ATP-activated decrosslinking and charge-reversal vectors for siRNA delivery and cancer therapy

Zhanwei Zhou; Qingyan Zhang; Minghua Zhang; Huipeng Li; Gang Chen; Chenggen Qian; David Oupicky; Minjie Sun

doi:10.7150/thno.26889

. 2018 Sep 9;8(17):4604–4619. doi: 10.7150/thno.26889

ATP-activated decrosslinking and charge-reversal vectors for siRNA delivery and cancer therapy

Zhanwei Zhou ¹, Qingyan Zhang ¹, Minghua Zhang ¹, Huipeng Li ¹, Gang Chen ¹, Chenggen Qian ¹, David Oupicky ^1,^2,^✉, Minjie Sun ^1,^✉

PMCID: PMC6160761 PMID: 30279726

Abstract

Stimuli-responsive polycations have been developed for improved nucleic acid transfection and enhanced therapeutic efficacy. The most reported mechanisms for controlled release of siRNA are based on polyelectrolyte exchange reactions in the cytoplasm and the degradation of polycations initiated by specific triggers. However, the degradation strategy has not always been sufficient due to unsatisfactory kinetics and binding of cationic fragments to siRNA, which limits the gene silencing effect. In this study, a new strategy that combines degradation and charge reversal is proposed.

Methods: We prepared a polycation (CrossPPA) by crosslinking of phenylboronic acid (PBA)-grafted 1.8k PEI with alginate. It was compared with 25k PEI, 1.8k PEI and 1.8k PEI-PBA on siRNA encapsulation, ATP-responsive behavior and mechanism, cytotoxicity, cell uptake, siRNA transfection, in vivo biodistribution and in vivo anti-tumor efficacy. The in vitro and in vivo experiments were performed on 4T1 murine breast cancer cells and 4T1 tumor model separately.

Results: The crosslinking strategy obviously improve the siRNA loading ability of 1.8k PEI. We validated that intracellular levels of ATP could trigger CrossPPA disassembly and charge reversal, which resulted in efficient and rapid siRNA release due to electrostatic repulsion. Besides, CrossPPA/siRNA showed strong cell uptake in 4T1 cells compared with 1.8k PEI/siRNA. Notably, the cytotoxicity of CrossPPA was pretty low, which was owing to its biodegradability. Furthermore, the crosslinked polyplexes significantly enhanced siRNA transfection and improved tumor accumulation. The high gene silencing ability of CrossPPA polyplex led to strong anti-tumor efficacy when using Bcl2-targeted siRNA.

Conclusion: These results indicated that the ATP-triggered disassembly and charge reversal strategy provided a new way for developing stimuli-responsive siRNA carriers and showed potential for nucleic acid delivery in the treatment of cancer.

Keywords: ATP-responsive, charge reversal, phenylboronic acid, siRNA delivery, cancer therapy

Introduction

RNAi technology has attracted tremendous interest among academic and industry investigators for its potential to treat many serious diseases ¹. Successful delivery vectors must be able to transport siRNA to the target site effectively and unload the cargo timely ².

In non-viral gene delivery, cationic polymers are generally used to form polyplexes with negatively charged siRNA by electrostatic interactions ³. However, polymer/siRNA polyplexes are thermodynamically stable, which greatly weaken the gene silencing efficiency because only the released siRNA could silence the target protein ⁴^,⁵. Hence, maintaining the balance between effective siRNA loading and controlled release plays a critical role in developing a siRNA delivery system. Many strategies have been utilized to facilitate controlled intracellular release of siRNA, including preparing stimulus-responsive polymers that degrade into fragments and therefore lower the charge density ⁶^,⁷^,⁸. Although this strategy could accelerate the release of siRNA, positive groups remaining in the short chains still interact with siRNA and affect gene silencing. In this study, a crosslinked cationic polymer was developed that could degrade into short fragments and achieve fast charge reversal from positive to negative with the stimulus of ATP (adenosine triphosphate) in the cytosol.

ATP, the most abundant ribonucleotide in cells, provides energy for cellular proliferation and metabolism ⁹. ATP is present in low concentrations (<0.4 mM) in the extracellular environment, but it is relatively concentrated within the cytosol (1-10 mM) ¹⁰^-¹³. Therefore, the intra/extracellular gradient of ATP inspired researchers to use it as a trigger for ATP-responsive nanocarriers ¹⁴^,¹⁵. Prof. Kataoka's group reported PBA-functionalized polyion complex (PIC) micelles for ATP-triggered release of siRNA. In that study, ATP was used as a trigger for breaking up the borate ester bond between PBA and siRNA and therefore promoting siRNA release ¹⁴. As we know, the negatively charged phosphate group in ATP also shows potential for drug delivery system design in tumor therapy. In this work, we desired to study the role of ATP's negative charge on triggering siRNA release. The pKa of ATP's primary and secondary phosphate groups are 1-2 and 6.5, respectively ¹⁶^,¹⁷ and the cytosol pH in tumor cells is around 6.8-7.2 ¹⁸^,¹⁹. Hence, the ATP is negatively charged in the biological environment (both intracellular and intracellular space).

ATP maintains negative charge in the cytosol, which further gave us the evidence that ATP binding in cytosol could reverse the carrier's zeta potential from positive to negative and should bring about the disassembly of polyplexes. The charge repulsion could trigger release of negatively charged therapeutic agent (such as siRNA, DNA or protein) from a cationic carrier. ATP, glucose and fructose were reported to have a unique ability to quickly form a boronic ester bond by dynamic chemical reaction with phenylboronic acid (PBA) due to their cis-1,2- or -1,3-diols ²⁰^,²¹. The binding between saccharides and PBA is highly sensitive to pH ²², other sugars ²³, and H₂O₂ ²⁴^,²⁵, making PBA moieties a suitable choice in stimuli-responsive drug delivery systems.

Taking these properties into consideration, we explored a crosslinked polymer based on PBA and saccharides, which was designed to remain stable in the blood circulation and disassemble in the cytosol by high intracellular concentration of ATP. The rapid rate of the reaction between PBA and ATP could accelerate the ATP binding progress and assist the disassembly of the polyplexes. The condensed negatively charged ATP around the polycations would reverse the charge from positive to negative, finally resulting in fast release of siRNA from carriers in the cytosol (Scheme 1). In addition, it has been reported that phenylboronic acid-modified nanoparticles can significantly enhance the tumor targeting delivery of anti-cancer drugs/gene, due to reaction of PBA with sialic acid (SA) over-expressed on the tumor cells ²⁶^,²⁷. Therefore, PBA shows potential to be used as a ligand for tumor targeting in this study.

Scheme 1 — **(A)** Schematic illustration of the formation of ATP-responsive charge reversal crosslinked polyplex. **(B)** Schematic illustration of tumor-targeted siRNA delivery by CrossPPA/siRNA polyplex: (a) tumor accumulation of CrossPPA/siRNA through passive and active targeting; (b) cell uptake of polyplex mediated by sialic acid; (c) endosomal escape of polyplex; (d) ATP-activated decrosslinking and charge reversal of CrossPPA, leading to siRNA release in the cytosol.

Herein, alginate was used as the diol linker to prepare crosslinked cationic polymers (CrossPPA) by forming borate esters between PBA and alginate. Low molecular weight polyethyleneimine (PEI 1.8k) was chosen as the cationic component for its low cytotoxicity. The crosslinking strategy condensed the charge of PEI 1.8k, improving its siRNA loading ability. PBA modification promoted cellular uptake and tumor targeting of the polyplex. Furthermore, intracellular ATP selectively triggered the release of siRNA. We also demonstrated a new mechanism of siRNA release from PBA-diol crosslinked polycations undergoing disassembly and charge reversal. The role of ATP's diols and phosphate group in accelerated siRNA release was clarified in this study. The crosslinked polyplexes effectively protected the siRNA from degradation in the serum, targeted the tumor, and released the cargo selectively in the cytosol. Our work demonstrated stimulus-responsive gene release by the combination of degradation and charge reversal, which shows strong potential for in vivo siRNA delivery and tumor therapy.

Methods

Materials, cell culture and animals

Branched polyethyleneimine (1.8k PEI and 25k PEI), alginate (270 kDa), 4-carboxyphenylboronic acid (PBA) and EDC.HCl (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) were purchased from Aladdin chemical regent company (Shanghai, China). Trypsin EDTA solution, penicillin streptomycin solution, RPMI-1640 medium and MTT were purchased from KeyGen biotech (Nanjing, China). The Annexin V-FITC/PI Apoptosis Detection Kit (Cat No.40302) and Super ECL Detection Reagent (Cat No.36208) were bought from Yeasen company (Shanghai, China). Fetal bovine serum (FBS) was purchased from Gibco (Thermo Fisher Scientific, USA).

siNC (negative control), FAM-siNC, cy3-siNC, siLuc (targeting luciferase) and siBcl2 (targeting Bcl2) were synthesized by GenePharma (Shanghai, China). Cy5-siNC was synthesized by RiboBio (Guangzhou, China). Bcl2-specific antibody (50E3) rabbit mAb, β-tubulin (9F3) rabbit mAb and HRP-linked anti-rabbit IgG were purchased from Cell Signaling Technology. The siRNA sequences are below:

siNC, sense strand, 5'-UUC UCC GAA CGU GUC ACG UTT-3'

siLuc, sense strand, 5'-GGA CGA GGA CGA GCA CUU CUU-3'

siBcl2, sense strand, 5'-GCA UGC GAC CUC UGU UUG AdTdT-3'

4T1 mouse breast cancer cells were obtained from ATCC (American Type Culture Collection). Female BABL/c mice (18-22 g) were bought from Yangzhou University. All the animal experiments were performed in compliance with the Guide for Care and Use of Laboratory Animals and were approved by China Pharmaceutical University.

Synthesis and characterization of PEI-PBA

4-Carboxyphenylboronic acid was conjugated to branched PEI 1.8k via amide bonds using carbodiimide chemistry. Briefly, 50 mg of 4-carboxyphenylboronic acid was dissolved in 10 mL mixture solvent (CH₃CH₂OH:H₂O = 1:1 v:v). Then, 200 mg of EDC.HCl was added into the mixture and reacted at room temperature for 1 h. The resultant solution was dropped into 100 mg of PEI 1.8k solution and kept stirring at room temperature for 24 h, followed by dialysis (MWCO = 1000 Da) against distilled water to obtain purified PEI-PBA. The chemical structure of PEI-PBA was characterized by 1H-NMR, 13C-NMR spectra and FTIR. The PBA substitution degree was calculated based on the ratio of phenyl on PBA to the primary amine on PEI by the 1H-NMR result.

Preparation of cross-linked polycations

CrossPPA (PEI-PBA-Alginate) was prepared based on borate ester formation between the PBA group on PEI-PBA and the diol group on alginate. The conjugation between PBA and diol was performed in neutral aqueous medium without any catalyst. A typical procedure is described as follows: PEI-PBA and alginate (PEI-PBA/alginate w/w ratios of 40:1, 20:1, 10:1, 5:1, 2.5:1 were optimized by luciferase silencing on 4T1 cells) were dissolved in HEPES buffer (pH 7.4) and stirred overnight at room temperature. The product was dialyzed (MWCO 1,000 Da) against distilled water for 48 h. Then, the CrossPPA polymer was lyophilized and stored at -20 ℃ for further preparation.

Formulation of siRNA polyplexes

siRNA solution (40 μg/mL in 10 mM HEPES, pH 7.4) was mixed with different concentration of polycations (25k PEI, 1.8k PEI, PEI-PBA and CrossPPA) to optimize the desired PEI/siRNA (w/w) ratios (w/w ratio means weight of PEI / weight of siRNA in the polyplex), where 1, 2, and 4 ratios were chosen for research. The w/w ratios of 1, 2, and 4 were equal to N/P of 7.4, 14.8, and 22.2. The mixture was vigorously vortexed for 30 s and incubated at room temperature for 30 min prior to use.

To evaluate polyplex formation, the polyplexes at w/w ratios of 0, 0.2, 0.5, 1, 2, and 5 were run on a 1% (w/v) agarose gel containing GelRed at 100 V for 15 min and siRNA was visualized by UV illumination. The w/w ratios of 0, 0.2, 0.5, 1, 2, and 5 were equal to N/P of 0, 1.48, 3.7, 7.4, 14.8, and 22.2.

Particle size and zeta potential of the siRNA polyplexes were determined by a dynamic light scattering (DLS) analyzer (Brookhaven) and a ZetaPlus zeta potential analyzer (Brookhaven). The polyplexes were prepared at different w/w ratios (1, 2, 4) and diluted with 10 mM HEPES; the siRNA concentration was 5 μg/mL.

ATP-responsiveness of PEI-PBA and CrossPPA polyplex

ATP-triggered release of siRNA from the polyplex was evaluated by agarose gel electrophoresis. siRNA-loaded polyplexes of 25k PEI, 1.8k PEI, PEI-PBA and CrossPPA were prepared at a w/w ratio of 4:1. After 30 min, each polyplex solution was divided into 2 parts, which were treated with 5 mM ATP or without ATP treatment, followed by agarose gel electrophoresis analysis immediately. To evaluate whether the siRNA release was time and ATP concentration dependent, each polyplex solution was treated with various concentrations of ATP (0 mM, 0.4 mM, 1 mM, 5 mM) and monitored at time intervals (0, 5, 10, 20, 40, 60, 90, 120 min) by Ribogreen assay ²⁸. For the release mechanism study, 5 mM dATP or adenosine were also incubated with PEI-PBA and CrossPPA polyplexes, followed by agarose gel electrophoresis analysis. The selectivity of PEI-PBA and CrossPPA polyplexes response to ATP in the cell, rather than phosphate and glucose in the blood, was also validated by incubation with 10 mM PBS (pH 7.4) containing 5 mM glucose for different times and was detected by agarose gel electrophoresis.

The morphological observations of CrossPPA/siRNA before and after 5 mM ATP treatment were obtained by H-600 transmission electron microscope (TEM) (Hitachi, Japan) after negative staining with 0.1 wt% sodium phosphotungstate solution.

The charge reversal of polycations and polyplexes after ATP incubation was measured using Zeta Plus. Different polycations of 25k PEI, 1.8k PEI, PEI-PBA and CrossPPA, polyplexes (w/w 4:1) of 25k PEI/siRNA, 1.8k PEI/siRNA, PEI-PBA/siRNA and CrossPPA/siRNA were respectively prepared in 10 mM HEPES solution with a PEI concentration of 200 μg/mL. An ATP stock solution was also prepared at a concentration of 50 mM. ATP solution was added into different polycation solutions to obtain different final concentrations of ATP (0, 0.1, 0.2, 0.4, 1, 2, 4, 5 mM), followed by zeta potential measurement.

Colloidal stability and serum stability

To evaluate the colloidal stability of the crosslinked polyplex, the polyplex was incubated at 4 °C and the particle size was monitored at time intervals by DLS.

For serum stability, polyplex was incubated in 10% FBS at 37 °C and the particle size was measured at different times. The replacement of siRNA by serum was also checked by agarose gel electrophoresis.

Cytotoxicity analysis and hemolysis assay

The cytotoxicity of different polycations (25k PEI, 1.8k PEI, PEI-PBA and CrossPPA) was measured by MTT assay. Briefly, the cell lines were incubated with increasing concentrations of the polymers for 24 h. Then, the MTT reagent (5 mg/mL in PBS) was added and the cells were incubated for an additional 4 h. The solutions contained MTT were carefully removed and DMSO was added to dissolve formazan, followed by measurement of the absorbance at 570 nm.

The cytotoxicity of polyplex was also evaluated. The polyplexes composed of siNC and 25k PEI, 1.8k PEI, PEI-PBA and CrossPPA were prepared at various w/w ratios and then diluted with FBS-free medium at a siNC concentration of 100 nM. After incubation with cells for 4 h, the old medium was replaced with 10% FBS-containing medium for another 20 h. The MTT assay was performed as described above.

For hemolysis assay, red blood cells (RBCs) were obtained from healthy rats and diluted with PBS (pH 7.4) at 2% concentration. Different polyplexes (w/w 4:1) were diluted with PBS (pH 7.4) to 1 mg/mL of PEI, then incubated with RBC solution at 37 °C for 2 h; water treatment was set as positive control and PBS as negative control. After incubation, the mixtures were centrifuged at 8000 rpm for 10 min. The supernatants were collected and the absorbance (570 nm) of each sample was detected by Synergy 2 Multifunctional Microplate Reader (BioTek, USA). The percentage of hemolysis was calculated according to the following formula: hemolysis ratio (%) = (A_sample - A_negative)/(A_positive -A_negative) × 100%.

Cell uptake and endosomal escape

4T1 cells were seeded into 24-well plates and cultured for 24 h. The next day, cells were treated with Free FAM-siRNA, 1.8k PEI/FAM-siRNA, PEI-PBA/FAM-siRNA or CrossPPA/FAM-siRNA (FAM-siRNA 1.33 μg/mL, w/w 4:1, diluted with RPMI-1640 medium) for 2 h. To block the sialic acid, 4T1 cells were pretreated with 50 mM free PBA for 30 min. After incubation, the cells were washed and collected, then detected by flow cytometry (BD FACSCalibur).

For the observation of endo/lysosomal escape, 4T1 cell lines were seeded in confocal microscopy dishes and cultured for 24 h at 37 °C, followed by incubation with 1.8k PEI/FAM-siRNA, 1.8k PEI-PBA/siRNA and CrossPPA/FAM-siRNA polyplex (FAM-siRNA 1.33 μg/mL, w/w 4:1, diluted with RPMI-1640 medium) for 3 h. Afterwards, the cells were washed with PBS twice and incubated with fresh medium for 0 and 3 h, followed by washing with PBS thrice and incubating with LysoTracker Red for 40 min at 37 °C. The cells were then washed with PBS twice and immediately observed using a confocal laser scanning microscope (Carl Zeiss LSM 700, Germany).

Intracellular ATP-dependent siRNA release

To verify the intracellular ATP-dependent siRNA release, low temperature (4 °C) and iodoacetic acid (IAA) at 37 °C were used to inhibit ATP production in the cells. 4T1 cells were seeded in confocal microscopy dishes at a density of 1×105 cells per well. After culturing for 24 h, the cells were incubated with cy3-/cy5-siRNA coloaded CrossPPA polyplex (both cy3-siRNA and cy5-siRNA were 100 nM, polymer/siRNA w/w = 4:1) at 37 °C for 4 h and then washed by PBS twice, following by incubation with medium (37 °C), low temperature and iodoacetic acid (IAA) for an additional 4 h. Subsequently, the 4T1 cells were washed by PBS twice and observed via CLSM. The observation parameters of CLSM were Cy3 λ ex/em = 550/570 nm, Cy5 λ ex/em = 550/670 nm.

Luciferase silencing in vitro

Luciferase silencing assay was performed on 4T1-Luc cell lines, which stably express luciferase. 4T1-Luc cells were seeded in 48-well plates at a density of 1×104 cells per well. After culturing for 24 h, luciferase-specific siRNA (siLuc) polyplexes (25k PEI/siLuc, 1.8k PEI/siLuc, PEI-PBA/siLuc and CrossPPA/siLuc) diluted with FBS-free or 10% FBS-containing medium were added and incubated for 4 h, followed by replacement with fresh medium for an additional 20 h. Then, the luciferase activity was measured by a microplate reader (BioTek Synergy2, America) using a luciferase assay kit (Beyotime, China).

Downregulation of Bcl2 protein, cell growth inhibition and cell apoptosis assay

4T1 cells were seeded in 12-well plates at a density of 1×105 cells per well. The next day, the cells were treated with PBS, Free siBcl2, 25k PEI/siBcl2, 1.8k PEI/siBcl2, PEI-PBA/siBcl2 and CrossPPA/siBcl2 at an siBcl2 concentration of 100 nM for 4 h, followed by incubation with medium for an additional 40 h. After that, protein of each group was collected and Bcl2 protein expression was detected by Western Blot. Cell growth inhibition was measured by MTT assay. Briefly, 4T1 cells (5×103 cells per well) were seeded in 96-well plates. The next day, the same treatment as in the Bcl2 silencing assay was performed. After incubation, cell viability was detected by MTT assay.

For cell apoptosis assay, the cells (1×105 cells per well) were seeded in 12-well plates. After incubation for 24 h, the cells were transfected with siBcl2 by the same method mentioned in the cell growth inhibition experiment and were detected using the Annexin V-FITC Apoptosis Detection Kit by flow cytometry (BD FACSCalibur).

Animals and tumor model

A 4T1 tumor model was established by subcutaneous injection of 4T1 cells (1×106) into selected positions. The tumor size was monitored by a vernier caliper, and the tumor volume (V) was calculated as V=L×W2/2, where L and W are the length and width of the tumor, respectively.

Pharmacokinetics study

The SD rats were randomly divided into 4 groups: Free FAM-siRNA, 1.8k PEI/FAM-siRNA, 1.8k PEI-PBA/siRNA and CrossPPA/FAM-siRNA. The mice were intravenously injected with different formulations (FAM-siRNA, 0.15 mg/kg). The blood samples were collected at time intervals and centrifuged at 10,000 ×g for 15 min. The fluorescence intensity of FAM-siRNA in the plasma was measured by Synergy 2 Multifunctional Microplate Reader (BioTek, USA).

In vivo biodistribution

For in vivo biodistribution study, the mice were divided randomly into 3 groups (n=3). Cy5-labeled siRNA was loaded into the polyplexes for indicating the siRNA distribution. Mice were administrated with 1.8k PEI/cy5-siRNA, 1.8 PEI-PBA/cy5-siRNA and CrossPPA/cy5-siRNA polyplexes separately by i.v. injection. Caliper IVIS Lumina II in vivo image system was utilized to monitor the distribution behavior of the polyplexes and images were obtained at 6 h, 12 h, 24 h after injection. After 24 h, the tumors and main organs were harvested from the mice, followed by fluorescence imaging.

In vivo anti-tumor study

The tumor-bearing mice were weighed and randomly divided into different groups (n=6) when the tumor volume reached 60 mm3, and treated with 150 μL of physiological saline, CrossPPA/siNC, 1.8k PEI/siBcl2, PEI-PBA/siBcl2 and CrossPPA/siBcl2 polyplexes on every 4 days intravenously. All polyplexes contained 20 μg siRNA/mice per injection. The tumor size and body weight of the mice were recorded every other day. After 20 days, the tumor was harvested and Bcl2 level of each tumor was analyzed by Western blot. Additionally, H&E staining and TUNEL analysis were also performed on tumor sections.

Statistical analysis

All quantitative data are expressed as mean ± S.D. unless otherwise noted. Data were analyzed using two-tailed Student's t-test for 2 groups. Probabilities less than 0.05 were considered significant.

Results and Discussion

Synthesis and characterization of PEI-PBA

The synthesis procedure of PEI-PBA is shown in Figure S1. Briefly, the carboxyl group of 4-carboxyphenylboronic acid was activated by EDC and reacted with the amine group of PEI. After reaction, the product was dialyzed against distilled water for 48 h to remove unreacted EDC and PBA. The structure of PEI-PBA was validated by 1H-NMR, 13C-NMR and FTIR. As shown in Figure S2A, phenyl proton signals of 4-carboxyphenylboronic acid groups occurred at 7.6-7.2 ppm and the ethylene proton signals of PEI was present at 3.0-2.0 ppm. The substitution degree of PBA to PEI was 20.6% as calculated from the peak integrations. Figure S2B exhibits the 13C-NMR result. The peak between 120-140 ppm was the signal of benzene on PBA. The characteristic IR absorption bands (Figure S2C) at 1369.4 cm-1 were attributed to B-O stretching, whereas the bands at 1545.9 cm-1 (C-O stretching) and 1533.4 cm-1 (-NH-CO- stretching) were the characteristic absorbances of the amide group of PEI-PBA ²⁹. Hence, the 1H NMR and FTIR spectra confirmed the conjugation of PBA to PEI.

Characterization of polyplex

The PEI-PBA/alginate w/w ratio was optimized by the luciferase silencing efficiency of CrossPPA/siLuc on 4T1 cells. As shown in Figure S3, w/w 10:1 showed the best performance. Therefore, 10:1 of PEI-PBA/alginate was chosen for further research. Particle size and zeta potential of the polyplexes were measured by dynamic light scattering (DLS) at w/w ratios of 1, 2 and 4. As shown in Figure 1A-B, the size decreased with the increase in w/w ratio, which could be ascribed to enhanced binding of the polycation and siRNA. Furthermore, both PBA modification and alginate crosslinking decreased the particle size. The zeta potential was enhanced with increased w/w ratio. PEI-PBA showed a higher zeta potential than PEI, which was attributed to the hydrophobicity of PBA. The modification of hydrophobic moieties on PEI promoted the formation of nanoparticles and the condensed surface zeta potential ³⁰. However, it decreased after crosslinking with alginate, which was attributed to the anionic property of alginate.

Polyplexes formation and characterization. **(A)** Particle size and **(B)** ζ potential of 1.8k PEI, PEI-PBA/siRNA, CrossPPA/siRNA polyplexes prepared at different w/w ratios. The w/w ratios of 1, 2, and 4 were equal to N/P of 7.4, 14.8, and 22.2. **(C)** siRNA encapsulation assessed by agarose gel electrophoresis of polyplexes prepared at different w/w ratios. The w/w ratios of 0, 0.2, 0.5, 1, 2, and 5 were equal to N/P of 0, 1.48, 3.7, 7.4, 14.8, and 22.2.

siRNA encapsulation was determined by agarose gel electrophoresis (Figure 1C). The effects of PBA modification and crosslinking on both polyplexes formation and siRNA loading were evaluated by electrophoresis. The result indicated that 25k PEI shows superior siRNA encapsulation ability than 1.8k PEI because of its higher molecular weight. Meanwhile, CrossPPA exhibited good siRNA loading ability, which was similar to that of 25k PEI. The crosslinking between PEI-PBA and alginate was thought to form a higher MW polycation and improve siRNA encapsulation consequently.

ATP-triggered release of siRNA

PBA can reversibly bind to different diols ³¹. The CrossPPA polycation formed based on chemical binding between PBA of the PEI-PBA and diols on the alginate. Additionally, the PBA could also bind with the sugar end group of siRNA. When the cross-linked polyplex reaches a diol-rich environment, the original structure could be dissociated by forming new borate ester bonds. Since the intracellular ATP concentration in tumor cells could reach as high as 5 mM ¹², much higher than the extracellular concentration, the effect of ATP on the CrossPPA polyplex disassembly was evaluated. As shown in Figure 2A, both PEI-PBA and CrossPPA groups showed burst release of siRNA from polyplexes with the treatment of 5 mM ATP. The release of siRNA from polyplexes treated with different concentrations of ATP was also monitored at time intervals. As shown in Figure 2B, within 120 min, relatively no siRNA released from 25k PEI/siRNA or 1.8k PEI/siRNA even when the ATP concentration was as high as 5 mM. In contrast, PEI-PBA/siRNA and CrossPPA/siRNA released almost all of their siRNA within 5 min, indicating the burst release behavior. Furthermore, when PEI-PBA/siRNA and CrossPPA/siRNA were treated with 1 mM ATP, 60% siRNA released from the polyplexes within 60 min. These results demonstrated that the release of siRNA from PEI-PBA/siRNA and CrossPPA/siRNA was time and ATP concentration dependent. The burst release of siRNA from CrosPPA/siRNA could be ascribed to the decrosslinking of PEI-PBA-alginate and PEI-PBA-siRNA or charge reversal of PEI-PBA.

We hypothesized that both diol and phosphate play important roles in the release of siRNA. Therefore, the detailed release mechanism was further studied to know the effects of diol and phosphate in siRNA release and the probable procedure of that. We proposed the following release mechanism (Scheme 1): ATP competed for binding with PBA and replaced alginate and siRNA, leading to decrosslinking of CrossPPA and PBA-siRNA. Next, the negatively charged ATP reversed PEI-PBA's positive charge, resulting in release of negatively charged siRNA from the carrier because of electrostatic repulsion.

The importance of ATP binding to PBA and the ATP negative charge for siRNA release was verified by adenosine and dATP incubation. Adenosine was used as an uncharged trigger and dATP was used as a diol-free trigger. As indicated in Figure 3A, for the PEI-PBA/siRNA group, uncharged adenosine could not trigger the release of siRNA, which proved the importance of the negative charge on ATP. In addition, diol-free dATP showed only a weak siRNA release, indicating that electrostatic competition alone was not able to trigger the release of siRNA. Thus, we can conclude that the dynamic chemical binding between PBA and ATP was the requirement for siRNA release from PBA-modified polycation. The CrossPPA polyplex showed a similar result as PEI-PBA/siRNA with dATP treatment, while it showed a different result under uncharged adenosine treatment. Some siRNA released from the crosslinked polyplex due to disassembly of the crosslinks with diol moiety treatment. Adenosine could destabilize the CrossPPA crosslinking and weaken the siRNA affinity to the carrier, leading to the release of siRNA. The result indicated that diol in ATP acted as the precondition and negative charge as the key factor for accelerated release of siRNA from CrossPPA/siRNA polyplex.

**(A)** Release mechanism studied by treatment with ATP, dATP and adenosine. **(B)** Morphological observation of CrossPPA/siRNA polyplex before and after 5 mM ATP treatment. **(C)** Charge reversal of 25k PEI, 1.8k PEI, PEI-PBA and CrossPPA with different amounts of ATP added. **(D)** Reaction scheme of the charge reversal of PEI-PBA with treatment of ATP.

Phosphate and glucose in the blood could also affect the stability of polyplexes. To evaluate the selectivity of PEI-PBA and CrossPPA to intracellular ATP, their stability in phosphate and glucose was also studied. As shown in Figure S4, no siRNA release was found from the 25k PEI/siRNA group after incubation with phosphate and glucose. However, obvious leakage of siRNA was detected in the 1.8k PEI/siRNA group, which was probably because of the weak binding of 1.8k PEI with siRNA. Interestingly, PEI-PBA/siRNA and CrossPPA/siRNA maintained their stability and avoided being destroyed by phosphate and glucose, proving their selective responsive to ATP.

Morphological observation was also performed on CrossPPA/siRNA polyplex before and after ATP treatment. After treatment with 5 mM ATP, the particle size increased to around 900 nm and the structure of the polyplex became loose and diffuse (Figure 3B). The added ATP could effectively react with PBA and force the disassembly of the crosslinks, making the structure become loose. In addition, the absorbed ATP moieties on PEI-PBA could interact with other PEI-PBA chains, which would promote aggregation of PEI-PBA-ATP, as indicated by the larger particle size.

From the data shown above, we hypothesized that PEI-PBA and CrossPPA accelerated charge reversal under ATP treatment. ATP was added to PEI-PBA and CrossPPA solutions to obtain environments with different concentrations of ATP. At higher ATP concentration, the zeta potentials of PEI-PBA and CrossPPA decreased sharply while 25k PEI and 1.8k PEI decreased smoothly (Figure 3C). Furthermore, the zeta potentials of PEI-PBA and CrossPPA reversed from positive to negative when the ATP concentration was higher than 1 mM. Thus, PBA modification was proved to make the cationic polymer more sensitive to ATP and achieve faster charge reversal compared with unmodified polymer, which was due to the violent reaction between PBA and ATP, forcing enrichment of ATP in the polyplex (Figure 3D). We also measured the charge reversal of polyplexes after incubation with ATP. As shown in Figure S5, the zeta potential of both 25k PEI/siRNA and 1.8k PEI/siRNA decreased smoothly after ATP was added, indicating that binding of ATP with the polyplex was difficult. However, 1.8k PEI-PBA and CrossPPA polyplexes showed sharp decreases and even reversal of their zeta potentials. This validated that ATP could effectively trigger the charge reversal of PEI-PBA, CrossPPA and their polyplexes.