Charge and Assembly Reversible Micelles Fueled by Intracellular ATP

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Biological and Medical Applications of Materials and Interfaces

Charge and Assembly Reversible Micelles Fuelled by Intracellular ATP for Improved siRNA Transfection Zhanwei Zhou, Chenzi Li, Minghua Zhang, Qingyan Zhang, Chenggen Qian, David Oupicky, and Minjie Sun ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b13300 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018

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ACS Applied Materials & Interfaces

Charge and Assembly Reversible Micelles Fuelled by Intracellular ATP for Improved siRNA Transfection

Zhanwei Zhou a, Chenzi Li a, Minghua Zhang a, Qingyan Zhang a, Chenggen Qian a, David Oupicky a, b, Minjie Sun a, *

a State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P. R. China b Center for Drug Delivery and Nanomedicine Department of Pharmaceutical Sciences University of Nebraska Medical Center Omaha, NE 68198, USA

* Corresponding authors: Prof. Minjie Sun, Phone /Fax: +86 25 83271098, Email: [email protected]

KEYWORDS: ATP fuelled trigger, cationic micelles, charge reversal, phenylboronic acid, siRNA delivery

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ABSTRACT Hydrophobic modification on polycations were commonly used to improve the stability and transfection efficiency of polyplexes. However, the improved stability often means undesired release of the encapsulated siRNA, limiting the application of cationic micelles for siRNA delivery. The current strategy of preparing bio-responsive micelles based on the cleavage of sensitive linkages between polycation and hydrophobic part was far from sufficient owing to the siRNA binding of the separated polycations from micelles, leading to the incomplete release of siRNA. In this study, we propose a new strategy by the combination of micelles disassembly

and

separated

polycations

charge

reversal.

FPBA

(3-fluoro-4-

carboxyphenylboronic acid) grafted PEI 1.8k (polyethyleneimine) as the polycations of PEIFPBA and dopamine (diols contained moiety) conjugated cholesterol (Chol-Dopa) as the hydrophobic part. The PFCDM micelles was assembled by PEI-FPBA and Chol-Dopa, based on the FPBA-Dopa conjugation. The prepared PFCDM showed strong siRNA loading ability and superior stability in the presence of PBS or serum. Besides, the PFCDM exhibited excellent ATP sensibility. The intracellular ATP could effectively trigger the disassembly of micelles and charge reversal of PEI-FPBA, resulting in the burst release of siRNA in the cytosol. With the property of extracellular stability and intracellular instability, PFCDM displayed good performance on in vitro and in vivo luciferase silencing on 4T1 cells. It should also be noted that the assembly of low molecular weight PEI was relatively safe to cells compared with 25k PEI. To sum up, the ATP fuelled assembly and charge reversible micelles gave examples for polyplexes to achieve better stability and on demand cargo release at the same time and shows potential to be used for in vitro and in vivo siRNA transfection.


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INTRODUCTION Over

the

past

decades,

numerous carriers

have

been developed

to

achieving

temporally/spatially controlled nucleic acid release.1-2 Among the delivery vectors, polycations were the most used materials by complexing with nucleic acid.3-4 Although great breakthrough has been obtained, there are also some problems of the polycations needed to be solved. The polycations, especially low molecular weight ones, showed weak nucleic acid condensing ability and poor stability in the blood circulation.5-6 They were easily destroyed by anions, such as phosphates, serum. These properties greatly hurdle the application of polycations on gene delivery. Many groups reported that the hydrophobic modification would improve the gene delivery by polycations.7-8 In our previous work, we synthesized cholesterol modified PAMD,9 which significantly enhanced the RNase stability when compared with cholesterol unmodified polyplexes (PAMD/siRNA). However, the improved stability also means uncontrollable release of siRNA in the cell. The current strategy for releasing gene at target site are mainly based on the strategy of conjugating a stimulus responsive linker between polycations and hydrophobic component. At target site, the cleavage of the linker would promote the disassembly of micelles and weaken the charge density. Unfortunately, this strategy sometimes was not sufficient because the separated polycations could still bind with nucleic acid, leading to the incomplete release of loaded gene.10-11 Therefore, how to avoid the undesired nucleic acid binding with separated polycations was of great importance for satisfactory nucleic acid release. In this study, we tried to propose a new strategy by the combination of disassembly of cationic micelles and charge reversal of separated polycations for accelerating nucleic acid release. Low molecular weight polyethyleneimine (PEI) was used as model polycations, cholesterol as the hydrophobic component and siRNA was used as the model nucleic acid for research.



Phenylboronic acid (PBA) could form dynamic borate esters with cis-diol contained molecules and has been widely used for drug delivery system.12-14 The boronate bond is easily formed and reversible in some specific conditions, such as acidic environment, diols abundant environment.15-16 Therefore, the reaction between PBA and diols met up the requirement of this study to prepare intracellular reversible micelles. We are desired to synthesis FPBA (3fluoro-4-carboxyphenylboronic acid) grafted PEI as the polycations. Dopamine (diols contained moiety) conjugated cholesterol (Chol-Dopa) as the hydrophobic part. The micelles could be assembled by the reaction between PBA and Dopa (Scheme 1). In our knowledge, adenosine triphosphate (ATP) is the most abundant ribonucleotide in cells, which reached 1-10 mM in the cytosol.17-19 ATP provides the energy for cellular proliferation and metabolism by the hydrolysis of triphosphate, which reflects the important role of triphosphate. For ATP-triggered release research, especially PBA based ATP sensitive system, most researchers focused on the diols group on ATP to trigger the breakage of PBAdiols linkage.10, 20 However, few researchers took the triphosphate for triggering cargo release. Another important knowledge should be taken into consideration is that ATP is negatively charged in the cytosol for the dissociation of protons on triphosphate.14, 21-22 Therefore, we made the hypothesis that the formation of boronate bond between PBA and ATP could reversal the charge of PEI-FPBA from positive to negative, forcing the release of siRNA by electrostatic repulsion.23-25 Besides, the PBA-ATP reaction would also trigger the disassembly of micelles by competing the PBA binding with Dopa, which would also decrease the charge density, assistant for accelerating siRNA release (Scheme 2). As designed, the formation of PFCDM (PEI-FPBA-Dopa-Chol micelles) could improve the stability of PEI-FPBA polyplex and hence enhance the siRNA transfection in FBS contained environment. Furthermore, once the PFCDM polyplex was delivered into the cells, the intracellular ATP would disturb the balance and reversal the charge and assembly of PFCDM, promoting the siRNA release. ATP, as the fuel, plays two roles on triggering siRNA ACS Paragon Plus Environment

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release. Diols and triphosphate are both important parts. The diols could promote the cleavage of the PBA-Dopa linkage, triggering the disassembly of micelles. In addition, the triphosphate would reverse the charge of the separated PEI-FPBA (Scheme 2). Apart from stabilities and release study, the in vitro and in vivo siRNA transfection efficiency of PEI-FPBA/siRNA and PFCDM/siRNA were compared in this study to exhibited the advantages of micelles.

Scheme 1. (A) Schematic illustration of the formation of PFCDM/siRNA cationic micelles. (B) Schematic illustration of intracellular siRNA delivery by PFCDM/siRNA: (a) Cell uptake of cationic micelles; (b) Endosomal escape of PFCDM/siRNA; (c) ATP triggered release of siRNA by the disassembly of PFCDM and charge reversal of PEI-FPBA.



Scheme 2. (A) Schematic illustration of ATP triggered breakage of FPBA-Dopa and the disassembly of PFCDM. (B) Schematic illustration of ATP activated charge reversal of PEIFPBA.

EXPERIMENTAL SECTION

Materials, cell culture and animals Branched polyethyleneimine (1.8k PEI and 25k PEI), 3-fluoro-4-carboxyphenylboronic acid (FPBA), dopamine, EDCHCl were purchased from Aladdin chemical regent company (Shanghai, China). Cholesteryl chloroformate was obtained from Macklin Biochemical Co., Ltd (Shanghai, China). MTT, DiO, DiI and GelRed were obtained from KeyGEN BioTECH (Nanjing, China). ACS Paragon Plus Environment

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siScr (scrambled siRNA), FAM-siScr, cy3-siScr, siLuc (targeting luciferase) and siEGFP (targeting EGFP) were purchased from GenePharma (Shanghai, China). The siRNA sequences were reported in our previous work.26 4T1 mouse breast cancer cells were purchased from American Type Culture Collection (ATCC). Female BABL/c mice (18-22 g) were bought from Yangzhou University. Synthesis and characterization of PEI-FPBA 3-fluoro-4-carboxyphenylboronic acid (FPBA) was conjugated to branched PEI 1.8k via amide bonds. Briefly, 184 mg (1 mmol) of FPBA was dispersed in 10 mL of mixture solvent, where the volume ratio of ethanol and H2O was 1:1. Then, 390mg (2 mmol) of EDCHCl and 230 mg (2 mmol) of NHS was added into the solution and reacted for 30 minutes. The resulted solution was dropped into 172 mg of PEI 1.8k solution and kept stirring for 24 h. After reaction, the mixture solution was dialyzed (MWCO = 1000 Da) against DW to remove the catalyzer or unreacted FPBA. The chemical structure of PEI-FPBA was characterized by 1

H NMR spectra and FTIR.

Synthesis and characterization of Chol-Dopa Cholesterol chloroformate (0.9 g, 2 mmol) and dopamine (0.306 g, 2 mmol) were separately dissolved in 20 mL mix solution (DCM : Methanol=1:1). Afterwards, the dopamine solution was injected into the cholesterol chloroformate solution drop by drop under N2 protection. After reaction for 24 h, the reaction mixture solution was washed with DW (2 10 mL) and extracted with DCM. The DCM layer was then dried with anhydrous Na2SO4. Finally, the DCM was removed to get a white powder. The purified product was obtained after separation with column chromatography. The chemical structure of Chol-Dopa was validated by 1H-NMR. Formation of cationic micelles



PEI-FPBA/Chol-Dopa cationic micelles (PFCDM) was prepared by solvent evaporation method.15, 27 Briefly, PEI-FPBA was dispersed in HEPES buffer (pH 7.4) with a concentration of 2.6 mg/mL (FPBA, 5 mM). Chol-Dopa was dissolved in 100 µL THF at various concentrations to obtain series Dopa/FPBA molar ratios of 0, 0.25, 0.5, 1, 2, 3, 4. The obtained Chol-Dopa solutions was slowly added to PEI-FPBA solutions under vigorous stirring for 24 h. At last the THF was eliminated by rotary evaporation to obtain the micelles solutions. The reaction between FPBA and Dopa was indicated by Alizarin Red S (ARS).28 To obtain the optimized ratios of Chol-Dopa/PEI-FPBA, the particle size of micelles was detected by dynamic light scattering (DLS). The morphological observation of PFCDM was obtained by transmission electron microscope (TEM) (H600, Hitachi, Japan) and the zeta potential was detected by Malvern Zetasizer Nano ZS (Malvern, UK). Formulation study of siRNA loaded polyplexes The siRNA loaded polyplexes were prepared by the mixing of siRNA solution with carrier solution. First, siRNA was dissolved in 10 mM HEPES solution (pH 7.4) at 40 µg/mL. PEI-FPBA or PFCDM was also diluted with HEPES solution to obtained a series of concentrations, according to the desired polycation/siRNA (w/w) ratios. The siRNA encapsulation was evaluated on agarose gel electrophoresis. Briefly, agarose gel was prepared at 1% (w/v) concentration and GelRed was added into the gel to indicate the siRNA. The prepared polyplexes at various w/w ratios were loaded on agarose gel at 20µL each well. After running for 15 min, the gel was imaged under UV illumination. Particle size and zeta potential of the polyplexes were also measured by Malvern Zetasizer Nano ZS (Malvern, UK). ATP-triggered siRNA release


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The ATP triggered release study was performed by agarose gel electrophoresis. PEIFPBA/siRNA and PFCDM/siRNA polyplexes were incubated with 4 mM ATP, Adenosine and dATP (deoxyadenosine triphosphate) separately. The polyplexes were incubated with the trigger solutions for 0.5 h before detection. The siRNA release study was also evaluated by fluorescence resonance energy transfer (FRET) technology. Cy3- and cy5- labeled siRNA were co-encapsulated within the polyplexes (FRET polyplex) at 1:1 ratio.29 The FRET ratios were calculated as Icy5/(Icy5 + Icy3) × 100%, where Icy5 and Icy3 are the ﬂuorescence intensities at 670 nm and 570 nm. The zeta potentials changes of PEI-FPBA, PFCDM and there polyplexes (PEIFPBA/siRNA, PFCDM/siRNA, w/w 8) after different ATP added were measure by Malvern Zetasizer Nano ZS. The ATP triggered disassembly of PFCDM/siRNA was also evaluated by FRET technology. Hydrophobic dyes DiO and DiI were coloaded in the PFCDM to form FRET micelles.30 The disassembly of the micelles was evaluated by the changing of FRET ratios which was calculated as IDiI/(IDiI + IDiO) × 100%, where IDiI and IDiO are ﬂuorescence intensities of DiI at 565 nm and DiO at 501 nm, respectively. Besides, the morphological changing of PFCDM/siRNA before and after 4mM ATP treatment was observed by TEM. Stability of polyplexes The stability of polyplexes in different environment (D.W, 10 mM pH 7.4 PBS, 10 mM glucose and 10% serum) were evaluated by monitoring the particle size at time intervals (0, 0.5, 1, 2, 4, 8, 12, 24, 36, 48 h). In addition, it was also checked by the changing of FRET ratio after various treatment. For serum stability, naked siRNA, PEI-FPBA/siRNA and PFCDM/siRNA were incubated in 10% FBS at 37 oC. After incubation, SDS was added into the solution to replace the encapsulated siRNA and then the siRNA was detected by agarose gel electrophoresis. ACS Paragon Plus Environment


Cytotoxicity analysis Cytotoxicity of carriers (25k PEI, PEI-FPBA and PFCDM) was studied by MTT assay. The 4T1 cells were incubated with serial concentrations of carriers for 24 h in the incubator. Then, the MTT reagent (5 mg/mL in PBS) was added (20 µL each well). The cells were incubated with MTT for 4 h, followed by replacing the solution with DMSO to dissolve the formazan. Next, the absorbance of each well at 570 nm was detected by microplate reader. To measure the cytotoxicity of polyplexes, siScr was loaded and polyplexes were prepared at various w/w ratios (1, 2, 4, 8). Then, the polyplex solutions were diluted with FBS free medium at siScr concentration of 100 nM. The cells were incubated with polyplexes for 4 hours. Afterwards, the old medium was replaced with 10% FBS contained medium for another 20 hours. The MTT assay was performed as described above. Cell uptake and endo/lysosome escape The cellular uptake study of polyplexes (PEI-FPBA/siRNA and PFCDM/siRNA) were carried on by confocal laser scanning microscope (CLSM) and flow cytometry (FCM). For observation of cell uptake by CLSM, 4T1 cells were cultured in confocal dishes for 24 h. Then, the cells were treated with PEI-FPBA/FAM-siRNA or PFCDM/FAM-siRNA for 2 h. After incubation, the cells were washed and fixed. Besides, the nuclei were stained with DAPI solution. Then, the cells were observed and imaged by CLSM (Carl Zeiss LSM 700, Germany). For the observation of endo/lysosomal escape, 4T1 cell were treated with PFCDM/FAMsiRNA polyplex for 3 h. After incubation, the polyplexes contained medium was replaced with fresh medium. The cells were cultured in the incubator for additional 0 or 3 h. Afterwards, cells were washed and the endo/lysosomes were stained with LysoTracker Red. After removing the LysoTracker and washed with PBS twice, the cells were observed by CLSM immediately. Intracellular ATP-triggered siRNA release ACS Paragon Plus Environment

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We used FRET technology to verify the ATP triggered siRNA release. Here, the cells were cultured at low temperature (4 °C) or with iodoacetic acid (IAA) at 37 °C to build low concentration of ATP model.31 Briefly, 4T1 cells were seeded in confocal microscopy dishes. The next day, cells were incubated with cy3-/cy5-siRNA coloaded PFCDM polyplex (both cy3-siRNA and cy5-siRNA was 100 nM, polymer/siRNA w/w=4) at 37 °C for 4 h. Afterwards, the cells were separately incubated with medium at 37 °C, 4 °C or with iodoacetic acid (IAA) at 37 °C for additional 4 h. Followed by washing the 4T1 cells with PBS twice and observed via CLSM. Luciferase silencing in vitro Luciferase silencing assay was performed on luciferase stably expressed cells (4T1-Luc). 4T1-Luc cells were cultured in 48-well plate. After 24 h, the cells were incubated with siLuc loaded polyplexes in the presence of different concentrations of FBS (0%, 10%, 30%) for 6 h. Next, the cells were incubated with fresh medium for additional 20 hours. The polyplexes of PEI-FPBA/siLuc and PFCDM/siLuc were prepared at w/w ratios of 2, 4, 8 and 12 separately. PEI 25k/siLuc prepared at w/w 2 was set as positive control. Afterwards, the luciferase contained solutions were incubated with luciferase kit and the luciferase activity was measured by a microplate reader (BioTek Synergy2, America). Enhanced green fluorescence protein silencing in vitro The siRNA transfection was also checked on EGFP (Enhanced Green Fluorescent Protein) stable expressed CHO cells (CHO-EGFP). CHO-EGFP cells were seeded in black 96-well plates. Later, different w/w ratios of siEGFP loaded polyplexes were prepared and CHO-EGFP cells were incubated with different formulations for 6 h. Then, the siEGFP contained medium were replaced with fresh medium and the cells were cultured for another 44 h. Here, the silencing efficiency of free siEGFP and PEI 25k/siEGFP (w/w 2) were also performed. The EGFP expression was measured by FCM and fluorescence microscopy



separately. The EGFP expression level was calculated as: The mean fluorescence intensity of sample group / The mean fluorescence intensity of PBS group × 100%. Luciferase silencing in vivo The 4T1-Luc mice model was constructed by injecting 4T1-Luc cells subcutaneously into the female BABL/c mice (1× 105 cells/mouse). The mice whose tumor size reached 200 mm3 were selected for experiment. Here, the mice were divided into 4 groups, 3 mice of each group: PBS group, PFCDM/siScr, PEI-FPBA/siLuc and PFCDM/siLuc (all of the polyplexes were prepared at w/w ratio of 8). The mice were intratumorally injected with different polyplexes solutions (100 µL, 1.2 mg/kg siRNA) on Day 0 and Day 1. Luciferase bio-images were taken on Day 0 and Day 2. Before imaging, mice were intraperitoneally injected with 200 µL D-luciferin (15 mg/mL) to make luciferase visible and the images were obtained by Tanon 5200 multi-imaging system. On Day 2, the luciferase in tumors was extracted and detected by luciferase assay kit after taken images.

Statistical analysis All quantitative data are expressed as mean ± SD, unless otherwise noted. Data were analyzed using two-tailed Student's t-tests for 2 groups.

RESULTS AND DISCUSSION

Synthesis and characterization of PEI-FPBA and Chol-Dopa The synthesis procedure of PEI-FPBA was shown in Figure 1A. The carboxyl of FPBA was activated by EDC. The primary amine group of PEI was then reacted with the activated FPBA. The PEI-FPBA product was purified by dialysis against distilled water to remove catalyzer and unreacted FPBA. The successful conjugation of FPBA on PEI was validated by


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1

H NMR. As shown in Figure S1, the peaks occurred at 7.6−7.2 ppm could be ascribed to the

phenyl proton signals of FPBA and the peaks appeared at 3.0−2.0 ppm belonged to the ethylene proton signals of PEI. Substitution degree of FPBA to primary amine on PEI was 39.4% as calculated from the peak integrations. The Chol-Dopa was obtained by the reaction between cholesterol chloroformate and dopamine. In 1H NMR result, the peak of 6-7 ppm was assigned to the phenyl proton signals of dopamine and the peak of 0-2 ppm was assigned to cholesterol, indicating the successful synthesis of Chol-Dopa.

Figure 1. Synthesis procedures of (A) PEI-FPBA and (B) Chol-Dopa.

Formation of cationic micelles The amphiphilic micelles (PFCDM) were formed by the hydrophilic cationic PEI-FPBA and hydrophobic Chol-Dopa via the reaction between PBA and diol. The driving force was relied on the unique features of PBA which could spontaneously react with diol at mild condition.



The successful formation of borate ester bond between FPBA and dopamine was evaluated by the fluorescence quenching of PEI-FPBA-ARS. ARS is common used reagent for indicating the reaction between PBA and diols. The fluorescence of ARS was only generated upon the formation of borate ester with PEI-FPBA.32-33 Chol-Dopa would compete the biding with PEIFPBA and lead to the fluorescence decreased. Therefore, we can evaluate the conjugation of Chol-Dopa on PEI-FPBA by the quenching of the fluorescence. As shown in Figure 2A, upon the increased Chol-Dopa added, fluorescence declined. The molar ratios of PEI-FPBA : Chol-Dopa was optimized by the particle size. As shown in Figure 2B, the particle size condensed with the increasing molar ratios from 0 to 1. The introduction of hydrophobic component condensed the nanoparticles. However, the micelles swelled with more Chol-Dopa


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added, which could be ascribed to that the excess hydrophobic ligand decreased the solubility of the

Figure 2. (A) Fluorescence spectra changing of PEI-FPBA-ARS solution with the addition of Chol-Dopa. (B) Particle size determination of PFCDM at various molar ratio of Dopa : FPBA. (C) Size distribution and morphology observation of PFCDM. (D) Zeta potential of PFCDM.

micelles. Therefore, we chose the ratio of 1:1 for further research. The morphology of the PFCDM was observed by TEM as sphere and the particle size was around 100 nm, which was a little smaller than the DLS result. The zeta potential was 56 mV as detected, which was



owing to the formation of micelles and the condensed positive charge in the surface of the particles.

Figure 3. (A) Particle size and (B) zeta potential of PEI-FPBA/siRNA, PFCDM/siRNA polyplexes characterization. (C) siRNA encapsulation detected by agarose gel electrophoresis.

Characterization of polyplex We firstly evaluate the particle size and zeta potential of the polyplexes at w/w ratios of 2, 4, 8 and 12. As shown in Figure 3A, the particle size of PEI-FPBA/siRNA swelled from 120 nm to 225 nm with the increasing w/w ratios from 2 to 12. The driving force of the formation of polycation/siRNA was electrostatic interaction. With the w/w ratio increased, the polycation was relative excess and the siRNA could not form compact particles with it, leading to the increased particle size. However, the size of PFCDM/siRNA seems to have no changing with the increased w/w ratios, because the micelles were mainly relied on the ACS Paragon Plus Environment

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formation of hydrophobic core of the amphiphilic PEI-FPBA-Dopa-Chol. Therefore, siRNA loading showed weak affect on the particle size. Zeta potential of PFCDM/siRNA was much higher than PEI-FPBA/siRNA at the same w/w ratio (Figure 3B), because the formation of micelles enhanced the charge density of polycations. The siRNA encapsulation was determined by agarose gel electrophoresis. As shown in Figure 3C, PEI-FPBA could encapsulated all of the siRNA at w/w ratio of 1. Delightedly, PFCDM improved the siRNA loading ability which could encapsulated all of the siRNA at w/w 1. The improved siRNA encapsulation was owing to the formation of micelles and the condensing of the charge density as shown in Figure 3B.

ATP-triggered release of siRNA The environmental diols could also react with FPBA and destroy the FPBA-Dopa binding. ATP, the most abundant ribonucleotide used in cells, contains diols moieties and can react with FPBA in the intracellular environment. Besides, ATP is negatively charged in the cell which inspired us to make the hypothesis that the conjugation of ATP on PEI-FPBA may reverse the charge of PEI-FPBA from positive to negative. Firstly, three triggers (ATP, dATP and Adenosine) were used to incubate with the polyplexes to evaluate the role of diols and phosphates on ATP. Here, ATP was the trigger containing diols and phosphates, dATP as the diols loss trigger and adenosine as the phosphates loss trigger. As shown in Figure 4A, ATP could effectively trigger the siRNA release of both polyplexes, nearly all of siRNA released from carriers. For PEI-FPBA/siRNA polyplex, neither the dATP nor the adenosine exhibited weak affection on polyplexes, showing as the dim bands after treatment. The importance of diols and phosphates on ATP triggered siRNA release was validated here. For PFCDM/siRNA polyplexes, dATP treatment micelles showed similar result as PEI-FPBA/siRNA while adenosine triggered more release of siRNA from PFCDM/siRNA than PEI-FPBA/siRNA. The adenosine could compete the ACS Paragon Plus Environment


binding with FPBA leading to the disassembly of micelles and weaken the siRNA loading ability of PFCDM/siRNA, resulting in the leakage of siRNA.

Figure 4. Charge and assembly reversible micelles fueled by ATP for controlled release of siRNA. (A) The siRNA release behavior of PEI-FPBA/siRNA and PFCDM/siRNA with the treatment of ATP, dATP and adenosine. (B) FRET ratio changing of cy3-and cy5-siRNA coloaded polyplexes with the treatment of ATP, dATP and adenosine. Data are shown as mean ± SD (n = 3). *p < 0.05. (C) ATP triggered charge reversal of PEI-FPBA/siRNA and


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PFCDM/siRNA. (D) ATP triggered the disassembly of PFCDM/siRNA. (E) Morphology observation of PFCDM/siRNA with or without the treatment of 4 mM ATP.

We also took FRET technology to evaluate the sensitivity of polyplexes to different triggers.34 The FRET only generated when cy3- and cy5-siRNA were coloaded in the polyplexes which means the decreased FRET ratio could indicate the siRNA release. As shown in Figure 4B, with the ATP treatment, FRET ratios of both polyplexes decreased sharply to about 20%. The decrease of FRET ratio of PFCDM/siRNA was larger than PEIFPBA/siRNA, which was in corresponding with the agarose gel result. Besides, dATP exhibited weak affection on both polyplexes. Recently, ATP triggered the cleavage of boronate bond and the disassembly of nanoparticles were the mainly mechanisms for PBA based drug/gene delivery system. From the above data we could find that the phosphates on ATP also played crucial role in triggering siRNA release. We measured the zeta potential of PEI-FPBA and PFCDM separately with increasing ATP treatment. As shown in Figure S2, the zeta potential of both groups dropped sharply and even reversed from positive to negative when the ATP concentration reached 4 mM. Next, we also checked whether the formation of polyplexes with siRNA would affect the charge reversal behavior. As shown in Figure 4C, the zeta potential changing of polyplexes showed similar trends as the polymer. It turned to be negative when the ATP concentration reached 4 mM. This result proved our hypothesis that ATP could effectively trigger the charge reversal of PEI-FPBA due to the formation of PEI-FPBA-ATP (Scheme 2B). The ATP triggered disassembly of the micelles structure was evaluated by FRET technology. Hydrophobic dyes DiO (donor) and DiI (acceptor) were coloaded into the PFCDM to form FRET micelles. The FRET signal was only generated when the two dyes were coloaded in the micelles. Thus, the decreasing FRET signal could represent the disassembly of micelles. As shown in Figure 4D, with the increasing ATP added into the ACS Paragon Plus Environment


micelles solution, the FRET ratio decreased sharply, indicating that ATP could effectively trigger the disassembly micelles. The morphology of PFCDM/siRNA with or without ATP treatment was also studied. As shown in Figure 4E, PFCDM/siRNA was sphere and exhibited core-shell structure of micelles. With 4 mM ATP treatment, the particles reached around 500 nm and displayed as two main structures: a) Some particles were still core-shell structure. However, the hydrophilic shell layer became thicker than untreated micelles. The thicker shell was probably ascribed to the absorption of hydrophilic ATP on the surface by FPBA-ATP reaction; b) The others were black solid particles as could be ascribed to the formation of new particles of PEIFPBA-ATP. Because the negatively charged phosphate on ATP may bind with positively charged PEI by electrostatic interaction, leading to the formation of new particles. Taken together, ATP was proved to act as fuel to trigger the release of siRNA from PFCDM/siRNA by the disassembly of the micelles and charge reversal of PEI-FPBA, both of which would accelerate the siRNA release. Stabilities of PEI-FPBA/siRNA and PFCDM/siRNA The stabilities of polyplexes in the presence of DW, PBS, glucose and serum were studied here. In the blood circulation, strong anions, high concentrations of glucose or serum were the main barriers that hurdle the successful siRNA delivery.35-37 The formation of nanomicelles was designed to improve the stabilities. As shown in Figure 5A, the DLS size of PEIFPBA/siRNA became larger with the incubation with PBS and serum, shown poor stability in PBS and serum. Comfortingly, PFCDM/siRNA exhibited excellent stability in the presence of both PBS and serum (Figure 5B). The PEI-FPBA/siRNA polyplex was mainly assembly by electrostatic attraction, therefore, it could be easily destroyed by the anions or negatively charged serum. However, the micelles were assembly by the formation of hydrophobic core, improving the stabilities in the negatively charged environment.


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The leakages of siRNA in the presence of DW, PBS, glucose and serum were validated by FRET. As shown in Figure 5C, the FRET ratio of PEI-FPBA polyplex decreased a lot with the treatment of both PBS and serum, 78% and 56% of each. In corresponding with the stability result, PFCDM/siRNA showed improve stability compared with PEI-FPBA/siRNA. The FRET ratios after PBS or serum treatment were 92% and 86% separately. The siRNA was easily to be

Figure 5. Stabilities evaluation of (A) PEI-FPBA/siRNA and (B) PFCDM/siRNA in the presence of DW, PBS, Glucose or Serum by DLS size monitoring. (C) FRET ratio changing of cy3-and cy5-siRNA coloaded polyplexes with the treatment of DW, PBS, Glucose or Serum. Data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01. (D) Serum stability of PEI-FBA/siRNA and PFCD/siRNA detected by agarose gel electrophoresis.



degradation by the RNase in the serum. Therefore, we further research the serum stability by agarose gel electrophoresis to evaluate the RNase degradation of siRNA in the polyplexes. As depicted in Figure 5D, naked siRNA was degraded within 2 hours with the incubation of serum. The polyplexes were also treated with serum and the sample were taken at time intervals. After incubation, SDS was added to replace the undegraded siRNA. We could find from the result that obvious degradation of siRNA in PEI-FPBA/siRNA group was observed, shown as weak band after 24 h incubation. However, for micelles group, the bands maintained strong within 24 h, exhibiting good siRNA protection effect. Therefore, the formation of micelles significantly improved the stability of polyplexes and effectively avoided the degradation of siRNA in the serum, which showed potential for in vivo siRNA delivery.

Cytotoxicity High molecular weight polyamines used to rise high toxicity. Here, low molecular weight PEI was thought to alleviate the toxicity on cells.38-40 We evaluated the cell toxicity of PEIFPBA and PFCDM on 4T1 cells, where 25k PEI was set as control. As shown in Figure 6A, the 25k PEI was so toxic to cells. The cell viability was lower than 50% when the concentration was only 20 µg/mL. Excitingly, both PEI-FPBA and PFCDM showed weaker toxicity on 4T1 cells, because low molecular weight PEI was more easily to be degraded in the cell. However, the PFCDM exhibited higher toxicity than PEI-FPBA, which could be owing to the strong positive charge on the surface of PFCDM. We next evaluated the cell toxicity of siScr loaded polyplexes to optimize the safety w/w ratios for transfection. The cell viability of both PEI-FPBA/siScr and PFCDM/siScr were over 90% even at w/w ratio reached 12 (Figure 6B). Therefore, we choose the w/w ratios of 2, 4, 8 and 12 for further research.


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Figure 6. (A) Cytotoxicity of 25k PEI, PEI-FPBA and PFCDM on 4T1 cells by MTT assay. (B) Cytotoxicity of PEI-FPBA/siRNA and PFCDM/siRNA on 4T1 cells at various w/w ratios.

Cell uptake The uptake of polyplexes on 4T1 cells were studied by CLSM and FCM. siRNA was labelled with FAM and encapsulated in polyplexes. As shown in Figure 7A, the green florescence of PFCDM/FAM-siRNA was obviously stronger than PEI-FPBA/siRNA, showing higher uptake ratio. The higher zeta potential of PFCDM may assist the cellular uptake of polyplexes. It was also validated by FCM in Figure 7B, the MFI (mean fluorescence intensity) of PFCDM group was extremely higher than PEI-FPBA group, approximately 2 folds of PEI-FPBA/FAM-siRNA. Therefore, the formation of micelles improved the cell uptake of PEI-FPBA/siRNA polyplexes. Endosomal/lysosomal escape The endo/lysosomal escape of PFCDM/siRNA was observed by CLSM. Endo/lysosomes were stained with red fluorescence dyes and siRNA labelled with FAM (green fluorescence dye). If the FAM-siRNA was trapped into the endo/lysosomes, the green signal would overlap with red signal, showing yellow color of the merged images. Once the FAM-siRNA escaped from the endo/lysosomes, the green signals would separate with the red signals. As shown in



Figure 7C, after 3 h incubation with polyplexes, the green signal of siRNA exhibited obvious overlap with the red endo/lysosomes signals, showing as strong yellow color in the merged images. However, after additional 3 h incubation, most of the green signal separated with the red signal. Besides, the green spots seemed to be diffused in the cell, validating the successfully endo/lysosomal escape. Intracellular ATP-dependent siRNA release Fluorescence resonance energy transfer (FRET) was applied to evaluate the intracellular ATP triggered release of siRNA from PFCDM/siRNA polyplex. In addition, the cells were incubated at a low temperature (4 °C) or in the presence of iodoacetic acid (IAA) at 37 °C to construct the low ATP model of 4T1 cells. Here, cy3 (green) was used as the donor and the cy5 (red) was used as the acceptor in the cy3-cy5 FRET pair. Cy3- and cy5-siRNA were coloaded within the FRET polyplex. When the FRET dyes were coloaded in the polyplex, the energy of cy3 could transfer to cy5, showing strong cy5 fluorescence. Correspondingly, the fluorescence of cy5 turns to be dim after siRNA released for the breaking up of the energy transmit. 4T1 cell lines were incubated for 4 h with FRET polyplex, then incubated with fresh medium or at ATP downregulation conditions for an additional 4 h. As shown in Figure 8, the normal cells with normal ATP concentration showed strong cy3 fluorescence signal and weak cy5 signal which indicated the successful disassembly of PFCDM/siRNA in the cells. However, after ATP depletion, the cells exhibited high cy5 signal which validated the good integrity of FRET polyplex. Thus, we could find that the release of siRNA in the cells was ATP-dependent. Without the fuel, the siRNA release was difficult in the cell.


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Figure 7. Cell uptake of PEI-FPBA/FAM-siRNA and PFCDM/FAM-siRNA in 4T1 cells (A) observed by CLSM, and (B) detected by FCM. Data are shown as mean ± SD (n = 3). ***p < 0.005. (C) Endosomal escape of PFCDM/FAM-siRNA in 4T1 cells 3 h, 6 h after incubation.



Figure 8. Intracellular ATP-dependent siRNA release. 4T1 cells were treated with PFCDM/cy3- and cy5-siRNA polyplex and observed by CLSM, incubating at 37 °C, 4 °C, and with iodoacetic acid (IAA) at 37 °C.

siRNA transfection and gene silencing in vitro From the above result we could find that the prepared micelles showed strong cell uptake on 4T1 cells, achieved successful endosomal escape and selectively release the siRNA in the ATP abundant cytosolic environment. We next evaluated the siRNA silencing ability of polyplexes. The siRNA transfection and gene silencing were evaluated on 4T1-Luc cells and CHO-EGFP cells. As shown in Figure 9A, with the increased w/w ratios from 2-12, the luciferase silencing efficiency improved of both polyplexes. In addition, PFCDM/siLuc showed better performance than PEI-FPBA/siLuc. The luciferase expression of 4T1-Luc cells was only 19.8% and 39.6% separately after PFCDM/siLuc and PEI-FPBA/siLuc treatment at w/w 12, much better than positive control of PEI 25k/siLuc. The advantages of cationic micelles were more clearly when the transfection was performed in 10% FBS and 30% FBS ACS Paragon Plus Environment

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contained medium (Figure 9B). The siRNA transfection efficiency decreased sharply of 25k PEI or PEI-FPBA group, which was because of the poor stability in serum validated in Figure 5. Comfortingly, the PFCDM/siLuc was relatively less affected by the FBS. The siRNA transfection efficiency was also evaluated on CHO-EGFP cells. We firstly use FCM to analyze the expression of EGFP by calculating the mean fluorescence intensity. The result shown in Figure 9C indicated the similar silencing efficiency as on luciferase of 4T1 cells. The silencing efficiency between PEI-FPBA/siEGFP and PFCDM/siEGFP showed significant difference at all of the measured w/w ratios. The EGFP expression was 37.2% and 15.6% separately with the treatment of PEI-FPBA/siEGFP and PFCDM/siEGFP at w/w 12. The fluorescence images shown in Figure 9D was corresponding with the result detected by FCM. To conclude, PFCDM/siRNA displayed excellent siRNA transfection efficiency in the presence or absence of FBS.



Figure 9. Luciferase silencing of PEI-FPBA/siLuc and PFCDM/siLuc in the absence of FBS. Data are shown as mean ± SD (n = 3). **p < 0.01. (B) Luciferase silencing of PEIFPBA/siLuc and PFCDM/siLuc in 10% FBS or 30% FBS contained medium. Data are shown as mean ± SD (n = 3). NS means not significant different, *p < 0.05, **p < 0.01, ***p < 0.005 vs PBS group (set as 100%). EGFP silencing mediated by PEI-FPBA/siEGFP and PFCDM/siEGFP at different w/w ratios (C) detected by FCM and (D) imaged by fluorescence microscopy, Scale bar = 400 µm. Data are shown as mean ± SD (n = 3). **p < 0.01.


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In vivo luciferase silencing The silencing ability of polyplexes was also studied in vivo. 4T1 tumor model was constructed on BABL/c mice and the polyplexes were injected when the tumor volume reached 200 mm3. As shown in Figure 10A, PFCDM/siScr didn’t downregulate the expression of luciferase, indicating that the carrier has no non-specific silencing on luciferase. When the mice were injected with PFCDM/siLuc, notable changing of the bioluminescence intensity was observed from the images. The bioluminescence intensity of each mice was also analyzed by image J (Figure 10B). The luciferase expressions after PEI-FPBA/siLuc and PFCDM/siLuc treatment were 51.2% and 19.6% separately. At Day 2, the tumors of each mice were collected. Afterwards, the luciferase was extracted and measured by luciferase kit. The detected result matched well with the bio-images analysis (Figure 10C). Therefore, the siRNA transfection ability of PFCDM/siRNA was also superior than PEI-FPBA/siRNA in vivo.



Figure 10. In vivo luciferase silencing. (A) Luciferase bio-images of 4T1-Luc tumor bearing mice on Day 0 and Day 2. (B) Semiquantitative analysis of luciferase expression of the bioimages using image J. (C) The luciferase expression analysis of ex-vivo tumor detected by luciferase assay kit. Data are shown as mean ± SD (n = 3). **p < 0.01, ***p < 0.005.


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CONCLUSION In this study, we developed a new siRNA delivery system which maintain the balance between extracellular stable and intracellular unstable statements. The formation of micelles PFCDM enhanced the siRNA loading ability of PEI-FPBA and improved the stabilities in the presence of PBS or serum. Furthermore, in the intracellular environment, the PFCDM underwent disassembly of micelles structure and charge reversal of separated PEI-FPBA fueled by ATP, both of which promoting the siRNA release. The ATP, as the fuel, played two roles here by the corresponding moiety on ATP of diols and triphosphates. The enhanced serum stability, cell uptake and intracellular ATP triggered siRNA release of PFCDM/siRNA finally resulted in the good performance on in vitro and in vivo siRNA transfection. This delivery system is easily constructed and pretty effective. In addition, it only exhibited weak cytotoxicity compared with 25k PEI. Thus, it held tremendous potential to overcome the problems of polycations and to be developed for siRNA transfection.

ACKNOWLEDGEMENTS This work was financially supported by the National Science and Technology Major Project (2017YFA0205400), the National Natural Science Foundation of China (No. 81573377), The Jiangsu Fund for Distinguished Youth (BK20170028).

Supporting Information.

1

H NMR spectra of PEI, PEI-FPBA and Chol-Dopa; ATP

triggered charge reversal of PEI-FPBA and PFCDM.



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Charge and Assembly Reversible Micelles Fueled by Intracellular ATP

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