and tedious synthesis and preparations. Therefore, a comprehensive solution covering all the dilemmas by a simple and convenient way would be really encouraging. A gene delivery system which was adaptive for different phases in the delivery process was highly desirable. In this study, an ultrasensitive pH triggered charge/size dual-rebound gene delivery system was constructed by a facile strategy to qualify the different requirements during the whole transportation process (Figure 1). Negatively charged therapeutic gene was complexed by PEI and PLG to form the gene loaded complex NPs, and the NPs were further tightened by PEG which had aldehyde groups at both of its terminals. The aldehyde groups of PEG could react with the amino groups of PEI to form Schiff bases in neutral or alkaline aqueous solution rapidly with high reaction efficiency, and the generated Schiff base bonds between PEG and PEI were stable in physiological pH 7.4 but labile and cleavable in acidic pH, including the slightly acidic tumor extracellular pH. The ultra pH-sensitive Schiff base bonds responded to acidic stimuli much faster than most reported acidic cleavable chemical bonds,25-28 which was helpful for the rapid detachment of PEG shielding. Benefit from the in situ Schiff base reaction, a pH-responsive PEG tightened gene delivery system with charge/size dual-rebound properties was obtained. This delivery system was expected to possess the following favorable properties: (1) a fast and efficient Schiff base reaction was exploited to in situ crosslink the gene complex particles in organic solvent-free system, which contributed a facile strategy to construct a gene delivery system with tunable PEG density and crosslinking 3


Nano Letters

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

degree. (2) PEG crosslinking could shield the surface positive charges and tighten the complex particles, leading to decreased cytotoxicity, improved stability and prolonged circulation, (3) PEG deshielding could be rapidly triggered by acidic tumor extracellular pH, which could accelerate the following tumor cellular uptake process. (4) dual charge/size ultrasensitively rebounding to higher positive potential and bigger size enhanced tumor cell uptake efficiency. The rational designed system was investigated through a series of experiments both in vitro and in vivo. The zeta potential and particle size, gene transfection, cytotoxicity and cell uptake of the delivery system were well characterized. The tumor accumulation and pharmacokinetics of the system were also investigated. Furthermore, a plasmid DNA (pDNA) which expressed small hairpin RNA (shRNA) that targeted vascular endothelial growth factor (VEGF) was used as the therapeutic gene to explore the anti-tumor therapeutic efficacy in vivo by monitoring the tumor volume and body weight of the mice. Pathology of the major organs was analyzed by hematoxylin-eosin (H&E) staining. The tumor angiogenesis inhibition was evaluated by immunofluorescence technique and photoacoustic imaging. VEGF gene expression was tested by real-time PCR (RT-PCR) and VEGF protein was analyzed by enzyme linked immunosorbent assay (ELISA) and western blot. These results verified clearly that the ultrasensitive pH-triggered charge/size dual-reboud gene delivery system had outstanding anti-tumor therapeutic efficacy and would have great potentials for cancer therapy in the future.

4


Page 4 of 20

Page 5 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

Figure 1. The schematic of the ultrasensitive pH triggered charge/size dual-rebound gene delivery system.

PLG was prepared according to the previous reported method by ring opening polymerization

of

N-carboxyanhydride

of

γ-benzyl-L-glutamate

(BLG-NCA)

with

N-hexylamine as the initiator.29 And 1H NMR spectra was exploited to characterize the structure of PLG (Figure S1, Supporting Information). The peaks of a~f were assigned to the initiator hexylamine. The peaks of g, h, and i were assigned to PLG. The molecular weight of PLG was calculated by integral area of a and g. The number-average molecular weight (Mn) of PLG was 2.1 KDa, with 16 degree of polymerization. The synthesis of aldehyde group modified PEG was according to the reported method with slight modification,30 and the final product was characterized by 1H NMR (Figure S2 and S3). The aldehyde groups of OHC-PEG-CHO could react with the amino groups of PEI to form Schiff base bonds in neutral or alkaline aqueous solution. The "click" reaction could happen on the surface of the 5


Nano Letters

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

NPs in situ to realize the crosslinking of PEG (Figure S2). And the Schiff base bonds were labile and cleavable in the slightly acidic tumor extracellular pH which was precisely conducive to the PEG detaching. The pH-sensitivity of the forming and cleavage of the Schiff base bonds was verified by 1H NMR in different pH values (Figure S4). The peak of aldehyde groups (at 10 ppm) completely disappeared in pH 7.4 which meant that all the aldehyde groups reacted with PEI to form Schiff base bonds. The slightly acidic pH 6.8 could rapidly cleave the Schiff base bonds and restored the signal of aldehyde groups in 1H NMR spectrum. This result sufficiently demonstrated the pH-sensitivity of the reaction, which responded more sensitively to pH stimuli than other reported pH cleavable chemical bonds. And this sharp feature was exploited to construct intelligent pH-responsive gene delivery system in this work. PEI/DNA (PD), PLG/(PEI/DNA) (G(PD)) and (PLG/PEI)/DNA ((GP)D) were prepared by mixing DNA, PEI or PLG aqueous solutions with equal volume in different orders. And the PEG crosslinked NPs were further prepared by simply adding different amount of OHC-PEG-CHO aqueous solution into the (GP)D to rapidly and efficiently obtain the PEG[(PLG/PEI)/DNA] which was abbreviated as P[(GP)D]. The zeta potentials and particle sizes of PD, G(PD), (GP)D and P[(GP)D] NPs were measured at different pH values (pH 7.4 and 6.8) and showed in Figure 2. For G(PD) and (GP)D, the introduction of negatively charged PLG (mass ratio, PLG:PEI=1:2) decreased the NPs' potentials compared with PD. Further shielding PEG on the surface of (GP)D effectively decreased its potential. More importantly, the potential of P[(GP)D] NPs was much higher in pH 6.8 than pH 7.4 (Figure 2A). The potential rebounding was attributed to PEG detaching from (GP)D under acidic environment. The particle sizes of G(PD) and (GP)D were bigger than PD, and further PEG crosslinking decreased their sizes in pH 7.4. Furthermore, their size changes showed different pH-dependent manners. The sizes of PD and G(PD) were insensitive to slight pH variations. But (GP)D could be effectively swelled by weak acidic pH, possessing an obvious bigger size in pH 6.8 compared with pH 7.4. It was worth noting that the size gap between pH 6.8 and 7.4 was amplified for P[(GP)D] (Figure 2B). This was caused by the following two reasons. The crosslinking of PEG on (GP)D surface could tighten its size in pH 7.4, and the detaching of PEG from P[(GP)D] would release (GP)D, restoring its size in pH 6.8. Thus a 6


Page 6 of 20

Page 7 of 20

shielding/condensing and pH-triggered charge/size dual rebound DNA loaded NP was developed by using in situ crosslinking strategy. At the same time, we monitored the speed of the shielding/condensing and charge/size rebound towards pH variations. The above process could happen within 5 min (Figure 2C, D). The morphologies of the NPs were observed by TEM (Figure 2E), which showed consistency with Figure 2B, further proving the pH-triggered size rebound behaviors. The ultrasensitive pH-triggered charge/size dual rebound NPs had lower zeta potential and smaller size during transportation in the blood, but higher zeta potential and bigger size in tumor tissue which helped to enhance cellular uptake. It was reported that the cellular uptake increased with the increasing size of the NPs to some extent,12 the pH-triggered dual rebound NPs were expected to have "stealthy" PEG shell and small size during the process of delivery, which would be benefit to long circulation. When the NPs accumulated in the slightly acidic tumor sites by EPR (enhanced permeability and retention) effect, the detaching PEG and rebounded charge/size would be helpful for further cell uptake.

Zeta potential (mV)

50 40

B

**

**

30 20 10 0

C

pH 7.4 pH 6.8

PD

G(PD)

(GP)D

D

30

20

10

(GP)D

P[(GP)D] pH 7.4

pH 6.8

**

**

150 100

PD

300

G(PD)

< 5min

(GP)D

P[(GP)D]

< 5min

250 200 150 100 50

P[(GP)D]

**

200

< 5min Particle size (nm)

< 5min

pH 7.4 pH 6.8

250

50

P[(GP)D]

40

0

300

Particle size (nm)

A

Zeta potential (mV)

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

Nano Letters

(GP)D

P[(GP)D] pH 7.4

7


P[(GP)D] pH 6.8

Nano Letters

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

E

Figure 2. (A) Zeta potential and (B) particle size of the NPs. (C, D) Charge/size dual-rebound property of the gene delivery system. (E) TEM images of the NPs (scale bar: 100 nm).

DNA transfection was carried out in CT26 cells and the luciferase plasmid DNA (pGL3-control) was used as the reporter gene. The 48 hours transfection efficiencies of (GP)D showed that the optimal mass ratio for PLG:PEI:DNA was 1.25:2.5:1 (Figure S5). The transfections of P[(GP)D] (with different PEG mass ratios) in different pH values were further tested. After PEG crosslinking, all the tested ratios showed significant transfection efficiency difference between pH 7.4 and 6.8 (Figure 3A). The detachment of PEG led to the exposure of more positive charges and increase of particle size, resulted in much higher transfection efficiency in pH 6.8 compared with pH 7.4. As the Schiff base reaction between PEI and PEG was very fast and efficient for in situ crosslinking, it was a chemical bench-free process, which provided a convenient method to adjust the PEG density in the gene delivery system. As showed in Figure 3A, the NPs with PEG:PLG:PEI:DNA mass ratio of 5:1.25:2.5:1 displayed the highest transfection efficiency and biggest differences between pH 6.8 and 7.4. So this optimal ratio was selected for following experiments. Cell uptake efficiency was evaluated by FCM (flow cytometry) and CLSM (confocal laser scanning microscopy). The results were shown in Figure 3B, C. The uptake efficiency of G(PD) in pH 6.8 was higher than that in pH 7.4, this was attributed to the increased potential in pH 6.8. The uptake efficiency of (GP)D in pH 6.8 was also higher than that in pH 7.4, this was attributed to the increased potential and size in pH 6.8. It could be concluded that acidic 8


Page 8 of 20

Page 9 of 20

pH could enhance the uptake efficiency for G(PD) and (GP)D. The enhancement of (GP)D was bigger than that of G(PD), this was caused by the bigger size change for (GP)D in different pH than that for G(PD). The uptake enhancement was further amplified for P[(GP)D] (Figure 3B). Because the shielding/condensing happened upon introduction of PEG onto (GP)D which leaded to decreased positive charges and sizes, resulting in dramatically reduced uptake efficiency. Furthermore, the acidic pH could induce stronger uptake efficiency rebound. For CLSM (Figure 3C and Figure S6), the cell nucleus was stained in blue by DAPI, and the cell membrane was stained in green with Alexa Fluor 488 phalloidin (AF 488). The Cy5 labeled DNA (Cy5-DNA) and Cy5 labeled PEI (Cy5-PEI) were respectively used for NPs preparation and intracellular tracking, and the red fluorescence was from Cy5-DNA or Cy5-PEI. (GP)D showed stronger red fluorescence in pH 6.8 than pH 7.4, and acidic pH could cause sharp red fluorescence contrast for P[(GP)D]. These results gave another

A

9

10

pH 7.4 pH 6.8

**

***

***

***

B

8

10

7

10

0

2.5

5

10

20

Mean fluorescence intensity

conclusive proof for the enhancement of charge/size rebound to cellular uptake efficiency.

RLU/mg 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

Nano Letters

1200 1000

pH 7.4

***

***

(GP)D

P[(GP)D]

pH 6.8

800 600 400 200 0

PD

G(PD)

P[(GP)D]=X/1.25/2.5/1

9


Nano Letters

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

C

Figure 3. (A) Transfection efficiency of P[(GP)D] (with various PEG mass ratios) at different pH values (7.4 and 6.8) in CT26 cells for 2 h. Data are shown as mean ± SD (n=3). (B) Mean fluorescence intensity of cellular uptake of PD, G(PD), (GP)D and P[(GP)D] at different pH values (7.4 and 6.8). (C) CLSM images of CT26 cells incubated with D, PD, G(PD), (GP)D and P[(GP)D] at different pH values (7.4 and 6.8), Cy5-DNA was tracked.

The cytotoxicity of the NPs was measured by MTT assay (Figure S7). PD was toxic to the cells, as it contained redundant positive charges, which might have an unfavorable influence on the cells. G(PD) and (GP)D were less cytotoxic than PD, the introduction of PLG had decreased the positive charge of PEI. When (GP)D was further shielded by PEG, the cytotoxicity was inhibited notably in pH 7.4, which would be helpful to decrease the damages to normal organs by systemic administration. The tumor accumulation of the different NPs was evaluated by ex vivo imaging on subcutaneous tumor model (Figure S8). The tumor-bearing mice were injected with the NPs via tail vein. The Cy5-DNA and Cy5-PEI were respectively used for NPs preparation and tracking. After 24 h, the tumors were excised and imaged. For PD and G(PD) groups, ignorable Cy5-DNA and Cy5-PEI fluorescence was observed in tumors. Cy5-DNA and Cy5-PEI fluorescence for (GP)D could be detected in tumor area, but the fluorescence was very weak. For P[(GP)D], both Cy5-DNA and Cy5-PEI showed the most effective 10


Page 10 of 20

Page 11 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

accumulation in tumor among all the groups. This result indicated that DNA and PEI in P[(GP)D] could be synchronously delivered to tumors. This could prove that DNA was stably complexed with PEI during delivery process after systemic injection. Pharmacokinetics was further studied by testing the Cy5-DNA concentrations in blood at fixed time intervals after the NPs were injected into the mice (Figure S9). The detected DNA concentration in blood decreased dramatically. D group showed lower DNA concentrations than the other groups. P[(GP)D] had highest DNA concentrations, which could be attributed to its long circulation feature. The as-prepared ultrasensitive pH-triggered charge/size dual rebound gene delivery has realized long circulation, high tumor cellular uptake and tumor accumulation, which was caused by PEG shielding, lower positive charges and smaller size in circulatory system, and PEG deshielding, higher positive charges and bigger size in acidic tumor site. This exciting result encouraged us to perform in vivo anti-tumor treatment. CT26 tumor-bearing mice were injected by PBS, D, PD, G(PD), (GP)D and P[(GP)D] via tail vein respectively. The pDNA which expressed shVEGF was used as the therapeutic gene, shVEGF could down-regulate the expression of VEGF. VEGF was crucial in tumor growth, infiltration and metastasis, it could stimulate the proliferation of endothelial cells and increase the tumor angiogenesis, thus inhibition of VEGF expression was beneficial for the treatment of tumor.31,32 The therapeutic gene injection dosage was 1 mg/kg body weight. The tumor volume and body weight were monitored every other day and the results were shown in Figure 4. The tumor volume increased rapidly for the PBS and D group, the other groups appeared effective antitumor therapeutic efficacy (Figure 4B). The P[(GP)D] group showed the most excellent therapeutic efficacy, and the tumors were much smaller than the other groups (Figure 4A). Moreover, it was worth mentioning that for P[(GP)D], no mice was died during the treatment process, but died mice were found for the PD, G(PD) and (GP)D groups. A relatively stable body weight (Figure 4C) was observed in the mice treated with the six groups, which revealed the good biocompatibility of the designed gene delivery system. To evaluate the safety of the NPs, the major organs (heart, liver, spleen, lung and kidney) and tumors were collected and examined by H&E staining (Figure 4D). The major organs of all the treatment groups showed normal histomorphology and had no pathological 11


Nano Letters

abnormality. For the tumors, extremely compact cell arrangement was observed in PBS group. After effective treatment, the tumor tissue morphology had changed and the tumor cell nuclei were dissolved. From the result, a great number of cell nuclei were dissolved for (GP)D and P[(GP)D] group, and the latter had most effective reduction for tumor cells. The tumor tissue sections were further detected for CD31 by immunofluorescence technique to assess the tumor angiogenesis inhibition (Figure 4E). The PBS and D group had a strong CD31 related fluorescence, which represented a horrible angiogenesis in tumor. PD, G(PD) and (GP)D group showed slight improvements on angiogenesis inhibition. It was encouraging to see that the P[(GP)D] exhibited significant reduction of angiogenesis. The tumor angiogenesis was a crucial factor for tumor development, the effective inhibition of tumor angiogenesis was helpful to eliminate the tumor cells for cancer therapy.

A

2000

C

3

PBS D PD G(PD) (GP)D P[(GP)D]

1600 1200

*** ***

800

*** ***

400

30

Body weight (g)

B Tumor volume (mm )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 20

25

PBS D PD G(PD) (GP)D P[(GP)D]

20

**

0

15 0

2

4

6

8

10

12

0

2

4

6

8

10

Time of treatment (day)

Time of treatment (day)

12


12

Page 13 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

D

E

Figure 4. (A) Images of the tumors at the end of the treatment. (B) Tumor volume changes of the mice administered with PBS, D, PD, G(PD), (GP)D and P[(GP)D] by intravenous injection. (C) Body weight changes of the mice during the treatment. (D) H&E-stained major organs and tumors from different treatment groups. (E) Immunofluorescence of CD31 for tumor tissue sections, CD31-positive vessels were stained green, and cell nuclei were stained blue.

The Hb and HbO2 in the blood could be detected by photoacoustic imaging, and used to reflect the location of blood vessels inside the tumors and further helped to estimate the 13


Nano Letters

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

anti-angiogenesis effect.33-35 The results were shown in Figure 5A, B. The Hb was detected as the blue signal, and the red signal was assigned to the HbO2 (Figure 5A). The signal was also quantified as the mean intensity (Figure 5B). The PBS group showed much stronger signals than the other groups, which represented a dense blood vessels existed in tumor. And P[(GP)D] group showed lower intensity of Hb and HbO2, which meant less blood vessels were in the tumor tissue, P[(GP)D] group could effectively suppress the tumor angiogenesis. The VEGF gene expression in mRNA level was measured by Real-time PCR (RT-PCR). As shown in Figure 5C, the relative quantity of VEGF mRNA of PBS group was set as 1. The VEGF mRNA decreased after the tumors were treated with the therapeutic gene loaded NPs. P[(GP)D] showed the most remarkable effect on the down-regulation of VEGF gene expression. The VEGF protein in the tumor tissues was evaluated by double antibody sandwich ELISA (Figure 5D). For PBS group, VEGF was detected as high as 11 pg/mg in the tumor tissues. After treated with the therapeutic gene-loaded NPs, the VEGF gradually decreased to different levels. In line with the other results, the P[(GP)D] group showed the most effective reduction by significantly decreasing the amount of VEGF to 3 pg/mg. This result further helped us to confirm the superior tumor inhibition of P[(GP)D] NPs. The VEGF content in the tumor tissues was also evaluated by western blot, and the result was shown in Figure 5E. Compared with PBS group, obvious reductions of VEGF protein were detected for G(PD), (GP)D and P[(GP)D] NPs. P[(GP)D] group showed most marked VEGF protein reduction in the tumors, which further reliably confirmed that the P[(GP)D] group was the most effective anti-tumor gene delivery system in our study.

14


Page 14 of 20

Page 15 of 20

A

2.8

B

Hb HbO2

Mean intensity

2.4 2.0 1.6 1.2 0.8 0.4 0.0

PBS

***

1.2

D

PD

D

*** ***

1.0

**

0.8 0.6

*

0.4 0.2 0.0

PBS

D

PD

G(PD)

(GP)D P[(GP)D]

***

12

VEGF (pg/mg tissue)

C Relative quantity (dRn)

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

Nano Letters

*** ***

10 8

*

6 4 2 0

) ] D G(PD (GP) P[(GP)D

***

PBS

D

E

15


PD

] ) D G(PD (GP) P[(GP)D

Nano Letters

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

Figure 5. (A) Photoacoustic imaging and (B) Mean intensity of Hb and HbO2 in tumors of different treatment groups. (C) Relative quantity of VEGF mRNA in the tumors treated by different groups. (D) ELISA of the VEGF protein in the tumors treated by different groups. (E) Western blot analysis of the VEGF protein in the tumors treated by different groups.

In conclusion, an ultrasensitive pH triggered charge/size dual-rebound gene delivery system was developed by a facile and practical strategy for efficient tumor treatment. The rational designed system was sufficiently investigated through a series of experiments both in vitro and in vivo. The gene delivery system possesseed shielding/condensing status in normal pH, and had rapid charge and size rebound in acdic tumor environment, this feature caused long circulation and high tumor accumulation. An excellent anti-tumor therapeutic efficacy was achieved for the anti-angiogenisis therapeutic gene loaded NPs with dramatic inhibition of the tumor volume and unaffected body weight. The effective inhibition of tumor angiogenesis was confirmed by immunofluorescence technique and photoacoustic imaging. VEGF mRNA was dramatically decreased by the dual-rebound gene delivery system and the VEGF reduction in protein level was verified by ELISA and western blot. The results clearly confirmed that the pH triggered charge/size dual-rebound gene delivery system had outstanding anti-tumor therapeutic efficacy and would have great potential in tumor treatment. Furthermore, our study had clearly verified that the aldehyde group modified PEG was a simple, convenient and facile shielding for positively charged NPs, and could be readily and extensively applied to other delivery systems. The active ligand will be introduced to the system for tumor targeting for further improvement.

ASSOCIATED CONTENT Supporting Information Supporting Information Available: [Experimental section, supplementary figures: 1H NMR spectra of the materials, the evaluations of transfection efficiency, CLSM, cytotoxicity, tumor accumulation and pharmacokinetics of the nanosystems are provided.] This material is available free of charge via the Internet at http://pubs.acs.org. 16


Page 16 of 20

Page 17 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected].

Author Contributions §

These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors are thankful to the National Natural Science Foundation of China (21474104, 51222307, 51321062, 51233004, 51403205, 51390484 and 51303173) for financial support to this work.

REFERENCES (1) Hatakeyama, H.; Akita, H.; Kogure, K.; Oishi, M.; Nagasaki, Y.; Kihira, Y.; Ueno, M.; Kobayashi, H.; Kikuchi, H.; Harashima, H. Gene Ther. 2007, 14, 68-77.

(2) Merdan, T.; Kopec̆ek, J.; Kissel, T. Adv. Drug Delivery Rev. 2002, 54, 715-758. (3) Morille, M.; Passirani, C.; Vonarbourg, A.; Clavreul, A.; Benoit, J.-P. Biomaterials 2008, 29, 3477-3496. (4) Tian, H.; Tang, Z.; Zhuang, X.; Chen, X.; Jing, X. Prog. Polym. Sci. 2012, 37, 237-280. (5) Liu, X.; Xiang, J.; Zhu, D.; Jiang, L.; Zhou, Z.; Tang, J.; Liu, X.; Huang, Y.; Shen, Y. Adv. Mater. 2015, 28, 1743-1752. (6) Kircheis, R.; Wightman, L.; Wagner, E. Adv. Drug Delivery Rev. 2001, 53, 341-358. (7) de Lima, M. C. P.; Simoes, S.; Pires, P.; Faneca, H.; Düzgüneş, N. Adv. Drug Delivery Rev. 2001, 47, 277-294. (8) Sun, Q.; Sun, X.; Ma, X.; Zhou, Z.; Jin, E.; Zhang, B.; Shen, Y.; Van Kirk, E. A.; Murdoch, W. J.; Lott, J. R. Adv. Mater. 2014, 26, 7615-7621. 17


Nano Letters

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

(9) Pearce, T. R.; Shroff, K.; Kokkoli, E. Adv. Mater. 2012, 24, 3803-3822. (10) Xu, C.-H.; Sui, M.-H.; Tang, J.-B. Chin. J. Polym. Sci. 2011, 29, 274-287. (11) Jin, E.; Zhang, B.; Sun, X.; Zhou, Z.; Ma, X.; Sun, Q.; Tang, J.; Shen, Y.; Van Kirk, E.; Murdoch, W. J. J. Am. Chem. Soc. 2013, 135, 933-940. (12) Liang, S.; Yang, X. Z.; Du, X. J.; Wang, H. X.; Li, H. J.; Liu, W. W.; Yao, Y. D.; Zhu, Y. H.; Ma, Y. C.; Wang, J. Adv. Funct. Mater. 2015, 25, 4778-4787. (13) Tasciotti, E.; Liu, X.; Bhavane, R.; Plant, K.; Leonard, A. D.; Price, B. K.; Cheng, M. M.-C.; Decuzzi, P.; Tour, J. M.; Robertson, F. Nat. Nanotechnol. 2008, 3, 151-157. (14) Dufort, S.; Sancey, L.; Coll, J.-L. Adv. Drug Delivery Rev. 2012, 64, 179-189. (15) Mishra, S.; Webster, P.; Davis, M. E. Eur. J. Cell Biol. 2004, 83, 97-111. (16) Hatakeyama, H.; Akita, H.; Harashima, H. Adv. Drug Delivery Rev. 2011, 63, 152-160. (17) Deng, C.; Jiang, Y.; Cheng, R.; Meng, F.; Zhong, Z. Nano Today 2012, 7, 467-480. (18) Guo, S.; Huang, L. J. Nanomater. 2011, 2011, 1-12. (19) Du, F.-S.; Wang, Y.; Zhang, R.; Li, Z.-C. Soft Matter 2010, 6, 835-848. (20) Du, J. Z.; Sun, T. M.; Song, W. J.; Wu, J.; Wang, J. Angew. Chem., Int. Ed. 2010, 122, 3703-3708. (21) Zhang, Z.; Zhang, X.; Xu, X.; Li, Y.; Li, Y.; Zhong, D.; He, Y.; Gu, Z. Adv. Funct. Mater. 2015, 25, 5250-5260. (22) Guo, X.; Wei, X.; Jing, Y.; Zhou, S. Adv. Mater. 2015, 27, 6450-6456. (23) Sun, C.-Y.; Shen, S.; Xu, C.-F.; Li, H.-J.; Liu, Y.; Cao, Z.-T.; Yang, X.-Z.; Xia, J.-X.; Wang, J. J. Am. Chem. Soc. 2015, 137, 15217-15224. (24) Sun, C. Y.; Liu, Y.; Du, J. Z.; Cao, Z. T.; Xu, C. F.; Wang, J. Angew. Chem., Int. Ed. 2016, 128, 1022-1026. (25) Ko, J.; Park, K.; Kim, Y.-S.; Kim, M. S.; Han, J. K.; Kim, K.; Park, R.-W.; Kim, I.-S.; Song, H. K.; Lee, D. S. J. Controlled Release 2007, 123, 109-115. (26) Liu, J.; Huang, Y.; Kumar, A.; Tan, A.; Jin, S.; Mozhi, A.; Liang, X.-J. Biotechnol. Adv. 2014, 32, 693-710. (27) Prabaharan, M.; Grailer, J. J.; Pilla, S.; Steeber, D. A.; Gong, S. Biomaterials 2009, 30, 5757-5766. (28) Bae, Y.; Fukushima, S.; Harada, A.; Kataoka, K. Angew. Chem. 2003, 42, 4640-4643. 18


Page 18 of 20

Page 19 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

(29) Xia, J.; Chen, J.; Tian, H.; Chen, X. Sci. China: Chem. 2010, 53, 502-507. (30) Gu, J.; Cheng, W.-P.; Liu, J.; Lo, S.-Y.; Smith, D.; Qu, X.; Yang, Z. Biomacromolecules 2007, 9, 255-262. (31) Liu, T.; Ye, L.; He, Y.; Chen, X.; Peng, J.; Zhang, X.; Yi, H.; Peng, F.; Leng, A. Exp. Mol. Pathol. 2011, 91, 745-752. (32) Inai, T.; Mancuso, M.; Hashizume, H.; Baffert, F.; Haskell, A.; Baluk, P.; Hu-Lowe, D. D.; Shalinsky, D. R.; Thurston, G.; Yancopoulos, G. D. Am. J. Pathol. 2004, 165, 35-52. (33) Song, W.; Tang, Z.; Zhang, D.; Yu, H.; Chen, X. Small 2015, 11, 3755-3761. (34) Mallidi, S.; Luke, G. P.; Trends Biotechnol. 2011, 29, 213-221. (35) Jose, J.; Manohar, S.; Kolkman, R. G.; Steenbergen, W.; van Leeuwen, T. G. J. Biophotonics 2009, 2, 701-717.

19


Nano Letters

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

An ultrasensitive pH triggered charge/size dual-rebound gene delivery system is designed via a facile and practical strategy. It can be constructed by an in situ chemical bench-free process and possesses long circulation and high tumor accumulation. The system shows outstanding anti-tumor efficacy and will have great potentials in tumor treatment.

20


Page 20 of 20

Size Dual-Rebound Gene

Recommend Documents